Stable biorelevant compositions and media for in vitro testing

Abstract

The invention provides significantly improved stabilised compositions for use in the preparation of in vitro biorelevant media that simulate mammalian fed or fasted gastrointestinal fluids. A method and pack that result in superior compositions have remarkably improved stability for preparing biorelevant media, reliably and reproducibly, after an extended period of storage at room temperature and above are disclosed. The resulting biorelevant media prepared from the stable composition have improved and consistent physiochemical properties for reproducible drug testing. The media are used in in vitro testing; for example, profiling, solubility, dissolution, permeability, pre-clinical and stability testing of pharmacological compounds / drugs and their dosage forms.

Description

[0001] STABLE BIORELEVANT COMPOSITIONS AND MEDIA FOR IN VITRO TESTING

[0002] Field of the Invention.

[0003] The present invention relates generally to stable biorelevant compositions and media produced therefrom for in vitro testing.

[0004] Background.

[0005] This invention is in the field of biorelevant compositions and biorelevant media for use in in vitro testing as described by Soderlind et al ("Simulating fasted human intestinal fluids: understanding the roles of lecithin and bile acids." Molecular pharmaceutics 7.5 (2010): 1498-1507). The biorelevant compositions used in this invention are manufactured as described in US8889189B2 and may be obtained accordingly by a skilled person. It should be noted, however, that Soderlind et al and US8889189B2 does not disclose the key parameters that characterise the pack, container and conditions to obtain stable biorelevant compositions and the media thereof described in the present invention.

[0006] The limitations of biorelevant compositions used to prepare biorelevant media are described by Krollik et al. (Krollik, Katharina, et al. "Increasing the robustness of biopharmaceutical precipitation assays-Part II: Recommendations on the use of FaSSIF." Journal of Pharmaceutical Sciences 111.1 (2022): 155-163). Problems with the stability and reproducibility of media prepared from scratch or media prepared from stored biorelevant compositions may adversely affect the quality and accuracy of in vitro drug tests. Biorelevant media prepared from scratch, aged or stored state of the art biorelevant compositions may result in variable colloidal particle size (e.g., micelles, mixed micelles, vesicles) and UV profile characteristics of aged media (see Figure 2: Graph A) that may also affect the consistency of experiments such as dissolution and precipitation tests, (see Figure 2: Graph C). Unstable biorelevant media can also affect the stability of oxidation sensitive labile drugs which can result in false negative stability findings.

[0007] To overcome the instability of state-of-the-art biorelevant compositions and corresponding media, Krolik’s recommendations were to either use fresh packs from the same batch or use double concentration of bile salt / lecithin compositions. However, the former solution is impractical and requires upfront purchase of unnecessarily large quantities of biorelevant compositions. Double concentrates also have limitations of still having UV responses that are too high and interfere with UV drug analysis and yet do not reflect the desired physiological concentrations of bile salts and lecithin found in the intestinal tract. The deleterious effect of high temperature storage on the stability of current biorelevant compositions are also highlighted by Krollik who demonstrates that storage of the compositions at 40°C for only 2 weeks and 4 weeks results in catastrophic and destructive changes (see Figure 1 : Table 3 and Figure 2: Graph B) to the composition and the subsequent biorelevant media prepared leading to irreproducibility of results.

[0008] In the 2018 review “The Limitations of Biorelevant Methods for Quality Control” by Grady et al., [Grady, Haiyan, et al. "Industry's view on using quality control, biorelevant, and clinically relevant dissolution tests for pharmaceutical development, registration, and commercialization." Journal of pharmaceutical sciences 107.1 (2018): 34-41], the limitations of biorelevant media are discussed. The media are used qualitatively to assess formulations - but notably not yet for quality control (QC) purposes.

[0009] Despite the widespread and increasing importance of biorelevant media, for example, in in vitro physiologically relevant dissolution tests, there is surprisingly no industrial nor pragmatic solution in the known prior art to overcome reproducibility challenges (e.g., unreliable precipitation results in two stage dissolution tests) and UV spectroscopy limitations imposed by state-of-the-art biorelevant media. These limitations prevent biorelevant media from being used reproducibly for direct UV measurements and were first identified by Kloefer et al. “Study of a standardized taurocholate-lecithin powder for preparing the biorelevant media FeSSIF and FaSSIF.” Dissolution Technol. 17.3 (2010): 6-13) but left unaddressed. Unpredictable UV changes make the prior art biorelevant media unsuitable for direct UV analysis and QC testing.

[0010] Despite these known limitations, prior art biorelevant media are being used for in vitro testing for example profiling, solubility, dissolution, pre-clinical, permeability and stability testing of drugs, biopharmaceutical drug products or microorganisms, pro-biotics or medical devices. W02020 / 201779A1 describes biorelevant compositions for use in preparing biorelevant media that can be used to simulate fed gastric fluid in in vitro drug testing.

[0011] The use of biorelevant media prepared from aged, or temperature stressed, commercially available biorelevant compositions in high density polyethylene (HDPE) containers; which is the type of container used commercially and widely used to protect biorelevant compositions; may adversely affect the results obtained, as described by the above-mentioned Krollik et al. (2022). The inconsistency in the resulting media may be seen by a change in particle size of the colloids in the biorelevant medium prepared from biorelevant compositions that are aged, when compared to the biorelevant medium prepared from fresh biorelevant compositions. Additionally, the UV spectra / characteristics may not be sufficiently reliable and reproducible to determine accurately drug content in the biorelevant media. This is especially challenging if the biorelevant medium is to be stored and used for testing up to 48hours. Furthermore, as described by Krollik et al, the stability of media prepared using prior art compositions is highly limited after first opening of the container.

[0012] Furthermore, the limited room temperature stability of biorelevant compositions held in HDPE containers require that the products are stored (long term) under refrigerated conditions; thereby seriously limiting industrial applicability and practicality. There is, therefore, a critical need for biorelevant compositions that have improved long term stability and can be stored at room temperature (25°C ± 2°C / 60% RH + 5% RH ) and above for extended periods of time, at least 6, preferably at least 12months, at least 18 or longer without there being any adverse effect on their performance when they are subsequently used to prepare biorelevant media for use in drug testing. Furthermore, given these compositions are shipped globally across different temperate and tropical zones they are often subjected to elevated temperatures for a prolonged period, more stringent stability e.g., 40°C ± 2°C / 75% RH + 5% RH for at least 3months and up to 6months are preferred. The stored compositions after first opening should not substantially change when compared with fresh compositions at any time during the intended shelf life.

[0013] It is an object of the present invention to provide a method of stabilising biorelevant compositions that overcomes the above-mentioned problems. A further object of the present invention is to provide a biorelevant composition having increased stability.

[0014] Summary of the Invention.

[0015] The present invention provides superior biorelevant compositions in selected containers and under specified conditions for preparing consistent test media. The parameters and thresholds essentially required for the methods and packs identified in this invention have not been disclosed in the known prior art.

[0016] Krollik remains silent on the pack, container and conditions to obtain stable biorelevant compositions and the media thereof described in the present invention.

[0017] A first aspect of the present invention provides a method of stabilizing a composition in a sealed container to provide a stable composition for at least 6months at 40°C, the composition comprising essentially at least one bile salt and / or acid and at least one phospholipid characterised in that: the composition has:

[0018] I. a mole ratio of bile acid and / or bile salt: phospholipid between 75:1 and <1 :1 ; wherein the bile acid and or / salt of the composition comprises:

[0019] II. at least one bile acid / bile salt selected from the group comprising:- sodium cholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium taurodeoxycholate, sodium glycodeoxycholate, sodium ursodeoxycholate, sodium chenodeoxycholate, sodium taurochenodeoxycholate, sodium glycochenodeoxycholate, sodium cholylsarcosinate, and sodium N- methyl taurocholate; and

[0020] III. the bile salt and / or acid has a purity of at least 80%w / w (on a dry weight basis); wherein the phospholipid of the composition comprises:

[0021] IV. at least one polyunsaturated phospholipid wherein the phospholipid content is at least 45%w / w phosphatidylcholine;

[0022] V. fatty acid chain(s) of the phospholipid contains at least 10%w / w polyunsaturated fatty acids; and

[0023] VI. wherein the said phospholipid is selected so that after dilution of the freshly prepared composition (containing bile salt and / or bile acid and phospholipid) to a phospholipid concentration of 0.75mM, the UV response at 234nm (10mm path cell length) is below 1.5AU (absorption units) from in particular t = 2hours up to 48hours after sample preparation; the method comprising the steps of: a) inserting the composition into a container with a gaseous mixture and / or at reduced pressure and sealing the container, wherein the molecular ratio of total phospholipid in the composition: oxygen inside said container immediately or soon after sealing is equal to 1000:100 or higher; preferably 1000:60 or higher, and b) selecting a container that when sealed will enclose the composition and the container has an oxygen transmission rate (OTR) at 40°C equal to or below 0.0250cc / sealed container / day x (multiplied by) the amount of phospholipid present in the composition in millimoles (mmol); and c) wherein the packaged composition, up to 3months; preferably up to 6months; especially up to 12months at 40°C provides, after first opening and diluting to a phospholipid concentration of 0.75m M in a predefined biorelevant phosphate buffer, exhibits a UV absorbance at 234nm (measured using a 10mm path length cell) that is below 2.0AU up to 2hours.

[0024] In preferred embodiments, the phospholipid is selected from one or a combination of USP egg phospholipids or USP NF Soybean Phosphatidylcholine. The composition may contain monoglycerides and / or a fatty acid(s). The method may further comprise adding to the composition inside the container one or more chelators, such as EDTA and / or desferrioxamine mesylate. The method may further comprise adding to the composition inside the container at least one further component selected from the group comprising:- a monoglyceride, a fatty acid, cholesterol, proteins and enzymes; wherein the mole ratio of each selected component: total unsaturated phospholipid in the composition is below 1 :1.

[0025] Preferably, the mole ratio of bile acid and / or bile salt: phospholipid in the composition is 50:1 and 1 :1.

[0026] The composition may be provided in the container with a gaseous mixture and may be provided under reduced pressure. In one embodiment, the composition is provided in the container under vacuum.

[0027] In preferred embodiments, the composition is in the form of a solid, powder or a cake. In embodiments, the method may comprise inserting the composition into a primary container and then inserting the sealed primary container into a secondary wrapping or container. Preferably, the secondary wrapping or container has a lower OTR than the primary container.

[0028] The method may further comprise opening the sealed container and using the composition contained therein for the preparation of media used for in vitro drug dissolution testing, drug solubility testing, pre-clinical drug testing, drug permeability and / or drug profiling.

[0029] A second aspect of the present invention provides a pack comprising a composition sealed in at least one primary container to provide a stable composition for at least 3months; preferably at least 6months; preferably at least 9months; especially at least 12months at 40°C, the composition comprising essentially at least one bile salt and / or acid and at least one phospholipid characterised in that: the composition has:

[0030] I. a mole ratio of bile acid and / or bile salt: phospholipid between 75:1 and <1 :1 ; wherein the bile acid and or / salt of the composition comprises:

[0031] II. at least one bile acid / bile salt selected from the group comprising:- sodium cholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium taurodeoxycholate, sodium glycodeoxycholate, sodium ursodeoxycholate, sodium chenodeoxycholate, sodium taurochenodeoxycholate, sodium glycochenodeoxycholate, sodium cholylsarcosinate, and sodium N- methyl taurocholate; and

[0032] III. the bile salt and / or acid has a purity of at least 80% w / w (on a dry weight basis); wherein the phospholipid of the composition comprises: IV. at least one polyunsaturated phospholipid and the phospholipid content is at least 45% w / w phosphatidylcholine; and

[0033] V. fatty acid chain(s) of the phospholipid contains at least 10% w / w polyunsaturated fatty acids; wherein the configured sealed container has: an oxygen transmission rate (OTR) at 40°C of: <0.0250cc / sealed container / day x (multiplied by) the amount of phospholipid present in the composition in millimoles (mmol); preferably <0.0125cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.0100cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.005cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.003cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); especially <0.001 cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol). and wherein the composition and any gaseous mixture inside the sealed container has the following characteristics: the molecular ratio of total phospholipid in the composition: oxygen of any gaseous mixture inside said container immediately after sealing is equal to 1000:100; preferably 1000:60 or higher.

[0034] In preferred embodiments, the composition in the sealed container provides, after first opening of the container stored for at least 3months; preferably at least 6months; preferably at least 9months; especially at least 12months; especially for at least 24months at 40°C and dilution of the composition in a predefined biorelevant phosphate buffer to a predefined phospholipid concentration of 0.75mM, a UV response at 234nm (1 Omm path cell length) that is below 2.0AU, preferably 1 .8AU from t=0 up to 2hours after sample preparation.

[0035] The composition inside the container may comprise at least one further component selected from the group comprising:- a monoglyceride, a fatty acid, cholesterol, proteins and enzymes; wherein the mole ratio of each selected component: total unsaturated phospholipid in the composition is below 1 :1 ; and / or one or more chelators, preferably being EDTA and / or desferrioxamine mesylate.

[0036] The primary container of the pack may be in the form of a bottle, pouch or sachet. The primary container may be a rigid container, the material of the container preferably being selected from HDPE, multilayered plastic (HDPE, UV layer, EVOH / HDPE, HDPE), aluminium (lacquered or uncoated) and / or glass. Alternatively, the primary container may be a flexible container, preferably selected from e.g., stick sachets, pouches, and / or sachets constructed from materials such as METPET (e.g. AlOx-coated PET), Silicon Oxide (SiOx)-coated PET, METBOPP or trilaminate aluminium (preferably at least 7 or at least 9 micrometre thickness), more preferably being sealed by suitable sealing means, such as heat sealing. The description of pouches includes sachets and stick sachets. The structure may be either stand up or preferably flat.

[0037] In embodiments, the pack may further comprise a secondary wrapping or a secondary container surrounding the primary container, the secondary wrapping or container having a lower OTR than the primary container.

[0038] A third aspect of the present invention provides the use of the pack according to the second aspect of the invention for preparing media from the composition contained in the sealed container for in vitro drug dissolution testing, drug solubility testing, drug permeability and / or drug profiling.

[0039] A fourth aspect of the present invention provides a method of preparing an aqueous medium containing at least one buffer component and at least one osmotic agent; the method comprising adding a composition from a pack according to the second aspect of the invention to the aqueous medium comprising the at least one buffer component and the at least one osmotic agent to form an in vitro test medium wherein the particle size (Z-average) of the said medium remains consistent and below 100nm.

[0040] A fifth aspect of the invention provides a method of determining solubility, permeability or dissolution of a pharmacological compound, comprising: preparing an aqueous medium containing at least one buffer component and at least one osmotic agent; adding a composition from a pack according to the second aspect of the present invention containing at least one bile salt and at least one phospholipid to the aqueous medium to form an in vitro test medium; adding at least one of a pharmacological compound, a physiological compound and a dosage form to the medium to determine a solubility.

[0041] Definitions and Terminology

[0042] Unless specifically indicated to the contrary, singular and plural terms used herein are interchangeable. Present tense may denote the past and vice versa.

[0043] Biorelevant compositions.

[0044] Biorelevant compositions essentially comprise at least one bile salt or bile acid with one or more phospholipids which are physiologically relevant surfactants present in the mammalian gastrointestinal fluids, in particular intestinal fluids.

[0045] Bile salt / Bile acid.

[0046] Bile acid and / or salt is a polar steroidal surfactant that is unconjugated (e.g. cholic acid) or conjugated with amino acids, for example glycine or taurine. Structurally they are classified as primary, secondary, and tertiary bile salts. Bile salts act as surfactants, emulsifying fats which facilitate digestive enzymes to break them down and the body to absorb them. They may also act as solubilizing components for examples lipophilic vitamins and drugs. They may also help eliminate waste products systemically, including cholesterol. This data is helpful for pre-clinical tests / assessments of drugs to understand how they are distributed within the io body. The term bile salt used here refers to at least one bile salt and may also refer to a bile acid. The term bile salt may be used interchangeably with bile acid.

[0047] Phospholipid.

[0048] The known biorelevant media thus comprise phospholipids, a group of ubiquitous amphipathic surfactants / lipids found, for example, in the intestinal fluids of mammals for solubilising (dietary) fat and lipophilic substances that facilitate digestion and drug absorption. A phospholipid structure consists of a glycerol backbone, with one (-lyso) or two fatty acid tails, and a phosphate group attached to the glycerol. There may be an associated molecule attached to the phosphate group, which is most commonly choline, but it may also be for example ethanolamine, serine, inositol or glycerol. If there is no associated molecule attached to the phosphate group, the phospholipid type is phosphatidic acid. In this invention the main phospholipid type is phosphatidylcholine. In this invention, lysophospholipids are included in the definition of phospholipids. The other nonphosphatidylcholine (e.g. phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol) phospholipids may be present in minor quantities. Other membrane lipids such as glycolipids and sphingolipids may also be present.

[0049] Phospholipids may be either saturated or unsaturated. This invention is focussed specifically on unsaturated phospholipids, more specifically polyunsaturated phospholipids. Unsaturation is due to the presence of one or more double bonds in the fatty acid tails. Isolated double bonds contain two or more double bonds (i.e. polyunsaturated) on at least one fatty acid tail of the phospholipid. If the fatty acid chain(s) of the phospholipid has two or more double bonds, it is referred to as polyunsaturated.

[0050] Fully saturated phospholipids are oxidatively more stable but are not suitable for simulating in vivo gastrointestinal fluids. Fully saturated phospholipids do not adequately reflect compositions found in vivo which contain unsaturated phospholipids. Using saturated phospholipids can lead to inaccurate in vitro information on interactions with drug molecules. The term phospholipid refers to one or more types of unsaturated phospholipids.

[0051] Double bonds and PUFAs.

[0052] Based on the relative position of the double bond in the overall molecule, they may be grouped into:

[0053] » Cumulated double bond: the fatty acid of the lipid has two successive double bonds on adjacent carbons (i.e. polyunsaturated). They are also called Allene.

[0054] ® Conjugated double bond: the fatty acid of the lipid has two double bonds (i.e polyunsaturated) separated by a single bond.

[0055] • Isolated double bond: the fatty acid of the lipid has at least two double bonds (i.e. polyunsaturated) separated by two or more single bonds.

[0056] PUFA: Polyunsaturated fatty acid means two or more unconjugated double bonds in the unsaturated fatty acid of one or both fatty acid chains of the phospholipid. Examples of PUFAs include for example linoleic acid, linolenic acid commonly found in soyabean phospholipid and for example arachidonic acid in egg phospholipids.

[0057] Lipid components.

[0058] Lipid components for example cholesterol and / or unsaturated fatty acids, such as oleic acid, and / or their salts; lipolytic products, for example unsaturated monoglycerides, such as glycerol mono-oleate; and / or unsaturated diglycerides; may be incorporated into biorelevant compositions to better simulate fasted and fed state biorelevant media.

[0059] Stability.

[0060] The biorelevant compositions according to this invention have improved stability. Stability in this context refers to the physicochemical stability of the biorelevant compositions by reference to the levels of PUFAs, conjugated double bonds and isolated double bonds of the phospholipids. The definition of stability also extends to (but not limited to): oxidative stability, reproducible UV profile, after dilution of the composition to 0.75mM of phospholipid (PL) in standardized phosphate buffer a low UV absorbance (below 2.0AU, below 1.8AU, below 1.5AU; below 1.25AU and below 1.0AU) up to 2hours and up to 48hours after sample preparation and substantially consistent particle size, as measurable parameters (for example Z- average and / or (Polydispersity Index (PDI)) of the biorelevant medium prepared therefrom. The absorption response at up to two-hour time point is important because this is the level above which it has been found effects colloidal stability. It is helpful to continue to monitor the UV measurement (for up to 48hours) because it is important to have a UV response that remains consistent and does not increase too much.

[0061] With media where the PL concentration is not 0.75mM, the UV test can also be applied to the PL concentration (and bile salt concentration) at which the medium is to be actually tested / used.

[0062] In this specification, this stabilization is achieved without additional antioxidants (other than those present as stabilisers in the phospholipid). Additional antioxidants can affect the measurements determined by the media because the space where lipophilic drugs can remain is occupied, thereby reducing apparent solubility of lipophilic drug or compound.

[0063] Oxidation.

[0064] In this invention, the terms oxidation, oxidative status, oxidative process, peroxidation, and auto-oxidation may be used synonymously.

[0065] In this invention, the terms unconjugated, non-conjugated or isolated double bonds may be used interchangeably.

[0066] Oxidation includes the first steps of oxidation, namely the rearrangement or destruction of isolated double bonds in unsaturated fatty acids with two or more double bonds. It covers the formation of conjugated double bonds in the total phospholipids content of the biorelevant composition.

[0067] Oxygen absorbers / scavengers.

[0068] Examples of oxygen absorbers / scavengers include ferrous; non-ferrous oxygen scavengers / absorbers which may be in sachets or cannisters or integrated into the container or closure and / or cap. The term absorber and scavenger can be used interchangeably.

[0069] Container.

[0070] The container is a sealed receptacle and closure that is used to store the stable biorelevant composition. The container may be either rigid or flexible.

[0071] Containers with the desired OTR may be either rigid or preferably flexible. Examples of flexible containers include a sealed sachet, pouch, or stick.

[0072] Oxygen Transmission Rate.

[0073] Oxygen transmission rate (OTR) is the (steady state) rate at which oxygen molecules (in cc) in air traverse through a defined surface area of solid material over time (typically 24hours) at atmospheric pressure. In this invention, the OTR refers to the OTR of the empty configured sealed container (including the closure). The OTR measurements in this invention were carried out using air (21 % oxygen v / v) at standard atmospheric pressure. Due to the potential differences in for example, sealing efficacy for the container and weaknesses in container construction, e.g. a laminate seal closure or sealed / folded edge of a pouch, an OTR of a material alone may be inaccurate for the container selection. The selection should be based on the OTR of the configured container with seal and closure (e.g. plug, cap, liner) A consistent OTR (low variability) of the sealed container is required so that long term stability in all containers is achieved. When selecting a container that meets the requirements multiple containers (e.g. 10) using the configuration of the sealed pack should be tested and the packs should be below the threshold OTR value.

[0074] Void space.

[0075] Void space is the volume of space that is present in the container after sealing and includes the space within the container after the product is filled / inserted.

[0076] After sealing, if the oxygen is not replaced with an inert gas, the void space contains air (21 %v / v oxygen) present in the gaseous mixture. If oxygen sachets or scavengers are not included after sealing, this oxygen will react with the phospholipid in the composition inside the sealed container. This will result in an undesirable increase of the UV response at 234nm when the composition is diluted to 0.75mM phospholipid in the standard phosphate buffer compared to fresh composition.

[0077] Gaseous mixture.

[0078] The gaseous mixture occupies the volume within the sealed container that is not taken up by the composition. An inert gas(es) may be used to displace oxygen from the gaseous mixture inside the container prior to sealing. Examples of inert gas or mixtures thereof include carbon dioxide, nitrogen, argon and / or helium. Preferably oxygen from the air is completely replaced with inert gas(es) but maybe present providing the oxygen level is below the threshold amount and the OTR is remarkably low, preferably less than 0.001 cc / sealed container / day. The threshold phospholipid to oxygen molecular ratio after or soon after sealing is 1000:100. Preferably the packaged composition containing the phospholipid has a threshold phospholipid to oxygen molecular ratio after or soon after sealing is >2000:1 .

[0079] Empty sealed container.

[0080] This is the sealed container without the composition which may be used for OTR determination.

[0081] Sealed container.

[0082] Sealed container is the container and closure used for inserting and storing the composition. The process of sealing involves isolating and enclosing the composition from the outside environment. Sealing can be carried out mechanically e.g. with a screw top cap with a liner or a screw cap. However, it is preferable to use a thermal or induction seal with an aluminium laminate seal liner (in case of bottles) or thermally seal aluminate laminate containers (e.g. sachets, stick sachets or pouches) or stoppering with a plug and crimping with an aluminium cap.

[0083] Sealing conditions. Sealing of the container and closure may be carried out with the gaseous mixture inside the container at atmospheric pressure, or alternatively reduced pressure. Preferably the container and closure are sealed under vacuum.

[0084] Pack.

[0085] Pack is the composition inside the sealed container that is stable at room temperature for at least 12months, preferably at least 18months, more preferably at least 24months; and especially at least 36months at room temperature; and at 40°C for at least 3months, preferably at least 6months, preferably at least 9months, more preferably at least 12months, especially at least 24months. The definition may also include a secondary container / packaging (when used) to lower the OTR of the primary sealed container.

[0086] (Total) phospholipid: oxygen ratio of gaseous mixture (after or soon after sealing).

[0087] This threshold value or ratio is the molecular ratio of total phospholipid to oxygen inside the container after sealing or soon after sealing. In the Examples, this ratio is referred to as “PL: oxygen ratio in gaseous mixture”. Typically, unless otherwise stated this is determined experimentally but may also be calculated. The phospholipid quantity in the container is determined in millimoles (mmol). The oxygen is the quantity (mmol) that is present soon after sealing. It is essential to be above this specified ratio. This ratio is 1000:100 or above, 1000:60 or above; 1000:50 or above; more preferably 1000:40 or above, more preferably 1000:25 or above, more preferably 1000:10 or above; more preferably 1000:5 or above, especially 2000:1 or above whilst achieving the UV target (equal to 1.5AU or below) at 234nm from in particular up to t = 2hours and up to 48hours after sample preparation and particle size measurements of medium (Z-average below 150nm, preferably equal to or below 100nm) which are consistent.

[0088] (Total) PL: oxygen ratio of gaseous mixture (at 40°C three).

[0089] The phospholipid quantity in the container is determined in millimoles (mmol). The oxygen is the quantity (mmol) that is present inside the void space of the sealed stored container at 3months. Typically, unless otherwise stated this is determined experimentally. This threshold value or ratio is the molecular ratio of total phospholipid to oxygen inside the sealed container after storage of the pack at 40°C for 3months 1000:60 or above; preferably 1000:25 or above whilst achieving the UV target (equal to 2.0AU or below; preferably 1.8AU or below from in particular up to 2hours and up to 48hours after sample preparation) at 234nm and particle size measurements of medium (with consistent and reproducible Z- average value which is below 150nm, preferably equal to or below 100nm).

[0090] Phospholipid to net oxygen molecular ratio (upon storage).

[0091] The phospholipid quantity is determined in millimoles (mmol). The net oxygen exposure is based on the molar amount (mmol) of oxygen present after sealing plus the amount (mmol) of oxygen that may permeate the container during storage at 40°C for at least 6months in contact with the phospholipid of the composition. Typically, unless otherwise stated this is determined using the OTR of the actual sealed container.

[0092] The amount (mmol) of oxygen after sealing may be calculated based on the oxygen present in the gaseous mixture of the void space (internal volume minus composition volume occupied) inside the container. The amount (mmol) of oxygen that enters the container during storage may be calculated based on the OTR of the configured sealed container (with closure) at 40°C and extrapolated to at least 3months; preferably at least 6months; preferably at least 9months; preferably at least 12months. If there is no oxygen in the gaseous mixture after sealing or soon after sealing, the molecular ratio of phospholipid in contact with oxygen in the sealed container during storage for at least 3months at 40°C should not exceed 1000:100.

[0093] Due to the consumption of oxygen by the phospholipid, this amount cannot be measured. This calculation may be used as a guide for selection of the sealed container and configuration required to stabilise the composition inside the container.

[0094] UV Measurement. In methanol

[0095] UV absorption measurement of phospholipid alone in methanol is used as part of the selection process for the phospholipid to ensure that the UV measurement of the freshly prepared medium will be of the appropriate standard.

[0096] UV of composition diluted in phosphate buffer

[0097] UV absorption of the composition (containing bile salt and phospholipid), diluted to a defined phospholipid (PL) concentration of 0.75mM in a specified phosphate buffer, can be used as a pre-test or quality check. This test helps verify that the phospholipid content is within specification. It can also confirm that the phospholipid remains within specification in the composition with bile salts and other components.

[0098] The UV absorbance at 234nm for this diluted composition is typically indicative of the overall quality of the medium prepared from the composition.

[0099] "UV absorbance" in this specification refers to the amount of ultraviolet (UV) light absorbed by the composition (containing bile salt and phospholipid), diluted to a defined phospholipid (PL) concentration of 0.75mM in a specified phosphate buffer at a wavelength of 234nm, measured using a 10mm path length cell. It can be used as a pre-test or quality check. UV absorbance is expressed in Absorbance Units (AU), where an increase in the absorbance indicates the phospholipid is changing and affecting the colloidal structures of the medium. For the purposes of this invention, the UV absorbance should be less than 2.0AU; preferably less than 1.8AU; preferably less than 1.5AU up to 2hours after the preparation of the sample to ensure colloidal structures are formed consistently. Also, the UV can be checked up to 48hours to ensure the stability of the composition.

[0100] UV of actual test medium

[0101] UV absorption at 234nm and other wavelengths (for example 264nm) of the medium with the desired concentrations of bile salt and phospholipid (prepared from unopened aged / stored compositions in sealed containers) may be evaluated to find out if the UV characteristics remain consistent and reproducible against medium prepared from fresh compositions. For FaSSIF medium the PL concentration in this test is the same as in the UV dilution test given the PL concentration in FaSSIF is 0.75mM. However, for compositions where the PL is not 0.75mM, the test should be carried out at the actual PL concentration used in the medium (along with bile salt concentration).

[0102] Additionally, the composition may contain other components / constituents, for example GMO and fatty acids.

[0103] Particle size measurements of medium.

[0104] The average particle size (expressed as Z-average) and distribution (expressed as polydispersity index (PDI)) of actual test media (i.e. at the intended bile salt and phospholipid concentrations) are determined by PCS (photon correlation spectroscopy). To examine quality of a medium, particle size should be determined at the concentration of defined bile salt(s) and phospholipid that is present in the medium and not the defined phospholipid concentration (at 0.75mM) for the UV. For example, FaSSIF-V2 medium has a STC concentration of 3mM and phospholipid concentration of 0.2mM. Therefore, particle size of the medium with this bile salt: phospholipid ratio should be measured.

[0105] Standardized / defined (biorelevant) phosphate buffer

[0106] "Biorelevant phosphate buffer / standardized / define phosphate buffer" in this specification refers to a phosphate buffer solution designed to closely approximate the aqueous conditions found in the small intestinal tract. This buffer contains: 0.420g NaOH, 3.438g of sodium phosphate monobasic (anhydrous), 6.186g of NaCI in 1 L adjusted to pH6.50. This defined buffer can also be produced by diluting a commercially available FaSSIF Buffer (FASBUF™) Concentrate. This buffer is used for dilution of the phospholipid composition to a phospholipid concentration of 0.75mM as well as preparation of actual biorelevant test medium.

[0107] Preparation of biorelevant media.

[0108] Biorelevant media may be prepared by adding the stable biorelevant compositions made according to the method of the invention or from packs of the invention to aqueous media, or vice versa. Aqueous medium is typically a buffer solution and osmolarity adjusting agents that may be prepared either from scratch or readymade or preferably using buffer concentrates described in WO 2019 / 239133.

[0109] Biorelevant media properties after preparation.

[0110] Biorelevant media are prepared from water dispersible biorelevant compositions with gentle agitation. Homogeneous media are prepared by opening the sealed container and adding the biorelevant composition to the appropriate buffer and / or or adding the buffer and / or water to the biorelevant composition. Particle size (for, e.g., Z-average and PDI using photon correlation spectroscopy) measurements of the reproducible biorelevant media are typically measured within 30 to 120minutes of preparation to allow for colloidal equilibration. Particle size up to 48hours of the medium preparation may also be used to establish quality and reproducibility of the medium.

[0111] Reopening.

[0112] Reopening is the reuse of the product (i.e. composition) after first opening the pack of composition inside the sealed container within at least one-month multiple times (for example more than 6 times) during this period.

[0113] Brief description.

[0114] Prior art biorelevant products / com positions must be stored refrigerated due to limited stability (for a maximum of 18months) and cannot be stored at ambient temperature for an extended period. These limitations have now been overcome using the disclosures of the current invention. Packs containing compositions in selected containers under defined conditions are stable at ambient and up to 40°C for up to 3years.

[0115] The invention provides a method and pack for stabilizing a composition in a sealed container for an extended period at room temperature (at least 9months, preferably at least 1year; more preferably at least 18months; more preferably still at least 24months; especially at least 36) and up to 40°C (at least 3months; preferably at least 6months; preferably at least 9months; more preferably at least 12months; especially at least 24months). The composition essentially comprises at least one bile salt and / or acid and at least one phospholipid.

[0116] The invention comprises configuring some, preferably all, of the following aspects:

[0117] I. a mole ratio of bile acid and / or bile salt: phospholipid in the composition between 75:1 and <1 :1 ;

[0118] II. selection of a bile acid and or / salt of the composition with at least 80% w / w purity (on a dry weight basis);

[0119] III. selection of the phospholipid with polyunsaturated fatty acids, comprising: a. selecting the phosphatidylcholine level; b. selecting the PUFA level; c. checking phospholipid UV response in methanol; d. determining the UV characteristics of the freshly manufactured composition when diluted to a defined phospholipid concentration of 0.75mM to ensure it is below 1 .5AU from in particular t = 2hours and up to 48hours after sample preparation;

[0120] IV. determining the starting quality of the biorelevant medium;

[0121] V. selecting an appropriate container and seal;

[0122] VI. selecting an appropriate fill volume defining a method of filling and sealing, gaseous mixture, optionally at reduced pressure or under vacuum;

[0123] VII. thereby providing a configured pack containing the composition and a gaseous mixture, optionally at reduced pressure, or alternatively under vacuum, in a sealed container;

[0124] VIII. testing the composition (bile salt / acid and phospholipid) in the stored pack (at the end of shelf life) after first opening by diluting to a defined phospholipid concentration of 0.75mM in a defined phosphate buffer to ensure it is below 2.0AU, preferably 1.8AU in particular up to 2hoursy IX. preparing biorelevant medium using the composition and determining the quality of the biorelevant medium at the end of shelf life.

[0125] Detailed Description.

[0126] The composition provided in the sealed container is characterised by a mole ratio of bile acid and / or bile salt: phospholipid in the composition between 75:1 and <1 :1 ; preferably 60:1 to <1 :1 ; more preferably 50:1 to <1 :1.

[0127] Examples of compositions for preparing media include, but are not limited to, for example: Fasted state simulated intestinal fluid (FaSSIF), Fed state simulated intestinal fluid (FeSSIF), FaSSIF-V2 and FeSSIF-V2.

[0128] Selection of the bile salt:

[0129] The bile salt for the composition is selected to reflect the bile acidZsalt(s) composition required.

[0130] Wherein the bile acid and or / salt of the composition comprises: i) at least one bile acid / bile salt selected from the group comprising:- sodium cholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium taurodeoxycholate, sodium glycodeoxycholate, sodium ursodeoxycholate, sodium chenodeoxycholate, sodium taurochenodeoxycholate, sodium glycochenodeoxycholate, sodium cholylsarcosinate, and sodium N- methyl taurocholate; and ii) each bile salt and / or acid has a purity of at least 80%, at least 85%, at least 90%, at least 94% w / w (on a dry weight basis).

[0131] Bile salts are in general preferred over bile acids. At least one bile salt may also be selected to reflect the different types of mammalian intestinal fluids. E.g. murine (e.g. rat or mouse), canine, porcine etc. Selection of phospholipid: wherein the phospholipid of the composition further comprises: i) at least one polyunsaturated phospholipid wherein the phospholipid content is at least 45% w / w phosphatidylcholine; at least 70%; at least 90%; at least 93%w / w (based on dry mass); ii) fatty acid chain(s) of the phospholipid contains at least 10% w / w; at least 15%; at least 20% polyunsaturated fatty acids; and iii) wherein the said phospholipid alone is dissolved at a phospholipid concentration of 0.75mM in methanol at 234nm (10mm cell path length) and the UV response is below 0.5, preferably 0.3, more preferably 0.1 (see UV determination of PL in methanol below for explanation); iv) wherein the said phospholipid is selected so that after dilution of the freshly prepared composition (containing bile salt and / or bile acid and phospholipid) to a phospholipid concentration of 0.75mM, the UV response at 234nm (10mm path cell length) is consistently below 2.0AU, preferably 1.8AU; preferably below 1.5AU from in particular t = 2hours and up to 48hours after sample preparation; (see below UV measurement of composition diluted in phosphate buffer for explanation).

[0132] PUFAs

[0133] Polyunsaturated fatty acid (PUFA) content is determined by a method for example GC-FID and involves establishing the fatty acid profile (cis-trans) (normalised in fat) and typically results are based on polyunsaturated fatty acids based on w / w of the total fatty acid. Methods such as those described in BS EN ISO 12966 may be used to determine PUFA content.

[0134] The polyunsaturated phospholipid source is selected from soyabean and / or egg, and / or milk, and / or soya and / or sunflower and / or oat, etc.

[0135] The polyunsaturated double bond content of the phospholipid depends on the source of the phospholipid, for example, egg phospholipid is above around 15%w / w and soya bean is above around 50%w / w, typically 53% to 83% of total phospholipid fatty acid content. It is to be understood that said phospholipid content may be also obtained by blending or combining phospholipids or lecithin from different sources or origin, including synthetic or semi-synthetic. The preferred phospholipids are USP egg phospholipids and / or USP-NF soyabean phosphatidylcholine. For USP-NF the polyunsaturated fatty content (linoleic and linolenic acids) of fresh phosphatidylcholine is between 53% and 83%, preferred is 65 to 75% PUFAs. It is a requirement that the % PUFA content of the PL in the composition after storage for at least 3months at 40°C remains substantially consistent (e.g. soya bean PL no more than 5%w / w; preferably 3%, more preferably 1 %), The change is based on the total fatty acid content after 3months at 40°C compared to the % PUFA content of the PL in freshly prepared composition.

[0136] UV determination of PL in methanol.

[0137] Determining the UV of phospholipid dissolved in methanol prior to preparation of the composition assists with the selection of the phospholipid to confirm the appropriate specific UV quality is met.

[0138] UV measurements are carried out by dissolving the phospholipid at a concentration of 0.75m M in methanol and determining the UV response at 234nm a 10mm Quartz cuvette from the baseline corrected absorbance by blank sample of methanol with the double beam UV-VIS spectrophotometer (Shimadzu UV- 1800).

[0139] UV of composition diluted in phosphate buffer.

[0140] This method is used to facilitate the selection of the phospholipid for the composition so that the UV profile is consistent and reproducible across a wide range of compositions with molecular ratios ranging from 75:1 to <1 :1 of bile salt: phospholipid.

[0141] UV measurements are carried out by measuring compositions diluted at a defined phospholipid concentration of 0.75m M in a standardised phosphate buffer at 234nm and 268nm in a 10mm path length Quartz cuvette from the baseline corrected absorbance by blank sample with the double beam UV-VIS spectrophotometer (Shimadzu UV-1800). Note Krollik used a path length of 1 mm for UV measurements.

[0142] 100mL of blank sample of the buffer can be prepared by adding 96.3g of purified water into 4.16g FaSSIF buffer concentrate.

[0143] Optical density.

[0144] Furthermore, when the biorelevant composition is diluted in preferably phosphate buffer to a phospholipid concentration of 0.75mM at room temperature, the optical density of the media is below 1 .OAU at 600nm (allowing for subtraction of the reading of the blank I buffer control without biorelevant composition).

[0145] Conjugation.

[0146] (i) The level of isolated double bonds of the phospholipid used to prepare the biorelevant composition / s can also be assessed by, for example, H-NMR can be used can identify isolated double bonds using a method as described by Knothe et al. "Determination of the fatty acid profile by 1 H-NMR spectroscopy." European Journal of Lipid Science and Technology 106.2 (2004): 88-96) or GC, prior to manufacture of the biorelevant composition, thereby ensuring that the phospholipid to be used conforms to the requisite content of isolated double bonds; in addition to:-

[0147] (ii) The amounts of conjugated double bonds can also be assessed, for example, by H-NMR or GC, thereby ensuring that the phospholipid / s to be used are of a good quality.

[0148] In another embodiment, the phospholipids of the fresh and aged biorelevant compositions may possess conjugated double bond phospholipid content that is less than 5mol% of the total phospholipids, preferably less than 2mol% of the total phospholipids and preferably less than 1 mol%, more preferably 0% of the total phospholipids. Preparation of the biorelevant composition.

[0149] Preferably the composition is prepared by dissolving or dispersing the bile salt and phospholipid in organosolvent, water-solvent solution, or water. The organosolvent is preferably a suitable hydrophilic volatile solvent, more preferably an alkanol, alkanol-water, more preferably still a mixture of ethanol and water may be used. Alternatively, the powder mixtures may be homogeneously dispersed in water, orwater-alkanol solution followed by removal of the solvent and / or water to yield a solid composition. Organosolvents such as methylene chloride, tetrahydrofuran, may be used with small amounts of water to dissolve the bile salt and lipid with the proviso that the water solvent mixture is monophasic (solution) and an o / w emulsion is not formed during solvent removal. The powder is prepared by removing the liquid using a method selected from solvent removal methods consisting of evaporation under ambient or elevated temperatures optionally under vacuum; granulation and drying with or without vacuum; solvent precipitation using an immiscible solvent, collecting the precipitate and removing solvents from the precipitate; lyophilisation; spray drying; spray granulation. The solid material may be inserted for example as powders, cakes or granules. Preferably the compositions are further sieved or screened to prepare free flowing or waxy powder compositions.

[0150] Filling.

[0151] The solid composition may also be filled / inserted into containers such as a vial or alternatively dried in situ, this latter approach is particularly helpful for small quantities of powder composition less than 5g, less than 1g, less than 100mg, less than 10mg.

[0152] UV of composition (fresh) diluted in phosphate buffer.

[0153] The UV measurement of the freshly prepared composition diluted to 0.75mM phospholipid in standardized phosphate buffer verifies the quality meets the defined target specification (less than 1.5AU; preferably less than 1.25AU; more preferably less than 1.0AU from in particular t = 2hours up to 48hours after sample preparation) and quality prior to or soon after inserting into the container, with further requirements needing to be met with regard to for example, configuring the container, closure and other conditions as further defined herein.

[0154] Particle size of biorelevant medium from fresh composition.

[0155] Measurements are typically performed on undiluted, unfiltered samples in 2.5-mL Polystyrene macro cuvettes (10mm width using a Zetasizer ZS (nano series, Malvern Instruments)) with non-invasive backscattering at an angle of 173°. Measurements are performed in triplicate at temperature of 22°C.

[0156] The average particle size (expressed as Z-average) and distribution (expressed as polydispersity index (PDI)) of actual test media (i.e. at the intended bile salt and phospholipid concentrations) prepared from freshly prepared composition prior to inserting into the container are determined by PCS (photon correlation spectroscopy) must be below 150nm; preferably 100nm.

[0157] To ensure the colloids of a selected medium are reproducible and consistent, particle size should be determined using the freshly prepared medium at the concentration of defined bile salt(s) and phospholipid that is to be used as the test media. For the sake of clarity, the phospholipid concentration of this test may not be the same as the concentration used for UV measurement of composition which is always at 0.75mM phospholipid. For example, FaSSIF-V2 medium has a STC concentration of 3mM and phospholipid concentration of 0.2mM. Therefore, these concentrations should be used to test particle size of the medium. Typically, a two-hour incubation after addition of the composition to the buffer may be required for the colloidal structures in the medium to equilibrate. Thereafter, in general the particle size should be substantially consistent and reproducible. Typically, measurements can be made at t = 0, 24 H and 48hours. Beyond this time, the media may be susceptible to microbial spoilage and are affected by the microbial burden of the water used.

[0158] Limiting the phospholipid to oxygen ratio.

[0159] Limiting the phospholipid to oxygen ratio involves for example, the gaseous mixture inside the container, ensuring the seal is robust, controlling sealing conditions (atmospheric pressure, reduced pressure or vacuum), minimizing headspace and minimizing the OTR by selecting a sealed container with consistent OTR and ensuring the OTR of the pack (the sealed container with composition) is robust at room temperature and 40°C.

[0160] The OTR of the whole pack is critical and, therefore, not only should the material be selected judiciously but also the seal, plug and / or closure. Furthermore, this may need to be considered together with for example the fill volume inside the container to limit the total phospholipid to oxygen ratio after sealing.

[0161] The composition may be inserted into a container with a gaseous mixture or alternatively under reduced pressure or vacuum and sealed within the container, wherein the molecular ratio of total phospholipid in the composition: oxygen of the gaseous mixture inside said container after sealing is equal to 1000:100 or above, 1000:60 or above; 1000:50 or above; more preferably 1000:40 or above, more preferably 1000:25 or above, more preferably 1000:10 or above; more preferably 1000:5 or above, especially 2000:1 or above.

[0162] The oxygen exposure should be adhered to and limited not only by the OTR but also by controlling the oxygen in the gaseous mixture (just before sealing) and / or the inclusion of an oxygen absorber.

[0163] Specifically, for every 1 mmol of phospholipid, the net oxygen exposure in the gaseous mixture in the sealed unopened container from sealing or soon after sealing up to at least 3months at 40°C; at least 6months at 40°C; at least 9months at 40°C and 12months at 40°C is limited to <0.100mmol of oxygen; <0.050mmol of oxygen; <0.040mmol of oxygen; <0.020mmol of oxygen; <0.010mmol of oxygen; <0.001 mmol of oxygen;

[0164] Assuming there is no oxygen in the gaseous mixture nor oxygen sachet(s) is included in the sealed container, using the threshold of phospholipid: net oxygen molecular ratio; >1000:100, the selected sealed container requires an oxygen transmission rate (OTR) at 40°C of:

[0165] <0.0250cc / sealed container / day x (multiplied by) the amount of phospholipid present in the composition in millimoles (mmol).

[0166] OTR of the sealed container.

[0167] The method further comprises I. sealing the composition and the gaseous mixture, optionally at reduced pressure or alternatively under vacuum in a sealed container having a defined oxygen transmission rate (OTR);

[0168] II. where the oxygen transmission rate (OTR) at 40°C is:

[0169] <0.0250cc / sealed container / day x (multiplied by) the amount of phospholipid present in the composition in millimoles (mmol); preferably <0.0125cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.0100cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.005cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.003cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); especially <0.001 cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol).

[0170] The OTR is determined at 40°C. The OTR of the empty whole sealed pack (with closure) may be determined by a standardized method for example as described in ISO-15105-2-2003.

[0171] In one embodiment, in the event that the OTR of the primary container is above the threshold value, a secondary wrapping or a secondary container such as a sachet or pouch which has a lower OTR than the primary packaging can be used, thereby effectively lowering the OTR of the primary container. However, the conditions inside the sealed primary container such as phospholipid to oxygen upon sealing still be adhered to.

[0172] After inserting the composition inside the container, the container is sealed to isolate the composition in the container. OTR measurements must not just focus on the material of the container but must consider the seal and any other closure for example a plug and / or cap.

[0173] Container.

[0174] Highly surprisingly, it has been found that biorelevant compositions in HDPE containers may still not provide satisfactory stability at room temperature, even either if blanketed with inert gas or when an oxygen absorbing sachet is added. This stability is even less so at elevated temperatures (40°C). Therefore, when stored at room temperature (at least 6months, at least 12months, at least 18months) and 40°C for an extended period (about at least 3months; at least 6months; at least 9months) prior art compositions in HDPE containers under nitrogen or in sachet, may not be used to prepare biorelevant media reproducibly.

[0175] The container is typically in the form of a bottle, pouch or sachet. In principle the container may be fabricated from a range of materials that satisfies the required OTR and are compatible with the composition and should not be limited to the materials described herein. A rigid container may be made from for example HDPE, multilayered EVOH, aluminium (lacquered or uncoated) or glass if the phospholipid to oxygen ratios after sealing and during storage are adhered to. Flexible containers such as stick sachets, pouches, or sachets constructed from METPET, METBOPP or trilaminate aluminium (at least 7 or at least 9 micrometre thickness) may be used and sealed by for example with heat.

[0176] To optimise the stability of the composition after opening, it is preferable to select a flexible container (e.g. sachet or pouch) with low OTR (pack and seal) that can minimise the void space because the air can be expelled by squeezing out the air and the container remains deformed. The pouch or sachet may be resealable with a resealable mechanism for example a zip lock and / or use sealable tape.

[0177] Sealed containers need to be carefully selected by determining the OTR. OTR reproducibility of the empty sealed containers should be carried out using, for example, 10 individual units. Surprisingly, significant variability has been identified for containers which those skilled in the art would assume to be ideal candidates for packaging.

[0178] This is why the closure and sealing method should also be carefully selected to ensure the OTR of the container is not affected. For example, it has been surprisingly found that even with UN approved aluminium containers typically used for hazardous materials and storage of oils, substantial variability of the OTR between the sealed containers was found. Careful selection will eliminate variability in stability of the composition when stored at room temperature at least 6months; at least 12months; at least 18months and / or 40°C for at least 3months; at least 6months; at least 9months; at least 12months. After filling and reducing the oxygen level, the container is preferably hermetically sealed. For rigid containers, the opening may be desirably sealed with an induction seal lined with an aluminium layer or an airtight impermeable closure. For flexible containers for example aluminium laminate pouches hermetically sealing is carried out with heat or induction.

[0179] Gaseous mixture (after or soon after sealing).

[0180] The gaseous mixture occupies the void space inside the sealed container with the composition. It is essential to control the gaseous mixture so that the phospholipid to oxygen mole ratio inside the sealed container is above 1000:100; preferably above 1000:60; more preferably above 1000:50; more preferably above 1000:40, more preferably above 1000:25, more preferably above 1000:10, more preferably above 1000:5, and ideally above 2000:1 after sealing or soon (= around up to two weeks if an oxygen scavenger or absorber is included) after sealing. The ratio selected is dependent upon the OTR of the sealed container selected at the start and after storage / desired end of life shelf life. Although a pack with a low OTR can afford a higher oxygen content, it is still preferable to start with the lowest level of oxygen feasible. This ratio can be transiently below the stated ratios if oxygen sachet(s) / scavenger(s) is / are included along with the gaseous mixture and composition. Incubation with oxygen absorber / scavenger will help remove oxygen before it reacts with the composition. The use of an oxygen absorber / scavenger with high oxygen removal capacity may also assist with limiting the exposure of phospholipid to oxygen originating from permeation through the sealed container. Absorbers / scavengers assist with limiting the threshold phospholipid: oxygen molecular ratio (1000:100 and above) soon after sealing and may also help with limiting the phospholipid to net oxygen molecular ratio (upon storage) to (1000:100 and above).

[0181] However, the approach of removal / lowering the oxygen exposure with an absorber is less preferred but it is included in the specification. It is less preferred because it is desirable to prevent the phospholipid of the composition being exposed to oxygen in the first place.

[0182] Gaseous mixture (at 3months at 40°C).

[0183] To achieve long term stability (>3months at 40°C), it is essential to continue to control the phospholipid to oxygen (in the gaseous mixture) mole ratio inside the sealed container. It is important to be below 1000:50 or above; more preferably 1000:40 or above, more preferably 1000:25 or above, more preferably 1000:10 or above; more preferably 1000:5 or above, especially 2000:1 or above inside the sealed container without a significant increase in the UV response compared with freshly prepared composition.

[0184] Fill volume.

[0185] One of the benefits of this invention is that it allows the filling of any volume / quantity of composition using a wide range of container types and volumes provided that e.g. the container OTR, the gaseous mixture and seal are carefully selected in addition to the quality and specific components of the composition. Depending upon the amount of phospholipid in the composition, the required oxygen transmission rate (OTR) at 40°C of the sealed container is selected from the following, wherein:

[0186] <0.0250cc / sealed container / day x (multiplied by) the amount of phospholipid present in the composition in millimoles (mmol); preferably <0.0125cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.0100cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.005cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.003cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); especially <0.001 cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol).

[0187] Pack.

[0188] A pack is manufactured by inserting / filling the freshly prepared composition inside suitable container with a closure and / or sealing or soon after manufacture.

[0189] A pack comprising a composition and a gaseous mixture, optionally at reduced pressure or alternatively a vacuum in a sealed container, the composition upon opening being for use in the preparation of media, the composition comprising essentially at least one bile salt and / or acid and at least one phospholipid. The composition with gaseous mixture in a sealed container has pack sizes that may include but are not limited to 5kg (bulk pack), below about 1 kg, below about 500g, below about 250g, below about 100g, below about 50g and below about 10g, below about 1g and below about 100mg (e.g. vial).

[0190] Preparation of biorelevant medium.

[0191] According to the present invention, there is also provided a method for the preparation of a biorelevant medium for testing which comprises the use of a biorelevant composition obtained from a pack of this invention, as described above. The method comprising opening the sealed container, removing the biorelevant composition from the container and adding the composition to a buffer (or in reverse order) containing osmolarity adjusting agent(s) (such as sodium chloride, potassium chloride and / or calcium chloride) or other aqueous media to prepare an in vitro test medium wherein the particle size of the said medium (Z- average) is below 150nm, preferably below 100nm and the Z-average and PDI of the said medium remain substantially consistent. UV of composition in the container (after first opening).

[0192] The composition in the sealed container provides, after first opening of the container (stored for at least after 3months at 40°C) and dilution of the composition in a defined biorelevant phosphate buffer to a defined phospholipid concentration of 0.75mM, a UV response at 234nm (10mm path cell length) that is below 2.0AU, preferably below 1 .8AU from in particular up to t = 2hours and up to 48hours after sample preparation.

[0193] Particle size of biorelevant medium prepared using composition in the container (after first opening).

[0194] To examine the colloidal quality of a medium prepared from a composition in a sealed container (after first opening), particle size should be determined at the concentration of defined bile salt(s) and phospholipid that is to be used as the test media. Again typically, a two-hour incubation after addition of the composition to the buffer may be required for the colloidal structures in the medium to equilibrate. Thereafter, in general the particle size should be substantially consistent and reproducible.

[0195] Reopening.

[0196] A further embodiment of the invention is to select a sealed container that will confer superior stability of the composition after first opening. If a flexible container (e.g. a sachet or pouch) with resealable properties (for example tape, zip lock) is selected, stored compositions will be even more stable and the resultant medium will have lower UV response at 234nm. Furthermore, the compositions will be more robust after multiple use after first opening.

[0197] Additional components.

[0198] If desired, the biorelevant composition inside the container may comprise at least one further component selected from the group comprising:- enzymes, such as proteolytic enzymes, for example pepsin (for simulated stomach fluid), bromelain, papain and / or lipases (for simulated intestinal fluids). These enzymes may be extracted from natural or microbially engineered sources. The mole ratio of each selected enzyme(s): total phospholipid in the biorelevant composition is dependent upon the activity / potency of the enzyme required in the dissolution medium.

[0199] If desired, the biorelevant composition inside the container may also comprise at least one further component selected from the group comprising:-

[0200] Lipolytic products of triglyceride digestion (namely, monoglyceride such as glycerol monooleate and / or fatty acid for example oleic acid and / or its salt), cholesterol, proteins and enzymes; wherein the mole ratio of each selected component: total phospholipid in the biorelevant composition is below 1 :1. Triglyceride may be present but in a minor proportion (i.e., less than 50%, less than 25%, less than 10% by weight of the overall glyceride content) and less than 5%, preferably less than 3% associated / originating from the phospholipid.

[0201] Chelator.

[0202] In this disclosure, a chelator is not classed as an antioxidant. An even more stable UV profile / characteristic of the stable biorelevant media may be maintained / obtained by the addition of a minimum level of chelator(s), for example EDTA, to either the aqueous medium, and / or buffer concentrate, and / or buffer used for preparation of the biorelevant medium, and / or incorporated into the biorelevant composition during manufacture. The level of chelator depends on the selected chelator and selected unsaturated phospholipid in the biorelevant composition. Typically, a molecular ratio less than 1 :10 of chelator to phospholipid is required. Preferred chelator / s is / are EDTA and / or desferrioxamine mesylate.

[0203] Unexpectedly, a chelator may further help stabilize particle size of the media (using different batches of stable biorelevant composition) up to and even beyond 48hours after preparation. Furthermore, the chelator also helps provide reproducible biorelevant media with substantially stable UV spectra for at least 48hours at room temperature; which is not possible using prior art biorelevant media. Reproducible biorelevant media prepared from stored and / or different batches of stable biorelevant compositions share similar particle size parameters (e.g., Z-average) compared to their freshly manufactured counterparts, as well as similar UV profiles. Thus, this avoids the need to prepare media from the same batch of biorelevant composition for reproducibility, as recommended by Krollik et al.

[0204] In one embodiment of the invention, the biorelevant composition inside the container includes one or more chelators. A particularly preferred chelator is EDTA and / or desferrioxamine. In one embodiment of the method, the buffer or other aqueous media used to prepare the biorelevant media includes one or more chelators. A preferred chelator is EDTA and / or desferioxamine.

[0205] The phospholipid to chelator molecular ratio in both of the above cases is typically 500:1 to 10:1 , preferably 200:1 to 25:1 , more preferably 100:1 to 30:1.

[0206] According to a still further embodiment of the present invention, wherein the composition in the sealed container (stored for 40°C for at least 3) includes one or more chelators provides, after first opening of the container and dilution of the composition in a defined biorelevant phosphate buffer to a defined phospholipid concentration of 0.75mM, a UV response at 234nm (10mm path cell length) that is below 2.0AU, preferably below 1.8AU, more preferably below 1.5AU, more preferably 1.25AU from in particular t = 2hours up to 48hours after sample preparation, and especially 1 .0AU or below from in particular t = 2hours and up to 48hours after sample preparation.

[0207] In another embodiment, reproducible biorelevant media prepared from stable biorelevant compositions according to the invention may also contain a chelator, for example EDTA. The concomitant addition of a chelator to the composition and / or the buffer solution or buffer concentrate surprisingly ensures that the spectrum of the biorelevant media remains substantially unchanged between about 220nm and 320nm, in particular at 234nm, even up to at least 48hours storage after preparation, even at 37°C. Furthermore, the mean particle size (Z- average) remains substantially similar. Use of medium.

[0208] The composition in the sealed container is used for preparing biorelevant medium for use in in vitro testing for various purposes, including for in vitro drug dissolution testing, drug solubility testing, drug permeability, quality control (QC) testing, pre- clinical drug testing and / or drug profiling. Also, for the testing of medical devices, probiotics and / or testing of one or more pharmacological compounds or dosage forms of the pharmacological compound.

[0209] The composition in the sealed container may also be used in a method of determining solubility, permeability or dissolution of a pharmacological compound, comprising: preparing an aqueous medium containing at least one buffer component and at least one osmotic agent; adding a composition from the pack of the invention containing at least one bile salt and at least one phospholipid to the aqueous medium to form an in vitro test medium; adding at least one of a pharmacological compound, a physiological compound and a dosage form to the medium to determine a solubility, permeability or dissolution characteristic of the pharmacological compound or dosage form.

[0210] In another embodiment of the present invention, testing for drug release may be carried out using USP dissolution apparatus, for example, USP 1 / 2, USP 3 or USP 4 using standard or mini-vessels or glass beakers.

[0211] In another embodiment of the present invention, testing for solubility may be carried out, for example, in beakers, vials or vials with in-built filters or well plates. The volume of medium used will depend upon the type of solubility experiment (kinetic or equilibrium) and drug amount available.

[0212] Biorelevant dissolution media as described, now provide a reliable and useful approach for reproducibly evaluating drug precipitation using two-stage / transfer biorelevant dissolution tests.

[0213] Analysis of media with drug or formulated drug. These improvements, for example, enable more accurate solubility and dissolution testing of a drug in the reproducible biorelevant media of the present invention using direct UV spectrophotometer measurement; and / or derivative UV spectrophotometry; and / or UV fibre optic probes which have hitherto not been possible using prior art biorelevant media due to lack of reproducibility. It also enables the possibility of more accurate solubility determination of compounds using HTS assays.

[0214] The present invention will now be described in more detail with reference to following examples and the accompanying drawings, in which:

[0215] Figure 1 and Figure 2 are illustrative of the prior art, taken from Krollik, Katharina, et al. "Increasing the robustness of biopharmaceutical precipitation assays-Part II: Recommendations on the use of FaSSIF." Journal of Pharmaceutical Sciences 111.1 (2022): 155-163);

[0216] Figure 3 is a graph illustrating dissolution of Ezetimibe 10mg tablets in FaSSIF medium; and

[0217] Figure 4 is a graph of the two-stage dissolution of Ketconazole (200mg) released 100% in FaSSGF medium.

[0218] EXAMPLES

[0219] By way of example only, the typical biorelevant compositions shown in the Examples below contain bile salt to phospholipid at a molecular ratio of 4:1 or 15:1. However, they equally apply to any bile salt: phospholipid ratio between 75:1 and <1 :1.

[0220] Examples 1 and 2 are comparative Examples of a Commercial Product.

[0221] Example 1 : This is a COMPARATIVE EXAMPLE of a Commercial Product.

[0222] Three commercial units of FaSSIF / FaSSGF / FeSSIF Powder (7g) in 50mL HDPE containers from three different batches were used in the following example (FFF- 1021 -A, FFF-1021-B, FFF-1021-C). The bottles contained moisture absorbers to keep the product dry but there no oxygen sachet was present.

[0223] Storage of commercial product FaSSIF / FaSSGF / FeSSIF Powder.

[0224] Two weeks after the date of manufacturing, the bottles of commercial product were removed from the fridge and stored at 40°C for 3months and 6months respectively.

[0225] Determining oxygen content.

[0226] The oxygen content was measured inside the sealed container just before opening after 3months and 6months of storage at 40°C. This value was used to calculate the average PL: oxygen molecular ratio inside the container.

[0227] Preparation of media.

[0228] 1 L of biorelevant medium (FaSSIF) was prepared by addition of 2.240g of the powder composition (t =0) to FaSSIF Buffer prepared by diluting 41.64g of a commercially available of FaSSIF Buffer concentrate with 961.1g of deionised water. Since this medium contains phospholipid at a concentration of 0.75mM, the same liquid can be used for both UV and particle size measurements.

[0229] UV of diluted composition.

[0230] UV measurements at 234nm of the compositions (stored unopened for 3months and stored unopened 6months at 40°C) were determined by diluting the compositions to PL concentration of 0.75mM in standardized phosphate buffer with sodium chloride at 234nm. Measurements were carried out at t =2hours, 24hours and 48hours after preparation.

[0231] Particle size determination of medium.

[0232] Particle size analysis (using PCS) of the media was carried out at t =2hours, 24hours and 48hours after preparation.

[0233] PUFAs.

[0234] The PUFA content of the aged commercial product was determined at 3months and 6months.

[0235] Results: Table 1 below: Oxygen and PL: oxygen ratio of gaseous mixture inside container (stored at 40°C 3months)

[0236] Table 2 below: UV of composition (stored at 40°C 3months) diluted to 0.75mM in phosphate buffer

[0237] Appearance of medium: clear and transparent liquid.

[0238] Table 3 below: Particle size and PUPA’s of medium prepared from composition (stored at 40°C 3months.

[0239] Table 3 below: PL: oxygen ratio of gaseous mixture (stored at 40°C 6months)

[0240] Table 4 below: UV of composition (stored at 40°C 6months) diluted to 0.75mM in phosphate buffer

[0241] Appearance of medium (prepared with composition stored 6months 40°C): clear and transparent liquid.

[0242] Table 5 below: Particle size of medium prepared from of composition (stored at 40°C 6months) and PUFA content NA = Not applicable too variable to report.

[0243] Conclusion.

[0244] After 3months of storage at 40°C, the commercial product showed insufficient stability: The UV response at 234nm of the biorelevant composition stored at 40°C diluted to 0.75m M of PL in a defined phosphate buffer was found to be more than 3.0AU at t=2hours and 48hours. This comparative example clearly shows that this high UV response means this is outside of the scope of the invention. This correlates well with the findings of Krollik et al.

[0245] The Z-average of the medium at 6months also had also changed compared to product stored at 3moths at 40°C. The PUFA content was about 63% of total fatty acid content after storage of the previously unopened product at 3months of storage at 40°C.

[0246] Similar results were found at 6months of storage at 40°C: The UV response at 234nm of the composition diluted to a PL concentration at 0.75mmol was found to be high (greater than 3.5AU) at 48hours. Furthermore, the medium was clear and the PUFA content of total fatty acid further decreased from 63% at 3months to about 59%.

[0247] The oxygen in the gaseous mixture within the container was 1000:262.

[0248] At 3months and 6months at 40°C the actual oxygen level inside the container just before opening was 0.15% and 20.6% (0.00425mmoles and 0.584mmoles) respectively. The phospholipid: oxygen ratio was found to be 1000:2 at 3months and 1000:262 at 6months. After storage at 6months at 40°C, the oxygen concentration subsequently increased again at 6months. Without being bound by explanation, it is likely that the low oxygen concentration at 3months in this example was likely to be attributed to the consumption of the oxygen by the phospholipid. The composition was already out of specification at 3months because UV absorbance of the diluted composition exceeded 3.0AU and the media prepared from the powder composition exhibited high particle size variability (at 3months and 6) suggesting a change in colloidal structures. Example 2.

[0249] This is a COMPARATIVE EXAMPLE of a Commercial Product.

[0250] Commercial Biorelevant product (FaSSIF-V2 Powder).

[0251] Storage.

[0252] A commercial bottle of FaSSIF-V2 Powder (55g) in 100mL HDPE container from a batch was used in the following example (V2FAS-0821-A)). The bottle contained a moisture absorber to keep the product dry but there was no oxygen sachet present. The product was test after storing for 3years at 4°C.

[0253] Preparation of medium.

[0254] 1 L of biorelevant medium (FaSSIF-V2) was prepared by addition of 1.790g of the powder product (t =0) to the defined phosphate buffer to yield a PL concentration of 0.2mM of PL and 3mM bile salt (sodium taurocholate).

[0255] UV of diluted Product.

[0256] The unopened product was stored for 3years at 4°C. After storage the container was opened, and the powder was diluted to 0.75mM phospholipid concentration (also containing 18.75mM of sodium taurocholate) by adding 8.950g of the powder to 1 L standardized / defined phosphate buffer. Diluting the product to this defined phospholipid concentration (0.75m M) allows the quality of the biorelevant composition to be assessed using a standardized concentration UV at 234nm. The aged product diluted in phosphate buffer were analysed at t =2hours, 24hours and 48hours by UV.

[0257] Particle size.

[0258] The particle size of the test media prepared from the product (aged) were analysed and compared at t =2hours, 24hours and 48hours PCS respectively. PUFAs.

[0259] The PUFA content of the aged commercial product was determined at 3years.

[0260] Results.

[0261] Table 6 below: UV of stored (3years at 4°C) composition (containing 0.75mM phospholipid and 18.75mM STC) diluted in defined phosphate buffer. Table 7 below: Particle size and PUFAs after 3years at 4°C FaSSIF-V2.

[0262] Appearance of medium: clear and transparent liquid.

[0263] After 3years of storage at 4°C, the stability of the product was insufficient: The UV response at 234nm of the stored powder at 4°C was found to be higher than 2.0AU at 2hours and greater than 3.0AU at 48hours.

[0264] Conclusion.

[0265] The unopened product when stored at 3years at 4°C was unstable because the UV response at 234nm when the composition was diluted to 0.75mM of PL was too high (greater than 2.0AU at 2hours and greater than 3.0AU at 48hours).

[0266] Example 3.

[0267] Testing biorelevant compositions in sealed HDPE containers with oxygen sachet and sealed HDPE containers under nitrogen.

[0268] Selection of the phospholipid.

[0269] UV of phospholipid in methanol, phosphatidylcholine (PC) content and PUFA determination.

[0270] 0.75mM of phospholipid was dissolved in methanol and the UV response (10mm path length) at 234nm was determined. The results are shown below.

[0271] The %w / w PC content (dry weight) of the phospholipid was taken from the Certificate of Analysis and PUFA content of the fresh phospholipid was determined.

[0272] Preparation of small test quantity of phospholipid bile salt(s) composition.

[0273] A small test quantity of biorelevant composition was prepared by preparing a solution of 7.4g sodium taurocholate and 2.5g of the phospholipid selected above (molar ratio of 4:1 ) dispersed in about 40.0g water. The resultant solution was lyophilised for 48hours to produce a biorelevant composition.

[0274] UV of diluted composition in phosphate buffer. The test composition was diluted to a phospholipid (PL) concentration of 0.75mM with standardized phosphate buffer and the UV absorbance at 234nm was determined (path length 10mm). The results are shown below.

[0275] Results.

[0276] Table 8 below: UV of phospholipid in methanol, PC content and PUFA determination.

[0277] Table 9 below: UV of pre-test composition diluted to 0.75mM in phosphate buffer.

[0278] The UV response at 234nm was below 2.0AU and even below 1 .OAU at between 2hours and 48hours and the media prepared from the fresh small test quantity composition was, therefore, satisfactory.

[0279] Preparation of biorelevant composition for storage tests.

[0280] Having selected the phospholipid, about 1000g of a bile salt phospholipid (PL) composition (biorelevant composition / composition) was prepared by freeze drying a solution of 743g sodium taurocholate and 257g of phospholipid (molar ratio of 4:1 ) dispersed in about 4000g water for 48hours.

[0281] Milling and filling.

[0282] Biorelevant composition in sealed HDPE container with oxygen sachet.

[0283] The composition manufactured for storage tests from above was milled. 7g (containing 2.3mmol of phospholipid) of the milled cake was filled into 50mL HDPE containers (actual internal 68.9ml, 63.5mL with powder, surface area of 0.01423 m2). An oxygen absorbing sachet sufficient to absorb 20mL of oxygen was weighed into the containers with an aluminium laminate liner sealed using heat induction and tightly closed with an HDPE cap. The OTR of the empty sealed HDPE container with aluminium laminate seal and cap was 0.236 cm3 / day at 40°C. The phospholipid: oxygen molar ratio of the gaseous mixture at the time of sealing was 1000:267. However, after about 1week of incubation post sealing, the phospholipid: oxygen molar ratio of the gaseous mixture was above 1000:4 without any significant increase in UV measurement at 234nm from the freshly prepared composition. This surprisingly suggests that the incubation with oxygen absorber can lower the oxygen within the container quickly enough after sealing before significant phospholipid oxidation can occur in the composition. Using the OTR of the sealed container, the net oxygen exposure molecular ratio in the gaseous mixture inside the sealed container was shown to be 1000:292 (which includes the presence of oxygen after sealing) at 3months.

[0284] Biorelevant composition in sealed HDPE container under nitrogen.

[0285] The composition manufactured for storage tests from above was milled. 7g (containing 2.3mmol of phospholipid) of the dried milled cake was filled into 50mL HDPE containers (actual internal 68.9ml, 63.5mL with powder, surface area of 0.01423 m2) under nitrogen with an aluminium laminate liner sealed using heat and tightly closed with an HDPE cap. The OTR of the empty sealed HDPE container with aluminium laminate seal and cap was 0.236cm3 / day at 40°C. The phospholipid: oxygen molar ratio of the gaseous mixture at the time of sealing was greater than 1000:1. Using the OTR of the sealed container, the net oxygen exposure molecular ratio in the gaseous mixture inside the sealed container was shown to be 1000:426 at 3months.

[0286] Preparation of medium.

[0287] The media (FaSSIF) were prepared following the same preparation procedure of Example using fresh and aged (for 3months and 6months at 40°C) composition.

[0288] 1 L of biorelevant medium (FaSSIF) was prepared by addition of 2.240g of the powder composition (t =0) to FaSSIF Buffer prepared by diluting 41.64g of a commercially available of FaSSIF Buffer concentrate with 961.1g of deionised water. As FaSSIF medium contains phospholipid at a concentration of 0.75mM, in this case, the same liquid can be used for both UV and particle size measurements.

[0289] UV of diluted composition.

[0290] UV measurement at 234nm of the compositions (stored unopened for 3months and stored unopened 6months at 40°C) in the two types of sealed containers (with oxygen absorber and under nitrogen respectively) were determined by diluting the compositions to PL concentration of 0.75mM in standardized phosphate buffer with sodium chloride at 234nm. Measurements were carried out at t =2hours, 24hours and 48hours after sample preparation.

[0291] Particle size determination of medium.

[0292] Particle size analysis (using PCS) of the media at t =2hours, 24hours and 48hours after preparation.

[0293] PUFAs.

[0294] The PUFA content of the fresh and aged compositions (6months) in sealed containers were also determined.

[0295] Results.

[0296] Freshly manufactured material.

[0297] Table 10 below PL: oxygen ratio of composition in gaseous mixture (after sealing and soon after sealing (1week)).

[0298] Table 11 below: UV of composition (fresh) diluted to 0.75mM in defined phosphate buffer.

[0299] Appearance of medium: opalescent liquid.

[0300] Table 12 below: Particle size of medium prepared from freshly manufactured material and PUFA’s.

[0301] Table 13 below: PL: oxygen ratio of composition In gaseous mixture of sealed HDPE containers with oxygen sachet (3months at 40°C).

[0302] Table 14 below: UV of diluted composition from sealed HDPE containers with oxygen sachet (3months at 40°C).

[0303] Table 15 below: Particle size sealed HDPE containers with oxygen sachet (3months at 40°C).

[0304] Appearance of medium: almost clear liquid.

[0305] Table 16 below: PL: oxygen ratio of composition in gaseous mixture of sealed HDPE containers with oxygen sachet (6months at 40°C).

[0306] Table 17 below: UV of diluted composition from sealed HDPE containers with oxygen sachet (6months at 40°C).

[0307] Table 18 below: Particle size and PUP As sealed HDPE containers with oxygen sachet (6months at 40°C).

[0308] Appearance of medium: clear and transparent liquid.

[0309] Table 19 below: PL: oxygen ratio of composition in gaseous mixture under nitrogen (after sealing).

[0310] Table 20 below: PL: oxygen ratio of composition In gaseous mixture under nitrogen (3months at 40°C). Average PL : 02 ratio |

[0311] Table 21 below: UV of diluted composition in sealed HDPE containers under nitrogen (3months at 40°C). Table 22 below: Particle size sealed HDPE containers under nitrogen (3months at 40°C).

[0312] Appearance of medium: almost clear liquid.

[0313] Table 23 below: PL: oxygen ratio of composition in gaseous mixture under nitrogen (6months at 40°C).

[0314] Table 24 below: UV of diluted composition In sealed HDPE containers under nitrogen (6months at 40°C). Table 25 below: Particle size sealed HDPE containers under nitrogen (6months at 40°C).

[0315] NA = Not applicable as results too variable.

[0316] Appearance of medium: clear and transparent liquid. Conclusion

[0317] After 3months and 6months of storage at 40°C, all stored compositions showed insufficient stability: The UV response of the biorelevant composition stored at 40°C when diluted to 0.75mM of PL (also containing 3mM sodium taurocholate) in a defined phosphate buffer at 234nm was found to be greater than 2.0AU and 3.0AU respectively at 48hours. At 6months the seal of one bottle (packed with oxygen sachet) was breached which was reflected by an even higher UV value than the other samples.

[0318] The Z-average of the medium prepared from compositions stored for 3months and 6months had significantly changed and the PDI values were highly variable. This was also reflected by physical appearance of the medium which had changed from opalescent when fresh to clear after storing at 40°C for 3months or 6months. At 6months, for this composition the PUFA content dropped to about 62% and 63% of total fatty acid content.

[0319] Although the PL to oxygen molecular ratio in the gaseous mixture in the container with oxygen absorbing sachet was high (1000:267) immediately after sealing, soon after filling and sealing (about 1 week of incubation (at room temperature)), the PL to oxygen molecular ratio equilibrated to around 1000:4. The oxygen content was determined experimentally with an Oxybaby®. This ratio soon after sealing was satisfactory. The threshold is above 1000:100, above 1000:60; above 1000:40; above 1000:25; above 1000:10; above 1000:5; and above 2000:1). Also, the UV measurement at 234nm was 1.0AU which is below the UV limit of 1.5AU soon after sealing suggesting there was no significant oxidation.

[0320] The actual oxygen level inside the container with the oxygen absorber sachet, measured just before opening at 3months, was 1.42%v / v (0.04028mmoles) which equated to a phospholipid: oxygen molar ratio inside the container of approximately 1000:18.

[0321] Similarly, at 3months the oxygen level inside the container filled with nitrogen just before opening was 0.89%v / v (0.02524mmoles) and the phospholipid to oxygen ratio was found to be 1000:11. However, after 3months of storage at 40°C, despite the low level of oxygen of the gaseous mixture of the container with either an oxygen absorbing sachet or filling under nitrogen, the biorelevant composition and the resultant media prepared from these compositions still showed insufficient stability and was out of specification because of high UV response (greater than 2.0AU) after dilution of the composition at a PL concentration of 0.75mM in the defined phosphate buffer at 48hours.

[0322] At 6months at 40°C, the oxygen level inside both containers, measured just before opening, has risen to 20.6%v / v (0.584mmoles) and the phospholipid: oxygen ratio was found 1000:262.

[0323] For long term storage at elevated temperature of 40°C, the compositions in sealed containers under the two conditions were unstable at 3months and 6months and do not meet the required parameter for UV absorption of the diluted composition (after storage), i.e. absorption value below 1.8AU after dilution of the composition to 0.75mMole of PL in defined phosphate buffer at 3months.

[0324] Example 4.

[0325] Compositions in sealed Aluminium, multilayered plastic (EVOH with HDPE) or HDPE containers.

[0326] Three different types of rigid containers (aluminium, rigid multilayered plastic (EVOH) and HDPE) under different conditions were tested.

[0327] Selection of the Phospholipid.

[0328] Phospholipid was selected as described in Example 3 was carried out and a biorelevant composition was prepared following the same preparation procedure of Example 3.

[0329] Milling and Filling.

[0330] Aluminium bottles.

[0331] The resultant dried biorelevant composition (cake) was milled and 7g (containing 2.3mmol of phospholipid) was filled into 50mL (nominal) UN approved aluminium bottles (56.87mL actual internal volume (empty), 51.4mL after inserting powder, calculated external surface area 0.0138m2) under nitrogen with the associated plastic PE plug and tightly closed with an HDPE cap with aluminium liner. The OTR range of the sealed aluminium containers (container, plug with cap) was surprisingly found to be highly variable between 3.6 to 29cm3 / m2 / day (calculated based on the variability of the configured containers) at 40°C and room temperature. The phospholipid: oxygen molar ratio of the gaseous mixture after filling and sealing was greater than 1000:1 which achieved the target ratio of 1000:100 and above.

[0332] Storage.

[0333] The aluminium bottles containing the powder under the nitrogen were stored at 40°C for 3months and 6months and room temperature for 6months. At 3months, the actual average phospholipid: oxygen molar ratio remained around 1000:1. However, after 6months the average actual phospholipid to oxygen of the gaseous mixture molecular ratio inside the aluminium bottles was found to be 1000:212. At room temperature for 6, variability was also found in the oxygen level between the samples (between 11.76% and 16.62%) and the average phospholipid: oxygen molar ratio inside the container was around 1000:140.

[0334] Rigid plastic multilayered bottle.

[0335] The resultant dried biorelevant composition (cake) was milled and 14g (containing 4.5mmol of phospholipid) was filled into 100mL extruded blow moulded bottles (6- layers including HDPE and EVOH) (actual internal 102.1 mL, 91.3mL with powder, surface area 0.0133m2) under nitrogen with an aluminium laminate seal and tightly closed with a cap using same multilayered plastic. The OTR of the 100 mL container, heat sealed aluminium laminate and cap was 0.002 cm3 / container / day at 40°C.The phospholipid: oxygen molar ratio of the gaseous mixture at the time of sealing was greater than 1000: 1 .

[0336] Storage.

[0337] The rigid plastic multilayered bottles containing the powder under the nitrogen were stored at 40°C for 3months and 6months. The PL: oxygen molar ratio inside the container just prior to opening was found to be 1000:4 at 3months. Using the OTR value, the phospholipid in contact with the net exposure of oxygen from the gaseous mixture that was in contact with in the sealed container at 3months at 40°C was around 1000:2 and 1000:4 after 6months at 40°C.

[0338] HDPE.

[0339] The resultant dried biorelevant composition (cake) was milled and 7g and 35g (containing 2.3 and 11.1 mmol of phospholipid) were filled into 50mL HDPE containers (actual internal 68.9ml, 63.5mL with powder, surface area of 0.01423 m2) under nitrogen with an oxygen sachet (capable of absorbing 20mL oxygen) and sealed with an aluminium laminate liner and tightly closed with an HDPE cap. The OTR of the sealed HDPE container with aluminium laminate seal and cap was 0.236cm3 / day at 40°C.

[0340] The HDPE bottles containing the powders (7g and 35g) under the nitrogen with oxygen sachet were stored at 40°C for 3months and 6months. The phospholipid: oxygen molar ratio of the gaseous mixture soon after sealing of both sets of bottles were greater than 1000: 1 .

[0341] Storage.

[0342] The HDPE bottles containing the 7g of powder under the nitrogen with oxygen sachet were stored at 40°C for 3months and 6months. The phospholipid: oxygen molar ratio of powder filled in 50mL HDPE containers surprisingly decreased to 1000:2 after 3months but after 6months the phospholipid: oxygen molar ratio was found 1000:262, the oxygen content was determined experimentally with an Oxy ba by®.

[0343] Using the OTR of the sealed container and considering the nitrogen filling and oxygen absorbing capacity of the sachet, the PL to net oxygen exposure molecular ratio in the gaseous mixture inside the sealed container were shown to be 1000:25 at 3months and 1000:450 at 6months.

[0344] The HDPE bottles containing the 35g of powder under the nitrogen with oxygen sachet were stored at 40°C for 3months and 6months. The phospholipid: oxygen molar ratio of powder filled in 50mL HDPE containers surprisingly decreased to 1000:1.5 after 3months but after 6months the phospholipid: oxygen molar ratio was found 1000:35, the oxygen content was determined experimentally with an Oxy ba by®.

[0345] Using the OTR of the sealed container and considering the nitrogen filling and oxygen absorbing capacity of the sachet, the PL to net oxygen exposure molecular ratio were shown to be 1000:5 at 3months and 1000:90 at 6months.

[0346] Preparation of medium.

[0347] See Example 3 for the preparation of the medium.

[0348] UV of diluted compositions. UV measurement at 234nm of the compositions (fresh, stored unopened for 3months and stored unopened 6months at 40°C) in the sealed rigid containers under the described conditions were determined by diluting the compositions to PL concentration of 0.75mM in standardized phosphate buffer with sodium chloride at 234nm. Measurements were carried out at t =2hours, 24hours and 48hours after preparation.

[0349] Particle size determination of medium.

[0350] Particle size analysis (using PCS) of the media prepared from the compositions were carried out at t =2hours, 24hours and 48hours after preparation of the media.

[0351] Results.

[0352] Table 26 below: PL to oxygen ratio of Aluminium bottles under nitrogen (fresh).

[0353] Table 27 below: UV of freshly manufactured material diluted to 0.75mM phospholipid.

[0354] Appearance of medium: opalescent liquid.

[0355] Table 28 below: Particle size of freshly manufactured material diluted to 0.75mM phospholipid.

[0356] Table 29 below: PL to oxygen ratio of Aluminium bottles under nitrogen 3months at 40°C.

[0357] Table 30 below: UV of diluted composition in Aluminium bottles under nitrogen 3months at 40°C. Appearance of medium: opalescent liquid.

[0358] Table 31 below: Particle Size and PUFAs of Aluminium bottles under nitrogen 3months at 40°C. Table 32 below: PL: oxygen ratio of composition in gaseous mixture inside aluminium bottles under nitrogen 6months at 40°C.

[0359] Table 33 below: UV of diluted composition in Aluminium bottles under nitrogen 6months at 40°C.

[0360] Appearance of medium: opalescent liquid. Table 34 below: Particle Size and PUFAs of Aluminium bottles under nitrogen 6months at 40°C.

[0361] Table 35 below: PL: oxygen ratio of composition In gaseous mixture Inside aluminium bottles under nitrogen 6months at 22°C.

[0362] Table 36 below: UV of diluted composition in Aluminium bottles under nitrogen 6months at 22° C.

[0363] Appearance of medium: Two opalescent liquids, one clear and transparent liquid.

[0364] Table 37 below: Particle Size and PUFAs of Aluminium bottles under nitrogen 6months at 22° C.

[0365] Surprisingly, the biorelevant composition in aluminium bottles showed some variability The UV response at 234nm of the biorelevant composition stored at 40°C diluted to a PL concentration of 0.75mM in a defined phosphate buffer was found to be greater than 2.0AU at 2 and 48hours after sample preparation in some containers. This study was also performed at room temperature which also showed high variability in the results which linked to the variable OTR (3.6 to 29cc / m2 (configured container)). This unexpected inconsistency was likely to be due to the unexpected variability of the sealed containers OTR.

[0366] Table 38 below: PL to oxygen ratio of gaseous mixture in rigid plastic multilayered under nitrogen 3months at 40°C.

[0367] Table 39 below: UV of diluted composition in rigid plastic multilayered under nitrogen 3months at 40°C.

[0368] Appearance of medium: opalescent liquid.

[0369] Table 40 below: Particle Size and PUFAs of rigid plastic multilayered under nitrogen 3months at 40°C.

[0370] Table 41 below: PL to oxygen ratio of gaseous mixture in rigid plastic multilayered under nitrogen 6months at 40°C.

[0371] Table 42 below: UV of diluted composition in rigid plastic multilayered under nitrogen 6months at 40°C.

[0372] Appearance of medium: opalescent liquid.

[0373] Table 43 below: Particle Size and PUFAs of rigid plastic multilayered under nitrogen 6months at 40°C.

[0374] After 3months and 6months of storage at 40°C, the biorelevant composition in a rigid plastic multilayered container showed sufficient stability. The UV response at 234nm of the biorelevant composition stored at 40°C diluted to 0.75m M of PL in a defined phosphate buffer was found to be less than 2.0AU; 1.8AU;1.5AU;1.25AU;1.0AU at t = 2hours and even at 48hours after sample preparation.

[0375] The Z-average, PDI and physical appearance of the medium did not change at 3months and 6months when compared to the medium prepared from powder at t = 0.

[0376] After 3months and 6months of storage at 40°C, the PUFA content remained stable at about 70% of total fatty acid content.

[0377] Table 44 below: PL to oxygen ratio of gaseous mixture in under nitrogen with oxygen sachet 3months at 40°C.

[0378] Table 45 below: UV of diluted composition in HDPE bottles 7g of powder under nitrogen with oxygen sachet 3 months at 40°C.

[0379] Appearance of medium: almost clear liquid.

[0380] Table 46 below: Particle size and PUFA’s HDPE bottles 7g of powder under nitrogen with oxygen sachet 3 months at 40°C.

[0381] Table 47 below: PL: oxygen ratio of composition In gaseous mixture inside container with 7g of powder under nitrogen with oxygen sachet 6months at 40°C.

[0382] Table 48 below: UV of diluted composition In HDPE bottles 7g of powder under nitrogen with oxygen sachet 6months at 40°C.

[0383] Appearance of medium: clear and transparent liquid.

[0384] Table 49 below: Particle size and PUFA’s bottles 7g of powder under nitrogen with oxygen sachet 6months at 40°C.

[0385] After 3months and 6months of storage at 40°C, the biorelevant composition (7g) had insufficient stability. The UV response at 234nm of the biorelevant composition stored at 40°C diluted in a defined phosphate buffer to 0.75m M of PL was around 2.0AU and 2.5AU respectively at 48hours.

[0386] The Z-average of the medium at 3months and 6months had significantly changed and the PDI values were highly variable, also the physical appearance of the medium had changed when compared to the medium prepared from powder composition at t = 0.

[0387] After 3months and 6months the PUFA dropped to about 68% and 65% respectively of total fatty acid content.

[0388] Table 50 below: PL: oxygen ratio of composition in gaseous mixture within HDPE bottles containing 35g of powder under nitrogen with oxygen sachet 3months at 40° C.

[0389] Table 51 below: UV of diluted composition In HDPE bottles 35g of powder under nitrogen with oxygen sachet 3 months at 40°C.

[0390] Appearance of medium: opalescent liquid.

[0391] Table 52 below: Particle size and PUFA’s ofHDPE bottles 35g of powder under nitrogen with oxygen sachet 3 months at 40°C.

[0392] Table 53 below: PL: oxygen ratio of composition in gaseous mixture inside sealed HDPE bottles 35g of powder under nitrogen with oxygen sachet 6months at 40°C.

[0393] Table 54 below: UV of diluted composition In HDPE bottles 35g of powder under nitrogen with oxygen sachet 6months at 40°C.

[0394] 15

[0395] Appearance of medium: opalescent liquid.

[0396] Table 55 below: Particle size and PUFA’s ofHDPE bottles 35g of powder under nitrogen with oxygen sachet 6months at 40°C.

[0397] After 3months at 40°C, the biorelevant composition (35g) was found satisfactory as the phospholipids: oxygen molar ratio was 1000:1.5. The UV response of the biorelevant composition prepared from compositions stored at 40°C diluted to 0.75mM of PL in a defined phosphate buffer at 234nm was around 1.4AU at 48hours.

[0398] After 6months at 40°C the UV response of the biorelevant composition diluted to 0.75mM of PL in a defined phosphate buffer was around 1.6AU at 48hours and the phospholipid: oxygen molar ratio was 1000:35. This is within the scope of the invention.

[0399] Conclusion:

[0400] Aluminium bottles.

[0401] Compositions stored in aluminium bottles after 3months and 6months at 40°C exhibited variable results. Just before opening, the actual oxygen level (averaged) inside the containers after 3months and 6months was found to be 0.05 and 20.6% (0.001 and 0.58mmoles) which equated to an average phospholipid to oxygen ratio of above 1000:1 and 1000:212 respectively.

[0402] Rigid plastic multilayered.

[0403] At 3months and 6months at 40°C the actual oxygen level inside the container just before opening was 0.42% (0.017mmoles) and the phospholipid to oxygen ratio was found 1000:4.

[0404] HDPE.

[0405] The biorelevant composition 7g in 50mL at 3months and 6months at 40°C, the actual oxygen levels inside the container just before opening were 0.14 and 20.6% (corresponding to 0.004 and 0.58mmoles) respectively. The phospholipid: oxygen ratio was found 1000:2 at 3months and 1000:262 at 6months.

[0406] The biorelevant composition (35g) in 50mL at 3months and 6months at 40°C, the actual oxygen levels inside the container just before opening were 0.42% and 20.6% (corresponding to 0.007 and 0.4mmoles) and the phospholipid: oxygen ratio was found 1000:1.5 at 3months and 1000:35 at 6months. This highlights the importance of selecting the fill volume to configure the pack / pack size.

[0407] Example 5.

[0408] Biorelevant composition in flexible containers.

[0409] The following biorelevant composition provided below contains bile salt to phospholipid at a molecular ratio of 4:1 . However, any composition with a bile salt: phospholipid mole ratio between 75:1 and <1 :1 could be selected.

[0410] Selection of the phospholipid.

[0411] 0.75 mM of phospholipid was dissolved in methanol and the UV response at 234nm was determined and found to be 0.09. The phospholipid selected had a 95% w / w phosphatidyl choline purity adhering to the monograph for USP Soyabean phosphatidylcholine but with a narrower polyunsaturated specification of between 65-75% w / w of phospholipid total fatty acid content. At time zero the polyunsaturated fatty acid content of the phospholipid was 70.4% w / w of the phospholipid total fatty acid content. This is the preferred specification of PL in this disclosure.

[0412] Selection of the bile salt.

[0413] The selected bile salt used in this Example was sodium taurocholate (>90% w / w purity, dry weight). However, one or more bile salt(s) and / or bile acid(s) (at least 80% w / w purity, dry weight) may be selected from the following list; sodium cholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium taurodeoxycholate, sodium glycodeoxycholate, sodium ursodeoxycholate, sodium chenodeoxycholate, sodium taurochenodeoxycholate, sodium glycochenodeoxycholate, sodium cholylsarcosinate, and sodium N- methyl taurocholate; preferably, sodium taurocholate; sodium glycocholate; sodium taurodeoxycholate; sodium glycodeoxycholate and sodium cholate.

[0414] Other non-essential components

[0415] The composition may contain at least one other physiologically relevant component such as glycerol monooleate, cholesterol and / or sodium oleate.

[0416] Preparation of biorelevant composition.

[0417] A biorelevant composition was prepared following the same preparation procedure of Example 3.

[0418] Milling as described in Example 3.

[0419] The resultant dried biorelevant composition (cake) was milled as described in Example 3.

[0420] Filling.

[0421] Barrier pouch (BoPET).

[0422] The resultant dried biorelevant composition (cake) was milled and 7g of the filled into a nominal volume 40mL (92.x123mm internal dimensions, surface area 0.0226m2) stand up pouch (110 micro BoPET) pouch, with 20mL absorption oxygen sachet and heated sealed under vacuum (about 2mBar). The OTR of this sealed pouch was 0.330cc / sealed container / day. The phospholipid: oxygen molar ratio of the gaseous mixture at the time of filling was greater than 2000:1. Storage.

[0423] The stand-up sachet containing the biorelevant composition was stored for 3months at 40°C.

[0424] METPET pouch.

[0425] The resultant dried biorelevant composition (cake) was milled and 7g of the filled into a nominal volume 40mL (16x24cm internal dimension, surface area 0.0768m2) METPET laminate sachet (composed by 14pm PET, 12pm METPET and 89pm PET) hermetically heated sealed under vacuum. The OTR of this thermally sealed pouch was less than 0.001 cc / configured container / day at 40°C. The PL: oxygen level was above than 2’000:1 .

[0426] Storage.

[0427] The METPET sachet containing the biorelevant composition was stored for 3months and 3years at 40°C.

[0428] Aluminium pouch.

[0429] The resultant dried biorelevant composition (cake) was milled and 7g of the filled into a nominal volume 40mL (100x50m internal dimension, surface area 0.010m2) aluminium laminate (7 micrometer aluminium thickness) sachet and hermetically heat sealed under vacuum (about 2m Bar). The OTR of this thermally sealed aluminium laminate pouch was less than 0.001 cc / configured container / day at 40°C. The PL: oxygen level was above 2’000:1 .

[0430] Storage.

[0431] The aluminium sachet containing the biorelevant composition was stored for 3months and 3years at 40°C.

[0432] METBOPP pouch.

[0433] The resultant dried biorelevant composition (cake) was milled and 7g of the filled into a nominal volume 40mL of the recyclable (145x65mm internal dimension, surface area 0.01885 m2) METPET laminate sachet (composed by 25pm MATTE PE, 18pm high barrier METBOPP and 89pm clear PE) and heated sealed under vacuum (about 2mBar). The OTR of this thermally sealed pouch was less than 0.001 cm3 / based on actual configured container at 40°C. The PL: oxygen molecular ratio inside the void space inside the sealed pouch was estimated to be above 2’000:1. Storage.

[0434] The METBOPP sachet containing the biorelevant composition was stored for 3months and 6months at 40°C.

[0435] PL: oxygen ratio of composition inside gaseous mixture of flexible containers (upon storage).

[0436] As the flexible pouches were sealed under vacuum, even upon storage the level of oxygen in the gaseous mixture was below the limit of detection. Therefore, the OTR was used to estimate the ingress of the oxygen into the pack during storage.

[0437] Preparation of medium.

[0438] Medium containing 0.75mM of PL and 3mM sodium taurocholate was prepared following the same preparation procedure of Example 3.

[0439] UV of diluted composition.

[0440] UV measurement at 234nm of the compositions in the various flexible containers were determined by diluting the compositions to PL concentration of 0.75mM in standardized phosphate buffer with sodium chloride at 234nm. Measurements were carried out at t =2hours, 24hours and 48hours after preparation.

[0441] Particle size determination of medium.

[0442] Particle size analysis (using PCS) of the media was carried out at t =2hours, 24hours and 48hours after preparation.

[0443] Results:

[0444] Table 56 below: UV of freshly manufactured material.

[0445] Appearance of medium: opalescent liquid.

[0446] Table 57 below: Particle size of freshly manufactured material.

[0447] Table 58 below: PL: oxygen ratio of composition in gaseous mixture inside BoPET pouch 3months at 40°C.

[0448] Table 59 below: UV of diluted composition in Barrier pouch (BoPET) with oxygen sachet 3months at 40°C.

[0449] Appearance of medium: clear and transparent liquid.

[0450] Table 60 below: Particle size of medium from composition in Barrier pouch (BoPET) with oxygen sachet 3months at 40°C.

[0451] Table 61 below: PL: oxygen ratio of composition In gaseous mixture Inside METPET pouch (3months at 40°C).

[0452] Table 62 below: UV of diluted composition in METPET pouch 3months at 40°C. Appearance of medium: opalescent liquid.

[0453] Table 63 below: Particle size of media and PUFA’s of composition in METPET pouch 3months at 40°C.

[0454] The composition in the thermally sealed METPET sachet sealed under vacuum was stored at 40°C for 03months at RT and 40°C. The PUFA content was found to be 73.08%.

[0455] Table 64 below: PL: oxygen ratio of composition In gaseous mixture Inside METPET pouch (3years at 40°C).

[0456] Table 65 below: UV of diluted composition in METPET pouch 3years at 40°C.

[0457] Appearance of medium: opalescent liquid.

[0458] Table 66 below: Particle size of METPET pouch 3years at 40°C.

[0459] Compositions in sealed METPET containers were stable for 3years at 40°C.

[0460] Table 67 below: PL: oxygen ratio of composition in gaseous mixture inside Aluminium pouch (3months at 40°C). Table 69 below: UV of diluted composition in of Aluminium pouch 3months at 40° C.

[0461] Appearance of medium: opalescent liquid.

[0462] Table 70 below: Particle size of Aluminium pouch 3months at 40°C.

[0463] Table 71 below: PL: oxygen ratio of composition In gaseous mixture Inside Aluminium pouch (6months at 40°C).

[0464] Table 72 below: UV of diluted composition in Aluminium pouch 6months at 40°C.

[0465] Appearance of medium: opalescent liquid.

[0466] Table 73 below: Particle size of Aluminium pouch 6months at 40°C. io

[0467] Table 74 below: PL: oxygen ratio of composition In gaseous mixture Inside Aluminium pouch 3years at 40°C.

[0468] Table 75 below: UV of diluted composition in Aluminium pouch 3years at 40°C.

[0469] 7U

[0470] Appearance of medium: opalescent liquid.

[0471] Table 76 below: Particle size and PUFA’s of Aluminium pouch 3years at 40°C.

[0472] Table 77 below: PL: oxygen ratio of composition In gaseous mixture Inside METBOPP pouch 3months at 40°C.

[0473] Table 78 below: UV of diluted composition in METBOPP pouch 3months at 40°C.

[0474] 5

[0475] Appearance of medium: opalescent liquid.

[0476] Table 79 below: Particle size of METBOPP pouch 3months at 40°C.

[0477] Table 80 below: PL: oxygen ratio of composition in gaseous mixture inside METBOPP pouch (6months at 40°C).

[0478] Table 81 below: UV of diluted composition in METBOPP pouch 6months at 40°C.

[0479] Appearance of medium: opalescent liquid.

[0480] Table 68 below: Particle size and PUFA’s of METBOPP pouch 6months at 40°C. 74.06 75.91 73.59 | 0.040 0.091 | 0.049 |

[0481] Results.

[0482] Vacuum sealing removes most of the air (at 2mbar with a minimal void space) with flexible containers results in very high PL: oxygen molecular ratios (>10’000:1 ), preferably > 5000:1 , more preferably >2000:1.

[0483] Compositions stored in the various sealed flexible containers for 3months of storage at 40°C were diluted to 0.75m M of PL diluted in phosphate buffer and analysed by UV at 234 nm. The biorelevant compositions prepared from powders packaged in various pouch materials exhibited acceptable stability, except for the sample stored in BoPET pouches. The UV response of the biorelevant composition stored at 40°C in BoPET pouches diluted in a defined phosphate buffer to 0.75mM of PL at 234nm was found to be 4.0AU, this was associated with higher oxygen transmission rate (OTR) associated with the BoPET material and inferior sealing. The opalescent appearance of the medium prepared from fresh composition at t = 0 had changed to a clear liquid medium (prepared from 3month stored composition in sealed container at 40°C). Due to the insufficient stability, further testing was stopped.

[0484] Subsequent analyses conducted after 6months and 3years of storage at 40°C confirmed that the biorelevant compositions packaged in METPET, aluminium, and METBOPP pouches diluted in a defined phosphate buffer to 0.75mM of PL demonstrated surprisingly exceptional stability and robustness at 40°C at 3months.

[0485] The compositions in METPET, aluminium, and METBOPP sachets diluted to 0.75mM of PL in phosphate buffer all yielded a UV response at 234nm around 1.0AU.

[0486] The Z-average, PDI and physical appearance of the medium prepared from compositions in aluminium pouches did not change at 6months and 3years when compared to the medium prepared from powder at t = 0. The PUFA content of aluminium pouch was unchanged at about 73% of total fatty acid content. The vacuum seal of METPET, aluminium, and METBOPP pouches / sachets was maintained and, therefore, the phospholipid to oxygen was calculated to be significantly above 2000:1 at 6months 40°C and aluminium also METPET pouches was found to be remarkably stable for 3years at 40°C. Due to the very low level of oxygen in the sealed container, it may be necessary to calculate the oxygen content based on vacuum / sealing pressure (mBar) using an estimated void space.

[0487] Conclusion

[0488] The solid compositions in sealed flexible containers (stored for 3months) when diluted to a PL concentration of 0.75m M yielded remarkably low UV responses at 234nm below 1.5AU; 1.25AU and even 1.0AU (with a 10mm path cell) after 48hours. Furthermore, the media prepared using the stored compositions in the sealed containers after first opening yielded consistent particle size compared to media prepared using fresh compositions. The compositions in the selected sealed flexible containers demonstrate remarkable stability at least 3months, at least 6months, at 9months and up to 36months at 40°C.

[0489] Example 6 Reopening data

[0490] Refrigeration after first opening the container

[0491] After opening for the first time after 3months of storage at 40°C, the sealed containers with compositions of Example 3 (HDPE), Example 4 (aluminium bottles) and Example 5 (aluminium pouches) were stored in the fridge. The containers were removed every day for 4weeks (exposing the container to air) and then resealed after 1 minute.

[0492] Small aliquots of powder from inside the container were withdrawn from the three containers at two weeks and four weeks after first opening the container and analysed for UV measurements and particle size.

[0493] Preparation of medium.

[0494] 0.75mM of PL contained 3mM sodium taurocholate medium was prepared following the same preparation procedure of Example 3.

[0495] UV of diluted composition.

[0496] UV measurement at 234nm of the sampled compositions in the three types of sealed containers were determined by diluting the compositions to PL concentration of 0.75mM in standardized phosphate bufferwith sodium chloride at 234nm. Measurements were carried out at t =2hours, 24hours and 48hours after preparation.

[0497] Particle size determination of medium.

[0498] Particle size analysis (using PCS) of the media prepared from the reopened compositions were determined at t =2hours, 24hours and 48hours after preparation.

[0499] Results.

[0500] HDPE bottle stored at 4°C.

[0501] Table 69 below: UV of diluted composition at 2weeks after first opening.

[0502] Appearance of medium: clear and transparent liquid.

[0503] Table 70 below: Particle size at 2weeks after first opening.

[0504] Table 71 below: UV of diluted composition at 4weeks after first opening.

[0505] Appearance of medium: clear and transparent liquid.

[0506] Table 72 below: Particle size and PUFA’s at 4weeks after first opening.

[0507] Aluminium bottle stored at 4°C.

[0508] Table 73 below: UV of diluted composition at 2weeks after first opening.

[0509] Appearance of medium: almost clear liquid.

[0510] Table 74 below: Particle size at 2weeks after first opening.

[0511] Table 75 below: UV of diluted composition at 4weeks after first opening.

[0512] Appearance of medium: almost clear liquid.

[0513] Table 76 below: Particle size and PUFA’s at 4weeks after first opening.

[0514] Aluminium pouch stored at 4°C.

[0515] Table 77 below: UV of diluted composition at 2weeks after first opening.

[0516] Appearance of medium: opalescent liquid.

[0517] Table 78 below: Particle size at 2weeks after first opening.

[0518] Table 79 below: UV of diluted composition at 4weeks after first opening. Appearance of medium: opalescent liquid.

[0519] Table 80 below: Particle size and PUFA’s at 4weeks after first opening.

[0520] After sampling from the reopened containers at 2 and 4weeks of storage at 4°C in HDPE containers, the biorelevant composition in the HDPE container showed insufficient stability: The UV response at 234 nm of the biorelevant composition stored at 4°C diluted to 0.75mM of PL in a defined phosphate buffer exceeded 2.0AU after 48hours.

[0521] The Z-average of the medium at 2 and 4weeks had significantly changed and the PDI values were highly variable also physical appearance of the medium had changed when compared to the medium prepared from powder composition at t = 0. The PUFA content dropped to about 65% of total fatty acid content.

[0522] Similar results were also found after re-opening at 2 and 4weeks of storage at 4°C in Aluminium containers. The biorelevant composition which exhibited insufficient stability after reopening: The UV response of the biorelevant composition stored at 4°C diluted to 0.75mM of PL in a defined phosphate buffer at 234 nm exceeded 2.0AU after 48hours.

[0523] However, the Z-average and PDI of medium at 2weeks and 4weeks did not change when compared to the medium prepared from powder at t = 0.

[0524] In contrast, the biorelevant composition stored in sealed aluminium pouches demonstrated excellent stability. The UV response at 234nm of the biorelevant composition stored at 4°C for 4weeks after first opening diluted to 0.75mM of PL in a defined phosphate buffer was below 1.8AU; 1.5AU; 1.25AU; 1.0AU after daily reopening over a period of four weeks.

[0525] The Z-average, PDI and physical opalescent appearance of the medium did not change at 2 and 4weeks when compared to the medium prepared from fresh powder composition at t = 0. The PUFA content of aluminium pouch was the same as fresh composition and found to be 70% of total fatty acid content. Conclusion

[0526] The flexible containers are not just stable during storage at 40°C for greater than 3months; 6months and even up to 3years but after re-opening for at least 4 weeks the compositions are more stable and produce superior media that have a lower UV and significantly less variable.

[0527] Example 7. In situ preparation.

[0528] 0.75 mM of phospholipid was dissolved in methanol and the UV response at 234nm was determined and found to be 0.09. The phospholipid selected had a 92% w / w phosphatidylcholine purity (on a dry weight basis) adhering to the monograph for USP Phosphatidylcholine. At time zero the polyunsaturated fatty acid content of the phospholipid was 70.4% w / w of the phospholipid total fatty acid content. The phospholipid was checked by using H-NMR to establish the level of conjugation of the fatty acid and found to be absent.

[0529] Preparation of biorelevant composition

[0530] Having selected the phospholipid about 1000g of a bile salt phospholipid composition (biorelevant composition) was prepared by freeze drying a solution 743g sodium taurocholate of 257g of phospholipid (molar ratio of 4:1) dissolved in about 4000g water.

[0531] Filling in situ and Sealing

[0532] 0.93g of the liquid was filled in situ into 10mL glass vials (actual volume11 mL, 0.00346m2 surface area) with closable stopper and sealed under vacuum. The resultant solution was lyophilised in the container (in situ) for 48hours to remove the water. Afterwards the resultant vials were sealed with aluminium crimp cap. The OTR of was <0.001 cm3 / day at 40°C. The fill volume was extremely low. With prior art approaches this could not be achieved at room temperature or higher temperature (40°C) for an extended period (at least 3months) without the composition yielding very high UV response. This high UV response and variability in particle size would hinder it is use for reliable direct UV solubility measurements or solubility measurements for HTS determinations.

[0533] In pre-experiments it was found that crimping of the cap was not always reliable and led to variability in the occasional vial during long term storage because the cap was not fully secure. This variability was eliminated by vacuum sealing the sealed crimped vial in an aluminium pouch to overcome the weakness and variability in the crimping.

[0534] Storage conditions

[0535] The glass vials were stored at 40°C for 3months. The PL: oxygen level was above than 1000:1. Furthermore, the glass vials sealed in the aluminium pouches at 40°C for 3months and the PL: oxygen level was consistently above 1000: 1 .

[0536] Preparation of medium

[0537] 0.1 L FaSSIF was prepared by addition of FaSSIF Buffer prepared by diluting 4.53g of a commercially available of FaSSIF Buffer concentrate with 100g of deionised water to glass vial contained powder equals to 100mL of FaSSIF medium (0.75mM of PL also containing 3mM sodium taurocholate).

[0538] UV of diluted composition

[0539] UV measurement at 234nm of the composition in container was determined by measuring the compositions diluted to PL concentration of 0.75mM in standardized phosphate buffer with the osmolarity adjusting agent sodium chloride. Measurements were carried out at t =2hours, 24hours and 48hours after sample preparation.

[0540] Particle size determination of medium

[0541] Particle size analysis (using PCS) was carried out on the medium at t =2hours, 24hours and 48hours after sample preparation.

[0542] The net amount of oxygen from the gaseous mixture that was in contact with phospholipid in the vacuum sealed container was calculated by adding the oxygen content in the container after sealing to the amount of oxygen entering / ingress the container (using the OTR of the sealed container) at 40°C after 3months. As the container was sealed under vacuum, the oxygen content in the container after sealing was zero and it was verified experimentally. The total amount of oxygen from the gaseous mixture that was in contact with phospholipid in the vacuumed sealed container was calculated by adding the oxygen content in the container after sealing with the amount of oxygen entering the container at 40°C after 3months and estimated at 1000:54. As there was a vacuum, the oxygen content in the container after sealing was zero and it was verified experimentally.

[0543] Results.

[0544] Table 81 below: UV of freshly manufactured material.

[0545] Appearance of medium: opalescent liquid.

[0546] Table 82 below: Particle size of freshly manufactured material.

[0547] Table 83 below: PL: oxygen ratio of composition in gaseous mixture inside container (after 3months storage at 40°C).

[0548] Table 84 below: UV of diluted composition (after 3months storage at 40°C).

[0549] Appearance of medium: opalescent liquid.

[0550] Table 85 below: Particle size and PUFA’s after 3months storage at 40°C.

[0551] After 3months of storage at 40°C, the biorelevant composition demonstrated satisfactory stability. The UV response at 234 nm of the biorelevant composition stored at 40°C diluted to 0.75mM of PL in a defined phosphate buffer from vials stored at 40°C was approximately 1 .OAU after 48hours. The Z-average, PDI and physical appearance of the medium remained unchanged compared to the medium prepared from the powder at time point t = 0. Additionally, the polyunsaturated fatty acids (PUFA’s) content was maintained at approximately 70% of the total fatty acid composition.

[0552] Conclusion.

[0553] The powder stored in the vial exhibited good stability despite the low fill volume, characterized by a low UV response.

[0554] At 6months at 40°C there no oxygen was present as the vacuum still remained intact and the phospholipid to oxygen ratio was above than 1’000:1 .

[0555] Furthermore, the glass vials where crimp was insufficient tightly sealed in the aluminium pouches overcame variability of imperfect crimping / sealing which occurred occasionally with some vials.

[0556] Example 8.

[0557] Phospholipid to oxygen ratio at start / upon sealing.

[0558] PL selection.

[0559] 0.75 mM of phospholipid was dissolved in methanol and the UV response at 234nm was determined and found to be 0.09. The phospholipid selected had a 95% w / w phosphatidylcholine purity adhering to the monograph for USP Phosphatidylcholine. At time zero the polyunsaturated fatty acid content of the phospholipid was 70.4% w / w of the phospholipid total fatty acid content.

[0560] Preparation of biorelevant composition.

[0561] A biorelevant composition was prepared following the same preparation procedure of Example 3.

[0562] Milling as described in Example 3.

[0563] The resultant dried biorelevant composition (cake) was milled as described in Example 3.

[0564] Filling.

[0565] The resultant dried biorelevant composition (cake) was milled and 4.5g aliquots were filled and hermetically sealed into 10ml_ glass containers with varying gaseous mixtures to achieve the following phospholipid: oxygen molar ratios of the gaseous mixture. At the time of filling the ratios were 1000:200, 1000:100, 1000:60, 1000:25, 1000:10 and 1000:1. Storage conditions.

[0566] The hermetically sealed glass containers with different molar ratios of phospholipid to oxygen in the gaseous mixtures were stored at 40°C for 1 month, 3months and some were also stored at 6months. The OTR of the hermetically sealed glass containers was <0.001 cm3 / day at 40°C.

[0567] Preparation of medium.

[0568] 100ml of FaSSIF media using each composition was prepared containing 0.75mM of PL and 3mM sodium taurocholate following the same preparation procedure of Example 3.

[0569] UV of diluted composition.

[0570] UV measurement at 234nm of the composition in each hermetically sealed container was determined by measuring the compositions diluted to PL concentration of 0.75mM in standardized phosphate buffer with the osmolarity adjusting agent sodium chloride. Measurements were carried out at t =2hours, 24hours and 48hours after sample preparation.

[0571] Particle size determination of medium.

[0572] Particle size analysis (using PCS) was carried out on each medium prepared from the various compositions at t =2hours, 24hours and 48hours after sample preparation.

[0573] Results.

[0574] Table 86 below: UV of freshly manufactured material.

[0575] Appearance of medium: opalescent liquid.

[0576] Table 87 below: Particle size of freshly manufactured material.

[0577] Table 88 below: UV of diluted composition in gaseous mixture (phospholipid: oxygen molar ratio 1000:200 after 1 and 3months storage at 40°C).

[0578] Appearance of medium: almost clear liquid.

[0579] Table 89 below: Particle size and PUFA’s phospholipid: oxygen molar ratio 1000:200 after 3months storage at 40°C.

[0580] Table 90 below: UV of diluted composition in gaseous mixture (phospholipid: oxygen molar ratio 1000:100 after 3months storage at 40°C).

[0581] Appearance of medium: almost clear liquid.

[0582] Table 91 below: Particle size of phospholipid: oxygen molar ratio 1000:100 after 3months storage at 40°C. Table 92 below: UV of diluted composition in gaseous mixture (phospholipid: oxygen molar ratio 1000:60 after 1 and 3months storage at 40°C).

[0583] Appearance of medium: opalescent liquid.

[0584] Table 107 below: Particle size of phospholipid: oxygen molar ratio 1000:60 after 3months storage at 40°C.

[0585] Table 108 below: UV of diluted composition in gaseous mixture (phospholipid: oxygen molar ratio 1000:25 after 3months storage at 40°C).

[0586] Appearance of medium: opalescent liquid. Table 109 below: Particle size of Oxygen molar ratio 1000:25 after 3months storage at 40°C.

[0587] Table 110 below: Particle size of phospholipid: oxygen molar ratio 1000:25 after 6months storage at 40°C. Table 111 below: UV of diluted composition in gaseous mixture (phospholipid: oxygen molar ratio 1000:10 after 3months storage at 40°C).

[0588] Table 112 below: Particle size of phospholipid: oxygen molar ratio 1000:10 after 3months storage at 40°C.

[0589] Table 113 below: UV of diluted composition in gaseous mixture (phospholipid: oxygen molar ratio 1000:1 after 3months storage at 40°C). Table 114 below: Particle size of phospholipid: oxygen molar ratio 1000:1 after 3months storage at 40°C.

[0590] After 3months of storage at 40°C, the gaseous mixture of the composition filled at a PL: oxygen molecular ratio of 1000:200 was unsatisfactory: The UV response at 234nm was too high (>3.00AU) at t = 2hours.

[0591] In contrast, the UV response of the composition stored at 40°C for 1 month (with a phospholipid: oxygen molar ratio in the gaseous mixture inside the hermetically sealed container was 1000:100) was found to be below 2.0AU after 2hours. Surprisingly, the Z-average, PDI and physical appearance of the medium remained unchanged compared to the medium prepared from the powder at time point t = 0 indicating that ageing had not resulted in variable colloids within the medium. Highly surprisingly, the UV response also did not change even after 3months storage at 40°C.

[0592] Similarly, the UV response of the composition (phospholipid: oxygen molar ratio was 1000:60) stored at 40°C for both 1 month and 3months were similar and gave the same UV responses at both storage time points. The Z-average, PDI and physical appearance of the medium also remained unchanged compared to the medium prepared from the powder at time point t = 0. The same behaviour was yet again observed with the composition (with a PL: oxygen in the gaseous mixture 1000:25). This indicates that if the oxygen level is carefully controlled, the increase of UV is only due to the consumption of exposed oxygen to the phospholipid.

[0593] These results also further supported by the results of differing phospholipid: oxygen molecular ratios using a variety of different sealed containers and fill volumes in the examples (for example HDPE).

[0594] These results were also further supported by the results of differing phospholipid: oxygen molecular ratios for example using a variety of different sealed containers, gaseous mixture, sachets and fill volumes in the examples (for example HDPE). The UV response of the diluted composition (with PL: oxygen molecular ratio at 1000:100) remained the same between 1 month and 3months in the hermetically sealed container. This supports that the phospholipid: net oxygen molecular ratio should be limited to 1000:100 or above. Therefore, for every 1 mmol of phospholipid, the net oxygen exposure in the gaseous mixture must be limited to around O.IOmmol of oxygen for the shelf life of the composition in the sealed container.

[0595] The oxygen exposure has to be adhered to and limited not only by the OTR but also by controlling the oxygen in the gaseous mixture (just before sealing) and / or the inclusion of an oxygen absorber.

[0596] Specifically, for every 1 mmol of phospholipid, the net oxygen exposure in the gaseous mixture in the sealed unopened container from sealing or soon after sealing up to at least 3months at 40°C; at least 6months at 40°C; at least 9months at 40°C and 12months at 40°C is limited to <0.100mmol of oxygen; <0.050mmol of oxygen; <0.040mmol of oxygen; <0.020mmol of oxygen; <0.010mmol of oxygen; <0.001 mmol of oxygen;

[0597] Assuming there is no oxygen in the gaseous mixture nor oxygen sachet(s) is included in the sealed container, using the threshold of phospholipid: net oxygen molecular ratio; >1000:100, the selected sealed container requires an oxygen transmission rate (OTR) at 40°C of:

[0598] <0.0250cc / sealed container / day x (multiplied by) the amount of phospholipid present in the composition in millimoles (mmol).

[0599] Conclusion.

[0600] Media were prepared using biorelevant composition with a phospholipid: oxygen molar ratio of the gaseous mixture was 1000:100 stored for 3months at 40°C wherein the UV of the composition diluted to 0.75mM PL in standardised phosphate buffer resulted in a UV response below 1.8AU up to 2hours after sample preparation. Therefore, it is essential to limit the molecular ratio of the total phospholipid amount in contact with total amount of oxygen (at the time of sealing within the sealed container) to 1000:100 or above prior to storing in at 40°C for at least 3months; at least 6months; at least 9months. At this threshold ratio after sealing, the OTR of the sealed container would have to be extremely low. It is still preferable to have a low PL to oxygen molecular ratio when sealing the container.

[0601] Example 9.

[0602] Inclusion of chelator: E.g Desferrioxamine.

[0603] 0.75mM of phospholipid was dissolved in methanol and the UV response (10mm path length) at 234nm was determined and found to 0.08.

[0604] The phospholipid has a 95% w / w phosphatidylcholine purity. At time zero the polyunsaturated fatty acid content of the phospholipid was 70.4% w / w of the phospholipid total fatty acid content.

[0605] Preferably a chelator may be incorporated into the composition during manufacturing. Alternatively, one or more chelator(s) may be added to the buffer or buffer concentrate used to prepare the medium prior to addition of the composition. The phospholipid to chelator molecular ratio is typically 500:1 to 10:1 , 200:1 to 25:1 , 100:1 to 30:1.

[0606] Preparation of biorelevant composition.

[0607] A biorelevant composition was prepared following the same preparation procedure of Example 3.

[0608] Milling and filling.

[0609] The resultant dried biorelevant composition (cake) was milled and filled as described in Example 4.

[0610] Storage.

[0611] The aluminium bottle containing the powder was stored at 40°C for 3months. The phospholipid: oxygen molar ratio was found to be 1000: 1 .

[0612] Preparation of medium.

[0613] 1 L FaSSIF was prepared by addition of 2.240g of the powder (t =0) to FaSSIF Buffer prepared by diluting 41 ,64g of a commercially available of FaSSIF Buffer concentrate with 961 ,1g of deionised water and 6.56mg of desferrioxamine mesylate as a chelator, final concentration of desferrioxamine mesylate was 0.01 mM.

[0614] The phospholipids to desferrioxamine mesylate molar ratio in FaSSIF medium was 0.75mM: 0.01 mM. UV of diluted composition.

[0615] UV measurement at 234nm of the composition were determined by diluting the compositions to PL concentration of 0.75mM in standardized phosphate buffer with sodium chloride at 234nm. Measurements were carried out at t =2hours, 24hours and 48hours after preparation.

[0616] Particle size determination of medium.

[0617] Particle size analysis (using PCS) of the media was carried out at t =2hours, 24hours and 48hours after preparation.

[0618] Similar experiments were carried out by adding the chelator to the powder prior during the process of preparing the composition. This inclusion did not affect the ageing of the composition and the container configuration still needs to be judiciously selected. Inclusion in the composition during manufacturing ensures the amount of chelator is accurately included.

[0619] Results.

[0620] Table 115 below: UV of freshly manufactured material without Desferrioxamine mesylate.

[0621] Appearance of medium: opalescent liquid.

[0622] Table 116 below: Particle size of medium without Desferrioxamine mesylate.

[0623] Table 117 below: UV of diluted composition after addition of 0.01 mM Desferrioxamine mesylate.

[0624] Appearance of medium: opalescent liquid.

[0625] Table 118 below: Particle size of medium after addition of 0.01 mM Desferrioxamine mesylate.

[0626] Table 119 below: UV of diluted composition in Aluminium bottles under nitrogen 3months at 40°C without Desferrioxamine mesylate.

[0627] Appearance of medium: opalescent liquid. Table 120 below: Particle size after addition of 0.01 mM Desferrioxamine mesylate. Table 121 below: UV of composition after addition of 0.01 mM Desferrioxamine mesylate. Appearance of medium: opalescent liquid.

[0628] Table 122 below: Particle size of medium after addition of 0.01 mM Desferrioxamine mesylate. S

[0629] At t = 0 and after three of storage at 40°C, the biorelevant composition exhibited minimal AUV (234 nm) from 2 to 48hours when 0.01 mM desferrioxamine mesylate was added to the biorelevant composition stored at 40°C diluted in a defined phosphate buffer to 0.75mM of PL. In comparison, a significantly higher AUV was observed over the same period when the biorelevant composition was analysed without the addition of desferrioxamine mesylate. It is important to note the chelator does not act as an antioxidant and does not help maintaining a low UV response during storage if the container is improperly configured. The Z-average, PDI and physical appearance of the medium remained unchanged compared to the medium prepared from the powder at time point t = 0.

[0630] Conclusion.

[0631] The addition of desferrioxamine mesylate to the biorelevant composition, at a molar ratio of 0.75 mM phospholipid to 0.01 mM desferrioxamine mesylate, resulted in a reduced differential in UV absorbance between 2hours and 48hours. A similar stabilizing effect was observed with the addition of EDTA, which also minimized UV changes over the same time period.

[0632] Example 10.

[0633] Testing compositions with different levels and types of phosphatidylcholine. Preparation of biorelevant composition.

[0634] Small quantities of biorelevant compositions were prepared by freeze drying solutions of 7.4g sodium taurocholate and 2.5g of the individual phospholipids: soyabean phospholipid with a minimum of 45%w / w soyabean phosphatidylcholine, soyabean phospholipid with a minimum of 70%w / w soya phosphatidylcholine, soyabean phospholipid with a minimum of 90%w / w soya phosphatidylcholine (prepared by blending) and egg phospholipids with a minimum of 94%w / w egg phosphatidylcholine dissolved in about 40g water. All solutions had a bile salt to phospholipid molar ratio of 4:1. The resultant solutions were lyophilised for 48hours.

[0635] Milling.

[0636] The resultant dried biorelevant compositions (cakes) were milled as described in Example 3.

[0637] Preparation of medium.

[0638] 0.75mM of PL contained 3mM sodium taurocholate medium was prepared following the same preparation procedure of Example 3.

[0639] Medium Analysis.

[0640] At each time point the stored biorelevant compositions were used to prepare 0.75mM of PL contained 3mM sodium taurocholate media with were analysed at t =2hours, 24hours and 48hours after preparation by PCS and UV.

[0641] Results. Table 123 below: UV of diluted freshly manufactured composition using soyabean phospholipid with a minimum of 45%w / w soyabean phosphatidylcholine.

[0642] Appearance of medium: clear and transparent liquid.

[0643] Table 124 below: Particle size of medium prepared from compositions with soyabean phospholipid with a minimum of 45%w / w soyabean phosphatidylcholine.

[0644] Table 125 below: UV of diluted freshly manufactured composition using soyabean phospholipid with a minimum of 70%w / w soya phosphatidylcholine.

[0645] Appearance of medium: clear and transparent liquid.

[0646] Table 126 below: Particle size of medium prepared from composition with soyabean phospholipid with a minimum of 70%w / w soyabean phosphatidylcholine. Table 127 below: UV of diluted freshly manufactured composition using soyabean phospholipid with a minimum of 90%w / w soya phosphatidylcholine (blended). Appearance of medium: almost clear liquid.

[0647] Table 128 below: Particle size of medium using composition with soyabean phospholipid with a minimum of 90%w / w soya phosphatidylcholine (blended).

[0648] Table 129 below: UV of diluted freshly manufactured composition using egg phospholipids with a minimum of 94% egg phosphatidylcholine.

[0649] Appearance of medium: opalescent liquid.

[0650] Table 130 below: Particle size of egg of medium using compositions using egg phospholipids with a minimum of 94% egg phosphatidylcholine.

[0651] Conclusion.

[0652] The different grades of phospholipids exhibited slightly different absorption at 234nm (at same PL concentration of 0.75mMoles). However, they all were within the target specification of 1.5AU for freshly prepared compositions.

[0653] Generally, the Z average of the media prepared using soyabean phospholipids with a lower PC content were smaller and the PDI were narrow.

[0654] The compositions would be suitable for inserting into sealed containers, as described in this specification.

[0655] Example 11.

[0656] Solubility and Dissolution Analysis of Ezetimibe and Ketoconazole in FaSSIF.

[0657] Solubility.

[0658] The composition in METBOPP Pouch (from Example 5) (stored for 6months at 40°C) was used to prepare the FaSSIF biorelevant medium using the FaSSIF medium recipe of Example 3.

[0659] Equilibrium solubility was carried out by incubating 10mg (excess) ezetimibe in 1 ml of FaSSIF in syringeless vials and determining the solubility until equilibrium was reached (n =3). Solubility measurements were performed at 3hours, 6hours and 24hours as described in Solubility Measurements <USP Chapter 1236>.

[0660] Solubility was determined by direct UV analysis at 232nm and subtracting the UV response of the medium.

[0661] Solubility.

[0662] Dissolution studies of “Ezetimibe 10mg tablets” were performed in FaSSIF medium prepared using the biorelevant composition of Example 5 (Composition in trilaminate Aluminium pouch). Single stage dissolution was carried out (n =6) in 900mL with USP II apparatus till 2hours.

[0663] Two stage dissolution.

[0664] To replicate how a drug behaves in the fasted stomach and then passage of the dosage form into the small intestine two stage dissolution (n =6) was performed. This involves carrying out dissolution in fasted simulated gastric fluid and then adding a x2 concentrate of FaSSIF.

[0665] FaSSGF and the two stage FaSSIF Concentrate were prepared using the composition stored in the Aluminium Pouches (from Example 5) (stored for 6months at 40°C) was used to prepare the FaSSIF biorelevant medium using the FaSSIF medium recipe of Example 3. The principle of the method is described by Krollik et al 2022.

[0666] Two different biorelevant composition were used to analyse “Ketoconazole 200mg tablets”. Two stage dissolution was carried out for 60minutes in 450mL of FaSSGF containing 0.02mM of PL and 0.08mM sodium taurocholate and subsequently converted to FaSSIF into 900mL of total medium with the USP II apparatus an additional 2hours.

[0667] Results.

[0668] Solubility.

[0669] The equilibrium solubility of Ezetimibe in FaSSIF was found to be 0.01 mg.

[0670] Dissolution.

[0671] The dissolution of Ezetimibe 10mg tablets in FaSSIF medium is shown in Figure 3 of the accompanying drawings. Ezetimibe was released 85% in the FaSSIF medium. Two stage Dissolution.

[0672] Ketconazole (200mg) was released 100% in the FaSSGF medium (0.02m M PL) and showed supersaturation behaviour after converting FaSSGF medium (0.02mL PL) to FaSSIF medium (0.75mM PL), as illustrated in Figure 4 of the accompanying drawings. After conversion to FaSSIF, variability in the results and supersaturation was found to be minimal.

[0673] Conclusion.

[0674] The In-vitro study conducted in media prepared from the biorelevant compositions stored in sealed containers provided insight into the dissolution of this poorly soluble drug. Ezetimibe released 85% in the FaSSIF medium, indicating that the drug is lipophilic and the drug product had good dissolution properties.

[0675] The basic drug Ketoconazole was released from its drug product in FaSSGF medium which indicates that drug is basic in nature. The drug remained largely in solution (supersaturated) and precipitation after 2 to 3hours was uniform.

[0676] Thus, the examples demonstrate that a combination of factors enable a satisfactorily stable composition to be provided. This stabilization is achieved without addition of additional antioxidants (other than those present as stabilisers in the phospholipid) which can affect the performance of dissolution media by occupying the space where lipophilic drugs can remain, thereby reducing apparent solubility of lipophilic drug or compound. The invention provides a method and pack for stabilizing a composition in a sealed container for an extended period (at least 3months; at least 6months; at least 9months; at least 12 months) at room temperature and up to 40°C by the selection of various parameters, including the components of the composition comprising at least one bile salt and / or acid and at least one phospholipid according to certain criteria and the composition being sealed within a container with a gaseous mixture and / or at reduced pressure or under vacuum to provide a molecular ratio of total phospholipid in the composition: oxygen inside said container immediately after sealing is equal to 1000: 100 or higher, and limiting the molecular ratio of the total phospholipid amount in contact with total amount of oxygen at both the time of sealing and from any entry of oxygen to the container to 1000: 100 or above for said at least 3months at 40°C in the unopened sealed container. The OTR of the sealed container should be within predefined limits to maintain the molecular ratio of total phospholipid: oxygen as defined above both immediately after sealing and for at least 3months thereafter at 40°C. Other factors may be selected to ensure the ratio is maintained during the shelf life of the composition, such as the type of closure, gaseous mixture, use of sachets, sealing, conditions (atmospheric, reduced pressure / vacuum) and fill volume. These are selected and carefully controlled so that the molecular ratio of the total phospholipid in contact with total exposed oxygen (i.e. from sealing and including the oxygen entry into the sealed container after storage for 6months at 40°C) is limited to 1000:100 or above at 40°C for at least 3months.

[0677] Preferably the method includes

[0678] (i) inserting the composition into a container with a gaseous mixture and / or at reduced pressure and sealing the container, wherein the molecular ratio of total phospholipid in the composition: oxygen inside said container immediately or soon after sealing is equal to 1000:100 or higher, and

[0679] (ii) sealing the composition in the container by sealing it in a container having an oxygen transmission rate (OTR) at 40°C.

[0680] (iii) wherein the configured sealed container has: an oxygen transmission rate (OTR) at 40°C of: <0.0250cc / sealed container / day x (multiplied by) the amount of phospholipid present in the composition in millimoles (mmol); preferably <0.0125cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.0100cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.005cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.003cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); especially <0.001 cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol). (iv) The composition in the sealed container preferably provides, after first opening of the container and dilution of the composition in a defined biorelevant phosphate buffer to a defined phospholipid concentration of 0.75mM, a UV response at 234nm (10mm path cell length) that is below 2.0AU at t = 2hours.

Claims

CLAIMS:1 . A method of stabilizing a composition in a sealed container to provide a stable composition for at least 3months at 40°C, the composition comprising essentially at least one bile salt and / or bile acid and at least one phospholipid, characterised in that: the composition has:I. a mole ratio of bile acid and / or bile salt: phospholipid between 75:1 and <1 :1 ; wherein the bile acid and or / salt of the composition comprises:II. at least one bile acid / bile salt selected from the group comprising:- sodium cholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium taurodeoxycholate, sodium glycodeoxycholate, sodium ursodeoxycholate, sodium chenodeoxycholate, sodium taurochenodeoxycholate, sodium glycochenodeoxycholate, sodium cholylsarcosinate, and sodium N- methyl taurocholate; andIII. the bile salt and / or acid has a purity of at least 80% w / w (on a dry weight basis); wherein the phospholipid of the composition comprises:IV. at least one polyunsaturated phospholipid wherein the phospholipid content is at least 45% w / w phosphatidylcholine;V. fatty acid chain(s) of the phospholipid contains at least 10% w / w polyunsaturated fatty acids; andVI. wherein the said phospholipid is selected so that after dilution of the freshly prepared composition (containing bile salt and / or bile acid and phospholipid) to a phospholipid concentration of 0.75mM, theUV response at 234nm (10mm path cell length) is below 1.5AU from 2hours up to 48hours after sample preparation; the method comprising the steps of: a) inserting the composition into a container with a gaseous mixture and / or at reduced pressure and sealing the container, wherein the molecular ratio of total phospholipid in the composition: oxygen inside said container immediately or soon after sealing is equal to 1000:100 or higher, and b) wherein the composition in the sealed container, after first opening of the container and dilution of the composition in a defined biorelevant phosphate buffer to a defined phospholipid concentration of 0.75mM, provides a UV response at 234nm (10mm path cell length) that is below 2.0AU, and below 1.8AU at 2hours after sample preparation.

2. The method according to claim 1 , further comprising:(i) sealing the composition and a gaseous mixture, optionally at reduced pressure or under vacuum, in a container having an oxygen transmission rate (OTR) at 40°C of: <0.0250cc / sealed container / day x (multiplied by) the amount of phospholipid present in the composition in millimoles (mmol); preferably <0.0125cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.0100cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.005cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.003cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol);especially <0.001 cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol).(ii) the composition, upon first opening of the container and dilution in a predefined biorelevant phosphate buffer to a phospholipid concentration of 0.75mM, exhibits a UV absorbance at 234nm (measured using a 10mm path length cell) that is: less than 2.0AU from 2hours after sample preparation; preferably less than 1 .8AU from 2hours after sample preparation.

3. The method according to claim 1 or 2, wherein the composition in the sealed container provides, after first opening of the container and dilution of the composition in a predefined biorelevant phosphate buffer to a predefined phospholipid concentration of 0.75mM, a UV response at 234nm (measured using a 10mm path cell length) that is below 2.0AU at 2hours after sample preparation.

4. The method according to any one of claims 1 to 3, wherein the phospholipid is selected from one or a combination of USP egg phospholipids or USP NF Soybean Phosphatidylcholine.

5. The method according to claim 1 to 4, wherein the composition contains monoglycerides and / or a fatty acid(s).

6. The method according to any one of the preceding claims, further comprising adding to the composition inside the container one or more chelators, preferably being EDTA and / or desferrioxamine mesylate.

7. The method according to any one of the preceding claims, further comprising adding to the composition inside the container at least one further component selected from the group comprising:-a monoglyceride, a fatty acid, cholesterol, proteins and enzymes; wherein the mole ratio of each selected component: total unsaturated phospholipid in the composition is below 1 :1.

8. The method according to any one of the preceding claims, wherein the composition is provided in the container under vacuum.

9. The method according to any one of the preceding claims, wherein the composition is a powder or a cake.

10. The method according to any one of the preceding claims, wherein the container comprises a primary container and the method further comprises inserting the sealed primary container into a secondary wrapping or container.

11. The method according to claim 10, wherein the secondary wrapping or container has a lower OTR than the primary container.

12. The method according to any one of claims 1 to 11 , further comprising adding a biorelevant phosphate buffer to the composition, or vice versa wherein the biorelevant phosphate buffer used for dilution of the composition to a phospholipid concentration of 0.75mM comprises a physiologically relevant aqueous buffer system selected to mimic intestinal conditions, and comprises 0.420g NaOH, 3.438g of sodium phosphate monobasic (anhydrous), 6.186g of NaCI in 1 L adjusted to pH 6.50.

13. The method according to any one of the preceding claims, further comprising opening the sealed container and using the composition for the preparation of media used for in vitro drug dissolution testing, drug solubility testing, drug permeability and / or drug profiling.

14. A pack comprising a composition sealed in a primary container to provide a stable composition for at least 3months at 40°C, the compositioncomprising essentially at least one bile salt and / or acid and at least one phospholipid characterised in that: the composition has:I. a mole ratio of bile acid and / or bile salt: phospholipid between 75:1 and <1 :1 ; wherein the bile acid and or / salt of the composition comprises:II. at least one bile acid / bile salt selected from the group comprising:- sodium cholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium taurodeoxycholate, sodium glycodeoxycholate, sodium ursodeoxycholate, sodium chenodeoxycholate, sodium taurochenodeoxycholate, sodium glycochenodeoxycholate, sodium cholylsarcosinate, and sodium N- methyl taurocholate; andIII. the bile salt and / or acid has a purity of at least 80% w / w (on a dry weight basis); wherein the phospholipid composition comprises:IV. at least one polyunsaturated phospholipid wherein the phospholipid content is at least 45% w / w phosphatidylcholine; andV. fatty acid chain(s) of the phospholipid contains at least 10% w / w polyunsaturated fatty acids; wherein the selected sealed container has an oxygen transmission rate (OTR) at 40°C of:<0.0250cc / sealed container / day x (multiplied by) the amount of phospholipid present in the composition in millimoles (mmol); preferably <0.0125cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol);more preferably <0.0100cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.005cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); more preferably <0.003cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol); especially <0.001 cc / sealed container / day x the amount of phospholipid present in the composition in millimoles (mmol).

15. The pack as claimed in claim 14, wherein the composition, when inserted and sealed in the container with a gaseous mixture and / or at reduced pressure has a molecular ratio of total phospholipid in the composition: oxygen inside said container immediately or soon after sealing is equal to 1000:100 or higher.

16. The pack as claimed in claim 15, wherein for every 1 mmol of phospholipid, the net oxygen exposure in the gaseous mixture in the sealed unopened container from sealing or soon after sealing up to at least 3months at 40°C; is limited to <0.100mmol of oxygen.

17. The pack as claimed in claim 14, 15 or 16, wherein the composition in the sealed container provides, after first opening of the container and dilution of the composition in a predefined biorelevant phosphate buffer to a predefined phospholipid concentration of 0.75mM, a UV response at 234nm (10mm path cell length) that is below 2.0AU, preferably below1 .8AU from in particular t = 2hours up to 48hours after sample preparation.

18. The pack according to any one of claims 14 to 17, wherein the composition inside the container comprises at least one further component selected from the group comprising:- a monoglyceride, a fatty acid, cholesterol, proteins and enzymes;wherein the mole ratio of each selected component: total unsaturated phospholipid in the composition is below 1 :1.

19. The pack according to any one of claims 14 to 18, wherein the composition inside the container includes one or more chelators, preferably being EDTA and / or desferrioxamine mesylate.

20. The pack according to any one of claims 14 to 19, wherein the primary container is in the form of a bottle, pouch or sachet.

21. The pack according to any one of claims 14 to 20, wherein the primary container is a rigid container, the material of the container preferably being selected from HDPE, multilayered plastic ((HDPE, UV layer, EVOH / HDPE, HDPE), aluminium (lacquered or uncoated) and / or glass.

22. The pack according to any one of claims 14 to 20, wherein the primary container is a flexible container, preferably wherein the flexible container is selected from stick sachets, pouches, and / or sachets constructed from METPET (e.g. AlOx-coated PET), METBOPP, Silicon Oxide (SiOx)-coated PET, or trilaminate aluminium (at least 7 or at least 9 micrometre thickness), preferably being sealed by suitable sealing means.

23. The pack according to any one of claims 14 to 22, further comprising a secondary wrapping or a secondary container surrounding the primary container, the secondary wrapping or container having a lower OTR than the primary container.

24. The pack according to any one of claims 14 to 23, wherein the container is a squeezable sachet that is resealable to enable lowering of the oxygen content prior to resealing.

25. The pack according to any one of claims 14 to 24, wherein an oxygen scavenger(s) / absorber(s) is enclosed within the sealed container to reduce oxygen content in the container after and sealing and during storage.

26. The pack according to any one of claims 14 to 25, wherein the composition in the sealed container is for preparing media used for in vitro drug dissolution testing, drug solubility testing, pre-clinical drug, drug permeability and / or drug profiling.

27. A method of preparing an aqueous medium containing at least one buffer component and at least one osmotic agent; the method comprising adding a composition from a pack of any one of claims 14 to 26 to the aqueous medium comprising the at least one buffer component and the at least one osmotic agent or vice versa to form an in vitro test medium wherein the particle size (Z-average) of the said medium remains consistent and below 100nm.

28. A method of determining solubility, permeability or dissolution of a pharmacological compound, comprising: preparing an aqueous medium containing at least one buffer component and at least one osmotic agent; adding a composition from a pack of any one of claims 14 to 26 containing at least one bile salt and at least one phospholipid to the aqueous medium or vice versa to form an in vitro test medium; adding at least one of a pharmacological compound, a physiological compound and a dosage form to the medium to determine a solubility.

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