Method for preparing plant-based squalene

Abstract

A method for producing a squalene composition having an enriched squalene content, particularly a pharmaceutical squalene composition, from a squalene-containing composition having a lower squalene content, particularly a plant-based oil having a lower squalene content, comprising the steps of: i) providing a squalene-containing composition having a lower squalene content; ii) subjecting the squalene-containing composition having a lower squalene content to a purification step at a temperature below the boiling point of squalene to obtain an intermediate composition having a certain squalene content; iii) performing a chromatography step using the intermediate composition having the squalene content; and iv) obtaining a squalene composition having an enriched squalene content. Furthermore, the present invention also provides a squalene composition having an enriched squalene content, particularly a pharmaceutical squalene composition, particularly a parenteral pharmaceutical composition, more preferably a vaccine and an oil-in-water emulsion comprising the squalene composition.

Description

[Technical Field]

[0001] A method for producing a squalene composition having an enriched squalene content, particularly a pharmaceutical squalene composition, from a squalene-containing composition having a lower squalene content, particularly a plant-based oil having a lower squalene content, comprising the steps of: i) providing a squalene-containing composition having a lower squalene content; ii) subjecting the squalene-containing composition having a lower squalene content to a purification step at a temperature below the boiling point of squalene to obtain a composition having an intermediate squalene content; iii) performing a chromatography step using the composition having the intermediate squalene content; and iv) obtaining a squalene composition having an enriched squalene content. Furthermore, the present invention also provides a squalene composition having an enriched squalene content, particularly a pharmaceutical squalene composition, particularly a parenteral pharmaceutical composition, more preferably a vaccine and an oil-in-water emulsion comprising the squalene composition.

[0002] Background technology Pharmaceutical squalene is used as a vaccine adjuvant and in pharmaceutical preparations. Currently, as described in International Publication No. 2011141819, squalene-containing oils from animal sources, such as shark liver, are used as starting materials for squalene production. This squalene is obtained in pharmaceutically pure quality through a distillation process. Thermal distillation always carries the risk of decomposition or isomerization of components due to the thermal distillation and vaporization of squalene that comes into contact with the inner surface of the distillation system.

[0003] The qualities required for pharmaceutical use are described, for example, in the European Pharmacopoeia 01-2020-2805. The extraction of industrial squalene from vegetable oils (olive oil, amaranthol, palm oil) is known from the literature: M. Azalia Lozano-Grande et al., "Plant Sources, Extraction Methods, and Uses of Squalene", International Journal of Agronomy, Vol. 2018, Article ID 1829160, p. 13, 2018. https: / / doi.org / 1 0.1 155 / 201 8 / 1829160, and Nisarg Gohil et al., "Engineering Strategies in Microorganisms for the Enhanced Production of Squalene: Advances, Challenges and Opportunities", Front.Bioeng.BiotechnoL, 201 9, 7. https: / / doi.org / 1 0.3389 / fbioe.201 9.00050.

[0004] Extraction and chromatographic purification techniques have been described, but these are not sufficient to produce pharmaceutically pure squalene as required according to the pharmacopoeias.

[0005] The object of the present invention was to develop a process that can provide squalene of pharmaceutical purity. Furthermore, the object of the present invention was to reduce the energy input of the process and to reduce the amount of organic solvent used. Furthermore, the object of the present invention was to provide a process that is compatible with plant-based sources of squalene. Furthermore, the present invention aims to develop a process that reduces the thermal stress applied to squalene.

[0006] Summary of the Invention Surprisingly, a hybrid process has been developed which utilizes a purification step that is neither a chromatographic purification step nor a distillation step, but a lower temperature purification step, in particular a temperature below the boiling point, in particular a filtration step or a chemical extraction step.

[0007] The method of the present invention is disclosed in claim 1 and the squalene composition is disclosed in claim 13. Further, the invention is disclosed in more detail in the dependent claims and further detailed in the specification. A pharmaceutical composition according to claim 14 and an oil-in-water composition according to claim 15.

[0008] MODE FOR CARRYING OUT THE INVENTION According to the present invention, there is provided a method for producing a squalene composition having an enriched squalene content from a squalene-containing composition having a lower squalene content, in particular a method for providing a pharmaceutical squalene composition, and optionally a squalene composition having an enriched squalene content obtainable by this method, the method comprising: i) providing a squalene-containing composition having a lower squalene content; ii) subjecting the squalene-containing composition having a lower squalene content to a purification step at a temperature below the boiling point of squalene, in particular below 100°C, optionally at a pressure of 0.01 to 100 bar, in particular below 75°C and optionally at a pressure of 0.01 to 10 bar, to obtain an intermediate composition having a squalene content; iii) performing a chromatography step using the intermediate composition having a squalene content; iv) obtaining a squalene composition having an enriched squalene content; Includes.

[0009] Preferably, the squalene-containing composition having a lower squalene content is a plant-based oil having a lower squalene content. Below the boiling point means below 275°C at 20 hPa or a lower temperature under a corresponding reduced pressure.

[0010] According to a first embodiment of the present invention, the squalene composition having an enriched squalene content is a pharmaceutical squalene composition. It is particularly preferred that the squalene-containing composition having a lower squalene content is amaranth oil.

[0011] Furthermore, ii) the purification step preferably comprises a physical and / or chemical purification step below the boiling point of squalene, such as a filtration step and / or a chemical extraction step, in particular a chemical extraction step with recycle of the extractant. A filtration step using membrane filtration is preferred. Particularly preferred purification steps include diafiltration and / or extraction steps, in particular the extraction step is a liquid-liquid extraction step and / or the extraction step comprises fluid extraction.

[0012] In another preferred embodiment of the present invention, the method comprises the steps of: i) providing a squalene-containing composition having a lower squalene content, the lower squalene content being less than 12% by weight, the total squalene content being 100% by weight; ii) subjecting the squalene-containing composition with a lower squalene content to a purification step at a temperature below the boiling point of squalene, in particular below 100°C, preferably below 75°C, optionally at a pressure of 0.01 to 100 bar, preferably 0.01 to 1 bar, to obtain an intermediate composition with a squalene content of at least 14% by weight of squalene, totalling 100% by weight of the intermediate composition; iii) performing a chromatography step using the intermediate composition having a squalene content; iv) obtaining a squalene composition having an enriched squalene content of 91% by weight or greater, the composition totaling 100% by weight; may include:

[0013] A newly developed method allows for the production of squalene purely from plant sources. Most preferred is amaranth oil, with an average squalene content of 1-10% by weight, which is first concentrated via diafiltration, particularly diananofiltration. This method allows for a significant reduction in solvent use compared to chemical extraction methods. The resulting squalene composition (intermediate composition) obtained from diafiltration contains approximately 20-50% by weight of squalene. This intermediate composition is further concentrated via a chromatography step, and the composition isolated from the chromatography unit preferably has pharmaceutical quality after removal of the mobile phase.

[0014] Optionally, the mobile phase or a composition containing a portion of the mobile phase can be treated with an adsorbent such as activated carbon or diatomaceous earth to finally remove the mobile phase, particularly the solvent. This method results in pharmaceutically pure squalene that meets the requirements of European Pharmacopoeia Monograph 01 / 2021:2805.

[0015] As an alternative to the combination of dia-nanofiltration and chromatography, a combination of extraction and chromatography can also be used. This can be fluid extraction or liquid-liquid extraction, including solvent extraction on the one hand and fluid extraction with CO2 extraction on the other. However, since nanofiltration-based diafiltration can be carried out without the use of additional solvents, especially organic solvents, the combination of diafiltration, especially dia-nanofiltration and chromatography, significantly reduces the use of solvents compared to extraction and chromatography. This results in an environmentally friendly method that uses significantly less organic solvent. A particularly preferred embodiment of this method is one in which the solvent used as the mobile phase and / or extractant can be reused. The used liquid alkane, especially hexane, n-hexane, and cyclohexane, can be reused after evaporation from the composition. Preferably, more than 90% of the liquid alkane used as the mobile phase and / or extractant can be reused in this method. By recycling a certain amount of solvent for the method, the amount of solvent used per year can be reduced.

[0016] The first purification step is used to separate the fatty acid esters contained in the squalene-containing composition having a lower squalene content and partially separate the phytosterols contained therein, while the remaining components are separated by chromatography and optional activated carbon filtration in the second step, yielding squalene of high pharmaceutical purity.

[0017] The term diafiltration or dia-nanofiltration describes a membrane-based method in which the solvent and, depending on the application, a portion of the components of the solution or suspension are exchanged. The starting composition, in this case a squalene-containing composition with a lower squalene content, from which additional components, particularly oily components, are exchanged, is circulated from a supply vessel. By adjusting the flow after the membrane, particularly after the membrane device, the transmembrane pressure (TMP), which acts as the driving force in diafiltration, is set. The molecular weight cut-off (MWCO) of the membrane is selected so that the molecules to be removed, in this case squalene, can pass through the membrane. Since the system operates as a closed system, the discharge of the permeate results in the reflux of the exchanged squalene-containing composition, thereby maintaining a constant circulation volume during diafiltration. When the same volume as that initially introduced leaves the system as permeate and is replaced by a new squalene-containing composition, the so-called diafiltration volume is achieved.

[0018] Chromatography is a chemical-physical technique for separating a mixture into its components. Typically, a mixture containing the components is dissolved in a fluid solvent, particularly a gas, CO2 in fluid extraction, or in a solvent in liquid-liquid extraction. According to the present invention, the fluid solvent or liquid hydrocarbon, particularly a liquid hydrocarbon, preferably a liquid alkane, is referred to as the mobile phase. The mobile phase carries the components through a system, such as a column, capillary, plate, or sheet, on which a material, called the stationary phase, is immobilized or contains the stationary phase. Because different components of a mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interaction with the surface sites of the stationary phase, the components pass through the stationary phase at different apparent velocities in the moving fluid, resulting in separation. Separation is based on differential partitioning between the mobile and stationary phases. Small differences in the partition coefficients of compounds result in differential retention on the stationary phase and thus affect separation.

[0019] ii) It is a further embodiment of the present invention that the purification step may comprise a membrane filtration step, in particular a membrane-based diafiltration step. It is more preferred that the membrane is a silicone-based membrane such as polydimethylsiloxane, which may further comprise a polyacrylonitrile layer and / or a polyethylene terephthalate layer. The membrane preferably has a molecular weight cut-off (MWCO) of 200-700 g / mol, preferably 280-600 g / mol. Since there is no standard procedure for determining MWCO, this value may be obtained differently. In this specific case, the rejection R of polystyrene (solute) with different molecular weights in toluene (solvent) can be considered. In this specification, R i is the exclusion of polystyrene of a certain molecular weight, and c p,i is the permeate concentration, and c R,i is the retentate concentration of this polystyrene.

number

[0020] Therefore, the molecular weight cutoff is described by the molecular weight of polystyrene that exhibits an exclusion R of 0.9.

[0021] According to an alternative embodiment, the extraction step may comprise a liquid-liquid extraction step in which the extractant is a mixture containing at least one liquid hydrocarbon and / or at least two liquid hydrocarbons, particularly at least a liquid alkane. The one or more alkanes may be selected from linear alkanes, branched alkanes, and / or cyclic alkanes, particularly alkanes selected from alkanes including pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane, and all isomers thereof, most preferably n-hexane, n-heptane, cyclohexane, cycloheptane, and / or mixtures thereof. Alternatively, the extraction step may comprise a liquid extraction in which the extractant is CO2. Liquid alkanes refer to liquids at temperatures below 100°C, particularly liquids at room temperature and 20 hPa.

[0022] According to a preferred embodiment, the purification step, particularly the extraction step, comprises a saponification step prior to the extraction step, preferably the liquid-liquid extraction step. The saponification step preferably comprises: a) contacting the squalene-containing composition having a lower squalene content with an alkali in an organic solvent, particularly an alkali solution, particularly an alkali solution of an alcohol, such as methanol, ethanol, or propanol; and b) obtaining a mixture. Optionally, in step c), the mixture obtained in b) is treated at elevated temperature, particularly by stirring and / or heating the mixture to a temperature in the range of 30 to 80°C, particularly 50 to 70°C, more preferably 50°C.

[0023] Optionally, in step d), the mixture may be cooled, and in step e), water, particularly distilled water, is added. Then, optionally, in step f), the mixture and the added water are heated to a high temperature, particularly 30 to 80°C, particularly 50 to 70°C, more preferably 50°C, and the mixture and water are treated with an extractant to obtain a mixture. Optionally, in step g), the mixture is separated into an aqueous phase and an organic phase containing the extractant and squalene, the two phases are separated, and the aqueous phase is optionally treated with the extractant again, and optionally step g) is repeated to obtain an organic phase, particularly containing the extractant and squalene. Preferably, in step h), the organic phases containing the extractant and squalene are combined and optionally contacted with water, particularly distilled water, and then i) the mixture is separated into an aqueous phase and an organic phase containing the extractant and squalene. Optionally, j) this organic phase containing the extractant and squalene is again contacted with water, in particular distilled water, and k) the resulting mixture is separated into an aqueous phase and an organic phase containing the extractant and squalene.Further, l) the organic phase containing the extractant and squalene obtained in step i) and / or k) is treated under vacuum and / or elevated temperature to remove the extractant and obtain an intermediate composition having a certain squalene content.

[0024] Optionally, alternatively, in step l), the organic phase containing the extractant, in particular the organic phase having a low content of extractant, is m) contacted with an adsorbent, in particular contacted with the adsorbent and stirred, and n) the adsorbent is removed from this organic phase containing the extractant, in particular by filtering it off. Thereafter, o) the organic phase containing the extractant is treated under vacuum and / or elevated temperature to remove the extractant and obtain an intermediate composition having a certain squalene content. Preferred adsorbents are diatomaceous earth or activated carbon. In certain preferred embodiments of the present invention, steps a) to k) are performed consecutively, or steps a) to o) are performed consecutively.

[0025] Treatment under vacuum and / or elevated temperature may include treatment at pressures in the range of from 0.1 mbar to less than 1 bar, preferably from 10 mbar to 600 mbar, more preferably from 100 mbar to 300 bar, and in particular at temperatures in the range from room temperature to 60°C, preferably from 30 to 50°C.

[0026] According to the present invention, the chromatographic step can be carried out with a stationary phase comprising silica, alumina, a diol-functionalized polymer or diol-functionalized silica, and / or hydroxylapatite, and in particular the stationary phase is selected from silica, alumina, a diol-functionalized polymer or diol-functionalized silica, and / or hydroxylapatite. In particular, the chromatographic step can be carried out using an intermediate composition having a certain squalene content, optionally with the addition of a mobile phase, and after evaporation of the mobile phase, a squalene composition having a concentrated squalene content can be obtained. In particular, in iii) carrying out the chromatographic step using an intermediate composition having a certain squalene content, it is preferred that the step be carried out using a mobile phase, in particular an intermediate composition and a mobile phase. Therefore, iii) can be carried out using an intermediate composition and an added mobile phase.

[0027] It is a further object of the present invention that the chromatographic step may be carried out using a mobile phase comprising at least one liquid hydrocarbon, in particular at least one liquid alkane, preferably at least one or more alkanes selected from alkanes including pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane, most preferably cyclohexane. Optionally, additionally or alternatively, the mobile phase may be selected from fluid solvents such as supercritical CO2.

[0028] Preferred liquid phases include compositions comprising an intermediate composition having a squalene content and, in particular, a liquid alkane as a mobile phase, where the content of the intermediate composition having a squalene content is 1 to 80% by weight, preferably 5 to 30% by weight, with the remainder being the mobile phase, in particular at least one liquid alkane, up to 100% by weight of the mobile phase. The liquid alkane is, in particular, cyclohexane. The liquid alkane may include the above-mentioned liquid alkanes and / or mixtures of at least two of them. Cyclohexane, n-hexane, n-heptane, cycloheptane, and / or mixtures thereof are most preferred.

[0029] It is a further object of the present invention that iii) after the chromatography step, at least one further step may comprise contacting the composition obtained from the chromatography step with at least one adsorbent, in particular the at least one adsorbent may comprise activated carbon, diatomaceous earth, silica, and / or a mixture of at least two of them.

[0030] According to a further embodiment of the method, a) the squalene-containing composition having a lower squalene content may have a squalene content of less than 12% by weight, in particular between 0.1% and 10% by weight or less, preferably between 5% and 10% by weight or less, totalling 100% by weight of the composition; and / or b) the intermediate composition having a squalene content may have a squalene content of at least 14% by weight, in particular from 15% to 90% by weight, preferably from 15% to 40% by weight, totalling 100% by weight of the intermediate composition; and / or c) Squalene compositions having an enriched squalene content, in particular pharmaceutical squalene compositions, may have a squalene content of 91% or more by weight, in particular 97% or more to 100% by weight, preferably 97% or more to 99.99% by weight, totalling 100% by weight of the composition.

[0031] Another embodiment of the present invention can include a diafiltration process, particularly a membrane filtration process, which can be carried out in a system comprising a feed vessel and at least one diafiltration device to which a feed is supplied, the feed being a squalene-containing composition with a lower squalene content, particularly the diafiltration device comprising a membrane, preferably a nanofiltration membrane, and from the at least one diafiltration device, a retentate, particularly at least one retentate, is obtained, which is recycled to the feed vessel, and from the at least one diafiltration device, a permeate, particularly at least one permeate, is obtained, the permeate being an intermediate composition with a certain squalene content. The diafiltration process is preferably carried out continuously in a closed system. The feed vessel preferably has a pressure of 100 mbar, which can be achieved by using an inert gas atmosphere, preferably nitrogen. In a preferred embodiment, the membrane is a silicone-based membrane such as polydimethylsiloxane, and the membrane optionally further comprises a polyacrylonitrile layer and / or a polyethylene terephthalate layer. The membrane preferably has a molecular weight cut-off (MWCO) of 200 to 700 g / mol, preferably 280 to 600 g / mol. The molecular weight cut-off is measured as described above.

[0032] It is preferred that at least one of the steps of the process of the present invention is carried out under an inert atmosphere, in particular under nitrogen, argon and / or a mixture of both. It is particularly preferred that the diafiltration step is carried out under an inert atmosphere.

[0033] The diafiltration unit is preferably supplied with a feed, in particular a squalene-containing composition, having an elevated temperature, in particular a lower squalene content, and a temperature of from 25°C to below the boiling point, in particular from 25 to 90°C, preferably from 30 to 70°C, most preferably from 30 to 60°C or 50°C, and / or the feed is supplied under a pressure of from 1 to 100 bar, in particular more than 20 bar, more preferably more than 50 bar. The pressure in the diafiltration unit, in particular the pressure across the at least one membrane, is in the range of 10 to 100 bar, preferably in the range of 30 to 80 bar, more preferably 50 to 70 bar, most preferably about 60 bar + / - 5 bar.

[0034] In a preferred embodiment, the method comprises at least three diafiltration devices, each of which is connected in series and combined in series or in parallel. When the diafiltration devices are connected in series, the first permeate from the first diafiltration device can be fed to the second diafiltration device, and the permeate from the second diafiltration device is an intermediate composition having a certain squalene content. The retentate from at least two diafiltration devices, particularly the first and second diafiltration devices, is fed to a feed vessel. The diafiltration process is preferably carried out continuously in a closed system. The device and / or method can be carried out using 1 or 2 to 100 diafiltration devices.

[0035] In a particularly preferred embodiment, the squalene-containing composition having a lower squalene content may be a composition containing plant-based squalene, preferably a composition containing plant-based squalene with a squalene content of 1 to 14% by weight, preferably 2 to 12% by weight, and more preferably 5 to 10% by weight. In a further embodiment of the present invention, the squalene-containing composition having a lower squalene content is most preferably a plant-based oil. Examples of plant-based oils that can be used include soybean oil, rice bran oil, olive oil, vegetable oils, particularly vegetable oil distillates, coffee oil, wheat germ oil, corn germ oil, palm oil, andiroba oil, oil from tomato residue, and amaranth oil. Amaranth oil is most preferred, particularly amaranth oil with a squalene content of 1 to 10% by weight, more preferably 5 to 9% by weight.

[0036] In an alternative embodiment of the present invention, a squalene composition having a squalene content of up to 25% by weight can be used. However, to obtain a squalene composition having a squalene content of 12 to up to 25% by weight, the squalene composition must be pre-concentrated in an additional process step not claimed in this application; therefore, a content of less than 12% by weight is preferred.

[0037] Unless otherwise indicated, all percentages stated herein are percent by weight.

[0038] Furthermore, a squalene composition having a concentrated squalene content prepared according to the method of the present invention is an embodiment of the present invention. The squalene composition is preferably a pharmaceutical squalene composition. Furthermore, an embodiment of the present invention is also a pharmaceutical composition comprising the squalene composition, preferably a pharmaceutical squalene composition, particularly a parenteral composition, more preferably a vaccine. Furthermore, an embodiment of the present invention is to provide an oil-in-water emulsion comprising the above squalene composition.

[0039] The method is preferably carried out in an apparatus which may comprise at least one feed vessel and at least one diafiltration device, in particular for carrying out step ii) of the method, the diafiltration device may comprise a membrane, preferably a nanofiltration membrane, more preferably a flat nanofiltration membrane, the at least one diafiltration device being capable of supplying a feed, the feed being a squalene-containing composition, in particular according to the method of the invention, having a lower squalene content, from the at least one diafiltration device a retentate is obtained which can be recycled to the feed vessel, and a permeate is obtained, the permeate being an intermediate composition having a certain squalene content. The flat membrane has a surface area of ​​1 cm in one plane. 2 ~1m 2 , preferably 10 cm 2 ~100cm 2 may have an area of

[0040] The apparatus is preferably a closed system, in which a composition having a lower squalene content is fed in a feed vessel and a permeate is obtained from at least one diafiltration unit, the permeate being an intermediate composition having a squalene content of 10 to 80% by weight, more preferably 20 to 80% by weight. The feed vessel preferably has a pressure of at least 100 mbar at room temperature, which is achieved by using an inert atmosphere, preferably nitrogen.

[0041] Example Measurement techniques and equipment: Gas chromatography: All percentages quoted herein are percentages by area and can be measured using gas chromatography (GC). GC techniques can be performed by injecting a sample of squalene in n-heptane or hexane into a gas chromatograph equipped with a flame ionization detector (FID). Analysis was performed on a 30 m x 0.32 mm x 0.50 mm capillary column held at 380°C for 2 minutes, then ramped at 12°C / min to 310°C and held there for 9 minutes. The injection port and FID are maintained at 300°C and 320°C, respectively. The identity of the squalene peak is established using GC / MS (gas chromatography with mass selective detection). Purity is reported as the area of ​​the squalene peak as a percentage of the sum of the areas of all peaks in the chromatogram.

[0042] Squalene retention time: approx. 19-19.80 min. Sample: 20.8 mg + n-heptane 1 mL Gas chromatograph type: Agilent GC 7890 Injector type: Split / splitless for capillary columns Column: Restek capillary column (30 m) Temperature: 380℃ Carrier gas: He 4.6

[0043] Flash chromatography: Chromatograph type: Buechi C-815 Column: Biotage SNAP KP-Sil, Flow rate: 30 mL / min, or see Examples. Equilibration: 15-25 min (15 mL / min), Continuous run time: 11.0 min, Temperature: RT Detector: UV1λ: 208nm, UV2λ: 220nm, UV3λ: 254nm, UV4λ: 320nm, UV scan 200-800nm.

[0044] Squalene content by gas chromatography: Amaranth oil (extract, squalene content 5-10% by weight): GC analysis: 6.1% area Concentrated amaranth oil (squalene content 15-40% by weight): GC analysis: 20.2% area Isolated squalene (squalene content 97-99% by weight): GC analysis: 99.8% by area

[0045] Squalene molecule: Boiling point: 275°C at 20 hPa [ka]

[0046] Example 1: Flash chromatography of amaranth oil 10 g (15 wt%) of amaranth oil was chromatographed on a 50 g silica gel cartridge (silica 60A, amorphous (irregular), average particle size 50 microns). The cartridge was passivated with ethyl acetate for 6.47 min. Equilibration was carried out with cyclohexane for 19.4 min. A 15 wt% solution was prepared from 10 g of amaranth oil. Flash chromatography was carried out using 66.6 mL of liquid, which was pumped in (flow rate: 15 mL / min). The pump was previously exposed to the liquid at 50 mL / min. Fractions from approximately 6.7 min to 8.7 min were collected and combined. Yield: 64%, 273.2 mg, GC: 98.1 area %

[0047] Example 2: Nanofiltration [Table 1]

[0048] Nanofiltration was performed as described below according to FIG.

[0049] Example 3A: Chromatography after diafiltration using nanofiltration membranes 6 g of the permeate obtained as permeate 2 was prepared as a 25 wt. % squalene solution in cyclohexane and chromatographed onto a cartridge (Biotage SNAP KP-Sil 50 g) at 30 mL / min (Büchi Pure C-815, 50 mL syringe, UV detector as mentioned below). The cartridge was conditioned with ethyl acetate at a flow rate of 15 mL / min for 6.47 min, followed by a cyclohexane wash at 15 mL / min for 22.64 min. 30 mL of the 25 wt. % squalene solution (permeate 2) was applied to the cartridge via syringe.

[0050] The fractions obtained at 2.1 to 2.7 minutes and 2.7 to 3.4 minutes were collected. The second fraction was evaporated under reduced pressure at 40 °C (product: 0.7788 g of yellow liquid) and analyzed by GC (product 20.5 mg + n-heptane 1 mL, GC: 99.3 area %).

[0051] Example 3B: Chromatography after diafiltration using nanofiltration membranes 10 g of the permeate containing 15 wt% amaranth oil was chromatographed at 30 mL / min, and 66.6 g of cyclohexane was added to the 10 g of amaranth oil and stirred with a magnetic stirrer to obtain approximately 84 mL of a clear yellow solution.

[0052] Flash chromatography (Büchi C-815, cartridge: Biotage SNAP KP-Sil 50 g, UV detector as mentioned below) was performed by passivation with 2 BV (bed volume) of ethyl acetate for 6.47 min (flow rate 30 mL / min) and equilibration with 6.99 BV of cyclohexane.

[0053] The fractions from 3.4 min to 4.9 min were combined and evaporated on a rotary evaporator (40° C., 130 mbar) to give an oily squalene composition of 97.1 area % squalene (GC). Product 0.93 g slightly yellow oil.

[0054] Example 4A: Post-Extraction Chromatography (Chemical Concentration) Flash chromatography of 40.8 g of 25 wt% crude amaranth oil on a 340 g cartridge (Biotage SNAP KP-Sil 340 g, Büchi C-815) was equilibrated with cyclohexane (3.5 BV) at a flow rate of 80 mL / min. The 40.8 g 25 wt% solution containing 122.4 g of cyclohexane was chromatographed at a flow rate of 80 mL / min for 2.3 min. To obtain the chromatogram on the Büchi apparatus, the flow rate was adjusted to 1 mL / min from 2.2 to 4.5 min. Before 2.2 min and after 4.5 min, the flow rate was 80 mL / min.

[0055] Three fractions between 8.76 and 14.71 minutes were collected and analyzed by GC. Fraction 4 was evaporated in a rotary evaporator under reduced pressure at 40°C. Product: 20.08 g, GC (21.4 mg product + 1 mL n-heptane): 98.2 area % squalene.

[0056] Example 4B: Extraction [Table 2]

[0057] 250.0 mL of methanol (technical grade) was placed in a 1-L flask at room temperature and stirred. 12.5 g of NaOH was added at room temperature and stirred until the NaOH dissolved. The solution was allowed to cool to room temperature, and 50 g of amaranth oil (Stuebener Kraeutergarten) was added. The mixture was heated to 60-65°C and stirred for 3 hours. Optionally, thin-layer chromatography (ethyl acetate in 9.8:0.2 hexane:KMnO4) was performed. The mixture was cooled to room temperature, and 300 mL of demineralized water was added, followed by stirring for 15 minutes. The mixture was heated to 50-55°C and extracted with 500 mL of hexane, and optionally, GC analysis was performed. The hexane phase was separated, and the aqueous phase was extracted with 500 mL of hexane at 50-55°C. The hexane phase was separated, combined with the first hexane phase at 50-55°C, and washed with 150 mL of demineralized water. From the separated hexane phase, hexane was removed on a rotary evaporator at 300 mbar to 10 mbar and 45° C. (crude product: 3.51 g).

[0058] To the obtained crude product, 250 mL of hexane and celite (diatomaceous earth) were added. Optionally, GC analysis can be performed before adding celite. Before adding celite, a clear yellow solution was obtained. The solution was stirred for 45 minutes and filtered through a glass frit, which was washed with 30 mL of hexane. The hexane was removed on a rotary evaporator at 45°C and 300 mbar to 10 mbar. 3.71 g of product was obtained (orange oily solid). GC analysis: 81.0 area % squalene. 1 H-NMR (600 MHz, CDCl): 75.97 wt% squalene. 1 H-NMR (600 MHz, CDCl3) analysis: squalene 22.43 mg and dimethyl terephthalate 19.49 mg

[0059] Chromatography The resulting product (3.71 g) was dissolved in 25 mL of n-hexane and stirred for 30 minutes. The solvent was evaporated on a rotary evaporator, leaving a cream-like solid. This was dissolved in 25 mL of cyclohexane and stirred with 2.5 g of silica (Porocell silica) for 30 minutes. The 25 wt. % solution was filtered through a No. 4 strainer and washed with cyclohexane. The filtrate was evaporated on a rotary evaporator, leaving a clear, yellow solution (oil). From this oil, a 25 wt. % solution was prepared with 25 mL of n-hexane and loaded onto a Biotage SNAP KP-Sil 340 g cartridge. The cartridge was equilibrated with 3.5 BV of cyclohexane for 22.6 minutes. 14 mL was injected onto the probe over 1:05 minutes. A 1 mL GC sample was analyzed from the resulting fractions. The product was a clear, colorless liquid containing 1.85 g of squalene (GC: 99.8 area %).

[0060] Example 5: Chromatography (30 mL / min) of CO2-extracted concentrated amaranth oil (Flavex amaranth seeds, 15% by weight) 3 g of Flavex was mixed with 17 g of cyclohexane to obtain a 25 wt% yellow solution. The mixture was stirred with a magnetic stirrer for 10 minutes. A slightly yellow, clear solution was obtained.

[0061] The cartridge was equilibrated with 3.5 BV of cyclohexane for 22.6 minutes. After the above, the resulting solution was injected into the cartridge (24 mL, application time: 0:48 min). 1 mL was used for gas chromatography. GC: 94.8 area% (GC RT[min]: 19.72 min) [Brief explanation of the drawings]

[0062] [Figure 1] FIG. 1 describes one alternative form of the method disclosing possible squalene content enrichment in the steps of the method. [Figure 2] FIG. 1 is a diagram illustrating a device 1 equipped with a diafiltration device 3.

[0063] Figure 1 shows a schematic diagram of the process, starting from plant-based amaranth oil with a typical squalene content of 5-10% by weight, for a total composition of 100% by weight. After a diafiltration step, carried out as a nanofiltration step, an intermediate composition with a squalene content of 15-40% by weight is obtained, resulting in a composition of 100% by weight. This composition is then subjected to a chromatography step, which, after removal of the mobile phase, results in a squalene composition with a concentrated squalene content. As an optional step, the mobile phase or a composition containing a portion of the mobile phase may be contacted with an adsorbent such as activated carbon, which may be carried out as a filtration step on activated carbon.

[0064] FIG. 2 illustrates an apparatus 1 comprising a diafiltration device 3 for nanofiltration.

[0065] Experimental Example - Part 1 - Concentration via Diafiltration Device 3 as Nanofiltration The tests were carried out in a continuously operating apparatus 1 (see Figure 2) in a closed circuit or with a total return. In principle, the apparatus 1 comprises a feed vessel 2 and a high-pressure circuit 4 with a flat channel test cell for testing sections of flat membranes in a diafiltration device 3.

[0066] Nitrogen is overflowed into the feed vessel 2, which is filled with amaranth oil (2 L), maintaining a slight nitrogen overpressure of about 100 mbar. From the feed vessel 2, the feed enters the high-pressure circuit 4 by means of a piston-diaphragm pump, which is used to feed the system. The pressure in the circuit is regulated to 50 bar by an adjustable pre-pressure regulator. This circuit contains a 84.5 cm 2 The flat channel test cell contains the flat membrane to be tested, which is installed before the test begins. The built-in membrane (silicone-coated PAN (polyacrylonitrile) PuraMem® Flux, molecular weight cutoff 280-600 g / mol) is overflowed in the circuit by a gear pump, and the temperature of the circuit (30-60 °C) is controlled by a heat exchanger connected to an external thermostat and / or cryostat. Furthermore, the device 1 contains corresponding sensors for pressure and temperature measurement in the circuit 4. There is also the possibility of sampling the feed, retentate, and permeate to determine the membrane selectivity. The permeate is obtained in flask 5.

[0067] In normal operation, both the permeate (oil enriched with approximately 10-50% squalene by weight), which is the flow that passes through the membrane, and the retentate, i.e., the excess of the piston-diaphragm pump's feed minus the permeate, which is again relieved via the pre-pressure regulator, are returned to the feed vessel. Since the components move through the system in a closed circuit, constant conditions at the membrane are set for the most practical measurements. The permeate mass flow rate is selectively indirectly determined by equilibrium. From this, the membrane permeance is given by the average pressure on the feed side of the membrane (p 供給物 +p 保持液 ) / 2) and the pressure on the permeate side of the membrane (p 透過液 ) divided by the applied transmembrane pressure and the installed membrane area.

[0068] Experimental Example - Part 2 - Chromatographic Purification of Concentrated Oil The intermediate composition containing approximately 10-50% squalene by weight in the vessel is diluted with cyclohexane to prepare a 25% solution by weight in a downstream vessel while stirring. A chromatographic apparatus equipped with at least one cartridge (SNAP KP-Sil 50 g) and a Büchi Pure C-815 flash system is used for chromatographic purification. The cartridge is preconditioned with ethyl acetate (15 mL / min) and washed with cyclohexane (15 mL / min for 22 min). Sample loading is performed using a 50 mL syringe. A 30 mL sample of the 25% solution is applied to the chromatography column. The column is run over a packed column at 30 mL / min of cyclohexane, and the main fraction is collected in a flask. The main fraction is completely concentrated on a rotary evaporator at a bath temperature of 40 °C, preferably under reduced pressure, to preserve squalene of high pharmaceutical purity (measured on an Agilent GC 7890 as described above). Optionally, an additional step may comprise contacting the squalene composition having an enriched squalene content and the mobile phase or at least a portion of the mobile phase in the container with an adsorbent such as activated carbon to obtain, after removal of the mobile phase, a composition having an enriched squalene content in the flask, in particular a pharmaceutical squalene composition.

Claims

1. 1. A method for producing a squalene composition having an enriched squalene content from a squalene-containing composition having a lower squalene content, comprising: i) providing a squalene-containing composition having a lower squalene content; ii) subjecting the squalene-containing composition having the lower squalene content to a purification step at a temperature below the boiling point of squalene to obtain an intermediate composition having a squalene content; iii) performing a chromatography step with the intermediate composition having said squalene content; iv) obtaining a squalene composition having the enriched squalene content; A method comprising:

2. 2. The method of claim 1, wherein the squalene composition having an enriched squalene content is a pharmaceutical squalene composition.

3. The purification step ii) comprises a diafiltration step and / or an extraction step, in particular the extraction step is a liquid-liquid extraction step or the extraction step comprises a fluid extraction, and the extractant is CO ２ 3. The method according to claim 1 or 2, characterized in that:

4. 4. The method according to any one of claims 1 to 3, characterized in that the purification step ii) comprises a membrane filtration step, in particular a membrane-based diafiltration step.

5. The membrane filtration step is performed using a silicon-based membrane and / or 5. The method of claim 4, characterized in that it is carried out with a membrane having a molecular weight cut-off (MWCO) of 200 to 700 g / mol.

6. 4. The method according to claim 3, characterized in that the extraction step comprises a liquid-liquid extraction step, and the extractant is a liquid hydrocarbon, in particular a liquid alkane, in particular the alkane is selected from alkanes including pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane, most preferably the alkane is n-hexane, cyclohexane, and / or mixtures thereof.

7. 7. The method according to any one of claims 1 to 6, characterized in that the chromatographic step is carried out on a stationary phase comprising silica, alumina, a diol-functionalized polymer or diol-functionalized silica and / or hydroxylapatite, in particular the stationary phase is selected from silica.

8. 8. The method according to any one of claims 1 to 7, characterized in that the chromatography step is carried out using a mobile phase comprising a liquid hydrocarbon, in particular a liquid alkane, preferably selected from alkanes including pentane, hexane, heptane, octane, nonane, decane, undecane and dodecane, most preferably cyclohexane.

9. 9. The method according to claim 1, wherein at least one further step after the chromatography step of iii) comprises contacting the composition obtained from the chromatography step with at least one adsorbent.

10. a) the squalene-containing composition has a lower squalene content of less than 25% by weight, preferably less than 12% by weight, in particular a squalene content of 0.1% to 10% by weight or less, preferably 5% to 10% by weight or less, the composition totalling 100% by weight; and / or b) the intermediate composition with a squalene content has a squalene content of at least 14% by weight, in particular from 15% to 90% by weight, preferably from 15% to 40% by weight, or from 25% to 40% by weight, of which the intermediate composition totals 100% by weight; and / or c) the squalene composition has a squalene content of the enriched amount, in particular a squalene content of 91% by weight or more, the total weight of the composition being 100%; 10. The method according to any one of claims 1 to 9, characterized in that

11. 11. The method according to any one of claims 3 to 10, characterized in that the diafiltration step, in particular the diafiltration step comprising a membrane filtration step, is carried out in a reaction system comprising a feed vessel and at least one diafiltration device to which a feed is supplied, the feed being the squalene-containing composition having the lower squalene content, in particular the diafiltration device comprising a membrane, preferably a nanofiltration membrane, and from the at least one diafiltration device a retentate is obtained which is recycled to the feed vessel, and from the at least one diafiltration device a permeate is obtained, preferably the permeate is an intermediate composition having the squalene content.

12. 12. The method according to any one of claims 1 to 11, wherein the squalene-containing composition having a lower squalene content is a composition comprising plant-based squalene, preferably a plant-based squalene-containing composition having a squalene content of 1 to 14% by weight, preferably 2 to 12% by weight, more preferably 5 to 10% by weight, and most preferably the squalene-containing composition having a lower squalene content is amaranth oil.

13. 13. A squalene composition having an enriched squalene content, particularly a pharmaceutical squalene composition, prepared according to any one of claims 1 to 12.

14. A pharmaceutical composition, particularly a parenteral pharmaceutical composition, more preferably a vaccine, comprising the squalene composition of claim 13.

15. 14. An oil-in-water emulsion comprising the squalene composition of claim 13.

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