Method for obtaining plant-based squalene

Abstract

The invention relates to a method for preparing a squalene composition, in particular a pharmaceutical squalene composition, having an enriched squalene content, from a squalene-containing composition having a lower squalene content, in particular from a plant-based oil having a lower squalene content, and to a method for preparing a squalene composition, in particular a pharmaceutical squalene composition, having an enriched squalene content. The present invention relates to a process for preparing a squalene-containing composition 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 and obtaining an intermediate composition having a squalene content, and iii) performing a chromatography step with the intermediate composition having a squalene content, and iv) obtaining a squalene composition having an enriched squalene content. Furthermore, the object of the present invention is also squalene compositions, in particular pharmaceutical squalene compositions, in particular parenteral pharmaceutical compositions, more preferably vaccines and oil-in-water emulsions comprising squalene compositions, with an enriched squalene content.

Description

Technical Field

[0001] A method for preparing a squalene composition, especially a pharmaceutical squalene composition, with an enriched squalene content from a squalene-containing composition having a lower squalene content, especially from a plant-based oil having a lower squalene content, the method 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 and obtaining a composition having a medium squalene content, and iii) performing a chromatography step on the composition having a medium squalene content, and iv) obtaining a squalene composition having an enriched squalene content. Furthermore, the object of the present invention also lies in a squalene composition having an enriched squalene content, especially a pharmaceutical squalene composition, especially a parenteral pharmaceutical composition, more preferably a vaccine and an oil-in-water emulsion containing the squalene composition. Background Art

[0002] Pharmaceutical squalene is used as a vaccine adjuvant and in pharmaceutical formulations. Currently, as described in WO2011141819A1, a squalene-containing oil from an animal source (e.g., shark liver) is used as the starting material for squalene preparation. The squalene is obtained in a pharmaceutically pure quality by a distillation process. Due to the thermal distillation and vaporization of squalene in contact with the inner surface of the distillation system, there is always a risk of component degradation or isomerization in the thermal distillation process.

[0003] The qualities required for pharmaceutical applications are described, for example, in Ph.Eur. 01-2020-2805. The extraction of industrial squalene from vegetable oils (olive oil, amaranthol, palm oil) is known from the following documents: M. Azalia Lozano-Grande et al., “Plant Sources, Extraction Methods, and Uses of Squalene”, International Journal of Agronomy, vol. 2018, Article ID 1829160, 13 pages, 2018. https: / / doi.org / 10.1155 / 2018 / 1829160, and Nisarg Gohil et al, “Engineering Strategies in Microorganisms for the Enhanced Production of Squalene: Advances, Challenges and Opportunities”, Front. Bioeng. Biotechnol., 2019, 7. https: / / doi.org / 10.3389 / fbioe.2019.00050.

[0004] Extraction purification techniques and chromatographic purification techniques are described, but these are not sufficient to prepare pharmaceutically pure squalene as required by the pharmacopoeia.

[0005] The object of the present invention is to develop a method capable of providing pharmaceutically pure squalene. In addition, an object of the present invention is to reduce the energy input of the method and reduce the amount of organic solvents used. Additionally, an object of the present invention is to provide a method adapted to plant-based squalene sources. Furthermore, a method for reducing the thermal stress applied to squalene should be developed. Summary of the Invention

[0006] Surprisingly, a combined method using purification steps at lower temperatures, particularly below the boiling point, has been developed, where the purification steps are not chromatographic purification steps and not distillation steps. In particular, the purification steps are filtration steps or chemical extraction steps.

[0007] The method of the present invention is disclosed in claim 1, and the squalene composition is disclosed in claim 13. In addition, the present invention is disclosed in more detail in the dependent claims and described in the specification. The pharmaceutical composition in claim 14 and the oil-in-water composition in claim 15. Detailed Description

[0008] According to the present invention, there is provided a method for preparing a squalene composition having an enriched squalene content from a squalene-containing composition having a lower squalene content, wherein the method particularly provides a pharmaceutical squalene composition and optionally provides a squalene composition having an enriched squalene content obtainable by the method, and wherein the method comprises the following steps:

[0009] i) providing a squalene-containing composition having a lower squalene content,

[0010] ii) subjecting the squalene-containing composition having a lower squalene content to a purification step at a temperature below the boiling point of squalene, particularly at a temperature below 100 °C, optionally at a pressure of 0.01 to 100 bar, particularly at a temperature below 75 °C, optionally at a pressure of 0.01 to 10 bar, and obtaining an intermediate composition having a squalene content, and

[0011] iii) performing a chromatography step on the intermediate composition having a squalene content, and

[0012] iv) obtaining a squalene composition having an enriched squalene content.

[0013] Preferably, the squalene-containing composition having a lower squalene content is a plant-based oil having a lower squalene content. Below the boiling point of squalene means below 275 °C at 20 hPa or a lower temperature at a corresponding reduced pressure.

[0014] According to a first embodiment of the present invention, the squalene composition having an enriched squalene content is a pharmaceutical squalene composition. Particularly preferably, the squalene-containing composition having a lower squalene content is amaranth oil.

[0015] Furthermore, preferably, the purification step in ii) 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, particularly a chemical extraction step with extractant recycling. The filtration step is preferably a filtration step using membrane filtration. A particularly preferred purification step comprises a percolation step and / or an extraction step, particularly, the extraction step is a liquid-liquid extraction step and / or the extraction step comprises fluid extraction.

[0016] In another preferred aspect of the present invention, the method may comprise: i) providing a squalene-containing composition having a lower squalene content, the composition having a squalene content of 12 wt% or less of squalene, wherein the composition totals 100 wt%, and

[0017] ii) 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 at a pressure of 0.01 to 1 bar, subject the squalene-containing composition having a lower squalene content to a purification step and obtain an intermediate composition having a squalene content of at least 14 wt% of squalene, wherein the intermediate composition totals 100 wt%, and

[0018] iii) Perform a chromatography step with the intermediate composition having a squalene content, and

[0019] iv) Obtain a squalene composition having an enriched squalene content, wherein the squalene content is equal to or greater than 91 wt%, wherein the composition totals 100 wt%.

[0020] The newly developed method allows to obtain squalene on a completely plant-based (vegetable) basis. Most preferably, amaranth oil having an average squalene content of 1 to 10 wt% is first enriched via percolation, in particular nano-percolation. Compared to chemical extraction methods, this method allows a significant reduction of solvents. The resulting squalene composition (intermediate composition) obtained by percolation contains approximately a squalene content of about 20 to 50 wt%. This intermediate composition is further enriched via a chromatography step, and the composition separated from the chromatography unit preferably has pharmaceutical quality after removal of the mobile phase.

[0021] Optionally, the composition comprising the mobile phase or a part of the mobile phase can be treated with an adsorbent such as activated carbon or diatomaceous earth and finally the mobile phase, in particular the solvent, is removed. This method prepares a pharmaceutically pure squalene that meets the requirements of Ph.Eur.Monograph 01 / 2021:2805.

[0022] As an alternative to the combination of nanofiltration and chromatography, a combination of extraction and chromatography can also be used. This can be fluid extraction or liquid-liquid extraction, which on the one hand includes solvent extraction and on the other hand includes fluid extraction with CO2 extraction. However, compared to the combination of extraction and chromatography, the combination of filtration (especially nanofiltration) and chromatography significantly reduces the use of solvents, because filtration based on nanofiltration membranes is carried out without the use of additional solvents (especially organic solvents). This forms an environmentally friendly method that uses significantly less undesirable organic solvents. A particularly preferred embodiment of the present method is that the solvent used as the mobile phase and / or extractant can be reused. The liquid alkanes used, especially hexane, n-hexane and cyclohexane, can be reused after they evaporate from the composition. Preferably, more than 90% of the liquid alkanes used as the mobile phase and / or extractant can be reused in the present method. Due to the reuse of the amount of solvent in the method, the amount of solvent used per year can be reduced.

[0023] The first purification step is used to separate the fatty acid esters contained in the squalene-containing composition with a lower squalene content and partially separate the contained phytosterols. By chromatography and optionally activated carbon filtration during the second step, the remaining components are separated and squalene with high drug purity is obtained.

[0024] The term "diafiltration" or "diananofiltration" describes a membrane-based process in which the solvent, as well as some components of the solution or suspension (depending on the application), are displaced. The starting composition, here the squalene-containing composition with a lower squalene content, is circulated from the feed container, and other components of the starting composition, especially the oily components, will be displaced. By adjusting the flow rate after the membrane, especially the flow rate after the membrane device, the transmembrane pressure (TMP) is set, which acts as the driving force in this diafiltration. The molecular weight cut-off value (MWCO) of the membrane is selected such that the molecules to be removed (here squalene) can penetrate the membrane. Since the system operates as a closed system, the discharge of the permeate causes the reflux of the displaced squalene-containing composition, which keeps the circulation volume constant during diafiltration. Once the initially introduced equal volume of material has left the system as permeate and has been replaced by a new squalene-containing composition, the so-called diafiltration volume has been reached.

[0025] Chromatography is a physicochemical technique used to separate a mixture into its components. Typically, the mixture containing the components is dissolved in a fluid solvent (e.g., in liquid extraction) especially a gas, according to the invention in CO2; or dissolved in a solvent as in liquid-liquid extraction. According to the invention, especially dissolved in a liquid hydrocarbon, preferably in a liquid alkane, the fluid solvent or liquid hydrocarbon is called the mobile phase. This mobile phase carries the components through a system, such as a column, capillary, plate, or sheet, on which a material called the stationary phase or containing the stationary phase is fixed. Since the different components of the mixture tend to have different affinities for the stationary phase and are retained for different durations according to their interactions with the surface sites of the stationary phase, the components pass through the stationary phase at different apparent velocities in the flowing fluid, resulting in their separation. This separation is based on differential partitioning between the mobile phase and the stationary phase. Minor differences in the partition coefficients of compounds result in differential retention on the stationary phase, thus affecting the separation.

[0026] Another embodiment of the invention is that the ii) purification step may include a membrane filtration step, especially a membrane-based diafiltration step. Further preferably, 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, more preferably 280 to 600 g / mol. Since there is no standard procedure for determining the MWCO, this value can be obtained in different ways. In this particular case, the rejection R of polystyrene (solute) with different molecular weights in toluene (solvent) can be considered. Here, R i is the rejection of polystyrene of a specific molecular weight, c p,i is its permeate concentration, and c R,i is the retentate concentration of the polystyrene.

[0027]

[0028] Then, the molecular weight cut-off is described by the molecular weight of polystyrene showing a rejection R of 0.9.

[0029] According to an alternative embodiment, the extraction step may include a liquid-liquid extraction step, wherein the extractant is at least one liquid hydrocarbon and / or a mixture comprising at least two liquid hydrocarbons, in particular at least liquid alkanes. The one or more alkanes may be selected from linear alkanes, branched alkanes and / or cycloalkanes. In particular, the alkanes are selected from alkanes including pentane, hexane, heptane, octane, nonane, decane, undecane and dodecane and all their isomers, and most preferably n-hexane, n-heptane, cyclohexane, cycloheptane and / or mixtures thereof. In an alternative, the extraction step may include a fluid extraction, wherein the extractant is CO2. Liquid alkanes refer to liquids at temperatures below 100 °C, in particular liquids at room temperature and 20 hPa.

[0030] According to a preferred embodiment, the purification step, in particular the extraction step, includes a saponification step, said saponification step being before the extraction step, preferably before the liquid-liquid extraction step. Preferably, the saponification step includes: a) contacting the squalene-containing composition having a lower squalene content with a base, said base being in particular a basic solution in an organic solvent, in particular a basic solution of an alcohol, said alcohol such as methanol, ethanol, propanol; and b) obtaining a mixture. And optionally, wherein in step c), the mixture obtained in b) is treated at an elevated temperature, in particular the mixture is stirred and / or heated to a temperature in the range of 30 to 80 °C, in particular 50 to 70 °C, more preferably a temperature above 50 °C.

[0031] And optionally, in step d), the mixture can be cooled, and in step e), water, in particular distilled water, is added. Then, optionally, in step f), the mixture and the added water are heated to an elevated temperature, in particular to a temperature in the range of 30 to 80 °C, in particular 50 to 70 °C, more preferably a temperature above 50 °C, and the mixture and water are treated with an extractant, and a resulting mixture is obtained. Optionally, g) the resulting mixture can be separated into an aqueous phase and an organic phase containing the extractant and squalene, the two phases are separated, and optionally, the aqueous phase is treated again with the extractant, and optionally step g) is repeated, in particular to obtain an organic phase containing the extractant and squalene. Preferably, h) the obtained organic phases containing the extractant and squalene are combined, and optionally contacted with water, in particular with distilled water; i) the resulting mixture can be separated into an aqueous phase and an organic phase containing the extractant and squalene. Optionally, j) the organic phase containing the extractant and squalene is contacted again with water, in particular with distilled water; k) the resulting mixture can be separated into an aqueous phase and an organic phase containing the extractant and squalene. Furthermore, l) the organic phase containing the extractant and squalene obtained in step i) and / or k) is treated under vacuum and / or at an elevated temperature to remove the extractant, and an intermediate composition with a squalene content is obtained.

[0032] Optionally, as an alternative, m) the organic phase containing the extractant, in particular the extractant with a reduced content, in step l) is contacted with an adsorbent, in particular contacted with the adsorbent and stirred; and n) the adsorbent is removed from the organic phase containing the extractant, in particular the adsorbent is filtered out. Then, o) the organic phase containing the extractant is treated under vacuum and / or at an elevated temperature to remove the extractant, and an intermediate composition with a squalene content is obtained. Preferred adsorbents are diatomaceous earth or activated carbon. In a particularly preferred embodiment of the invention, steps a) to step k) are carried out continuously, or steps a) to step o) are carried out continuously.

[0033] The treatment under vacuum and / or at an elevated temperature can include treatment at a pressure in the range of 0.1 mbar to below 1 bar, preferably 10 to 600 mbar, more preferably 100 to 300 mbar, in particular at a temperature in the range of room temperature to 60 °C, preferably in the range of 30 to 50 °C.

[0034] According to the present invention, the chromatographic step can be carried out using a stationary phase comprising silica, alumina, a diol-functionalized polymer, or diol-functionalized silica, and / or hydroxyapatite. In particular, the stationary phase is selected from silica, alumina, a diol-functionalized polymer, or diol-functionalized silica, and / or hydroxyapatite. In particular, the chromatographic step can be carried out using an intermediate composition having a squalene content, optionally with a mobile phase added, and a squalene composition having an enriched squalene content can be obtained after evaporation of the mobile phase. Particularly preferably, in step (iii) of carrying out the chromatographic step with the intermediate composition having a squalene content, the step is carried out using a mobile phase, in particular using the intermediate composition and the mobile phase. Thus, step (iii) can be carried out using the intermediate composition and the added mobile phase.

[0035] Another object of the present invention is that the chromatographic step can be carried out using a mobile phase comprising at least one liquid hydrocarbon, in particular at least one liquid alkane, preferably the at least one or more alkanes are selected from alkanes including pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane, and most preferably cyclohexane. Optionally, in addition or as an alternative, the mobile phase can also be selected from fluid solvents such as supercritical CO2.

[0036] Preferably, the liquid phase comprises a composition comprising an intermediate composition having a squalene content and a liquid alkane, in particular as the mobile phase, wherein the content of the intermediate composition having a squalene content is 1 to 80 wt%, preferably 5 to 30 wt%, and the remainder is the mobile phase, in particular at least one liquid alkane, totaling 100 wt% of the mobile phase. The liquid alkane is in particular cyclohexane. The liquid alkane can include the above liquid alkanes and / or a mixture of at least two of them. Most preferably, it is cyclohexane, n-hexane, n-heptane, cycloheptane, and / or a mixture thereof.

[0037] Another object of the present invention is that after the chromatographic step (iii), at least one additional step can include contacting the composition obtained from the chromatographic step with at least one adsorbent, in particular the at least one adsorbent can include activated carbon, diatomaceous earth, silica, and / or a mixture of at least two of them.

[0038] According to another embodiment of the method, a) the squalene-containing composition having a lower squalene content can have a squalene content of 12 wt% or less of squalene, in particular 0.1 wt% to equal to or less than 10 wt%, preferably 5 wt% to equal to or less than 10 wt% of squalene, wherein the composition totals 100 wt%, and / or

[0039] b) The intermediate composition having a squalene content may have a squalene content of at least 14 wt% of squalene, particularly equal to or greater than 15 wt% to 90 wt%, preferably equal to or greater than 15 wt% to 40 wt% of squalene, wherein the intermediate composition totals 100 wt%, and / or

[0040] c) The squalene composition having an enriched squalene content, particularly a pharmaceutical squalene composition, may have a squalene content equal to or greater than 91 wt%, particularly equal to or greater than 97 wt% to 100 wt%, preferably equal to or greater than 97 wt% to 99.99 wt% of squalene, wherein the composition totals 100 wt%.

[0041] Another embodiment of the present invention may include a percolation step. In particular, the percolation step may include a membrane filtration step, and the membrane filtration step may be carried out in a system comprising a feed container and at least one percolation device, the system being supplied with a feed, wherein the feed is a squalene-containing composition having a lower squalene content. In particular, the percolation device includes a membrane, preferably a nanofiltration membrane, and a retentate, particularly at least one retentate, is obtained from the at least one percolation device and recycled to the feed container, and a permeate, particularly at least one permeate, is obtained from the at least one percolation device, wherein the permeate is an intermediate composition having a squalene content. The percolation step is preferably carried out continuously in a closed system. The feed container preferably has a pressure of 100 mbar, which is obtained by using an inert gas atmosphere, preferably nitrogen. In a preferred embodiment, the membrane is a silicon-based membrane, such as polydimethylsiloxane, and the membrane optionally further includes 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, more preferably 280 to 600 g / mol. The molecular weight cut-off is measured as described above.

[0042] Preferably, at least one step of the method of the present invention is carried out under an inert atmosphere, particularly under nitrogen, argon, and / or a mixture of the two. Particularly preferably, the percolation step is carried out under an inert atmosphere.

[0043] The percolation device is preferably supplied with a feed having an elevated temperature. In particular, the feed, especially a squalene-containing composition having a lower squalene content, has a temperature of from 25 °C to below the boiling point temperature, especially 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 at a pressure of equal to or greater than 1 bar to 100 bar, especially above 20 bar, more preferably above 50 bar. The pressure in the percolation device, especially the pressure applied to 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, and most preferably about 60 bar in the case of + / - 5 bar.

[0044] In a preferred embodiment, the at least one permeate obtained from the at least one percolation device comprises at least three percolation devices, which are combined one after another in a continuous connection manner or side by side in a continuous connection manner. In the case where the percolation devices are combined one after another, the first permeate obtained from the first percolation device can be fed into the second percolation device, wherein the permeate obtained from the second percolation device is an intermediate composition having a squalene content. The retentates from at least two percolation devices, especially the first and second percolation devices, are fed into the feed container. The percolation step is preferably carried out continuously in a closed system. The device and / or the method can be carried out with 1 or 2 to 100 percolation devices.

[0045] In another embodiment of the present invention, the squalene-containing composition having a lower squalene content can be a plant-based squalene-containing composition in a particularly preferred embodiment. Preferably, it is a plant-based squalene-containing composition having a squalene content of 1 to 14 wt%, preferably 2 to 12 wt%, more preferably 5 to 10 wt%. Most preferably, the squalene-containing composition having a lower squalene content is a plant-based oil. The plant-based oils that can be used include soybean oil, rice bran oil, olive oil, vegetable oils, especially distillates of vegetable oils, coffee oil, wheat germ oil, corn germ oil, palm oil, andiroba oil, oil from tomato pomace, and amaranth oil. Most preferably, it is amaranth oil, especially amaranth oil having a squalene content of 1 to 10 wt%, more preferably 5 to 9 wt%.

[0046] In an alternative embodiment of the present invention, a squalene composition having a squalene content of at most 25 wt% can be used. However, in order to obtain a squalene composition having a squalene content of 12 to 25 wt%, the squalene composition needs to be pre-enriched by additional method steps not claimed in the present application, and thus a content of 12 wt% or less is preferred.

[0047] Unless otherwise specified, any percentages described herein are percentages by weight.

[0048] In addition, a squalene composition having an enriched squalene content prepared by the method according to the present invention is an embodiment of the present invention. The squalene composition is preferably a medicinal squalene composition. In addition, an embodiment of the present invention is also a pharmaceutical composition comprising the squalene composition, preferably a medicinal squalene composition, particularly a parenteral composition, more preferably a vaccine. In addition, an embodiment of the present invention is to provide an oil-in-water emulsion comprising the above-mentioned squalene composition.

[0049] The method is preferably carried out in a device which may comprise at least one feed container and at least one filtration device, particularly for carrying out step ii) of the method. In particular, the filtration device may comprise a membrane, preferably a nanofiltration membrane, more preferably a flat nanofiltration membrane, wherein the at least one filtration device may be supplied with a feed, wherein the feed is a squalene-containing composition having a lower squalene content, particularly according to the method of the present invention, and wherein a retentate and a permeate can be obtained from the at least one filtration device, the retentate can be recycled to the feed container, and the permeate is an intermediate composition having a squalene content. The flat membrane may have an area in one plane of 1 cm 2 to 1 m 2 , preferably 10 cm 2 to 100 cm 2 in area.

[0050] The device is preferably a closed system, in which a composition having a lower squalene content is supplied in the feed container, and a permeate can be obtained from the at least one filtration device, wherein the permeate is an intermediate composition having a squalene content. Wherein the squalene content is preferably 10 to 80 wt%, more preferably 20 to 80 wt%. The feed container preferably has a pressure of at least 100 mbar at room temperature, and the pressure is obtained by using an inert atmosphere, preferably nitrogen.

[0051] Examples

[0052] Measurement techniques and devices:

[0053] Gas chromatography:

[0054] All percentages cited in this text are percentages by area % and can be measured using gas chromatography (GC). The GC technique can be carried out by injecting a squalene sample in n - heptane or hexane into a gas chromatograph equipped with a flame ionization detector (FID). The analysis was performed on a 30m×0.32mm×0.50mm capillary column, which was maintained at 380°C for 2 minutes and then ramped at 12°C per minute to 310°C and held at 310°C for 9 minutes. The injection port and FID were maintained at 300°C and 320°C respectively. The identification of the squalene peak was established using GC / MS (gas chromatography using a mass selective detector). The purity was reported as the percentage of the area of the squalene peak to the total area of all peaks in the chromatogram.

[0055] The retention time of squalene is about 19 to 19.80 minutes, sample: 20.8mg + 1mL n - heptane

[0056] Type of gas chromatograph: Agilent GC 7890

[0057] Type of injector: split / splitless for capillary column

[0058] Column: Restek capillary column (30m)

[0059] Temperature: 380°C

[0060] Carrier gas: He 4.6

[0061] Fast chromatography:

[0062] Type of chromatograph: Büchi C - 815

[0063] Column: Biotage SNAP KP - Sil, flow rate: 30 mL / min or see examples,

[0064] Equilibration: 15 to 25 minutes (15 mL / min), run time: 11.0 minutes, temperature: room temperature

[0065] Detector: UV1 λ: 208nm, UV2 λ: 220nm, UV3 λ: 254nm, UV4 λ: 320nm, UV scan 200 to 800nm.

[0066] Squalene content according to gas chromatography:

[0067] Amaranth oil (isolated, squalene content 5 to 10 wt%): GC analysis: 6.1 area %

[0068] Enriched amaranth oil (squalene content 15 to 40 wt%): GC analysis: 20.2 area %

[0069] Isolated squalene (squalene content 97 to 99 wt%): GC analysis: 99.8 area %

[0070] Squalene molecule: Boiling point: 275 °C at 20 hPa

[0071]

[0072] Example 1: Flash chromatography of amaranth oil

[0073] 10 g of amaranth oil (15 wt%) was chromatographed on a 50 g silica column (Silica 60A, amorphous (irregular), average particle size 50 µm). The column was passivated with ethyl acetate for 6.47 minutes. It was equilibrated with cyclohexane for 19.4 minutes. A 15 wt% liquid was prepared from 10 g of amaranth oil. Flash chromatography was performed with 66.6 mL of the liquid, and the liquid was injected by pump (flow rate: 15 mL / min). The pump was pre-treated with the liquid at 50 mL / min. The fractions collected from about 6.7 minutes to 8.7 minutes were combined. Yield: 64%, 273.2 mg, GC: 98.1 area %

[0074] Example 2: Nanofiltration

[0075] Squalene (area %) GC (wt %) Feed 11.0 6.51 Feed 0 11.0 6.88 Permeate 1 20.0 12.83 Permeate 4 19.9 13.38 Retentate 1 10.7 6.81 Retentate 4 10.2 6.49

[0076] According to Figure 2 , nanofiltration was carried out as described below.

[0077] Example 3A: Chromatography after diafiltration with a nanofiltration membrane

[0078] 6 g of the permeate obtained as permeate 2 was prepared as a 25 wt% squalene solution in cyclohexane and chromatographed through a column (Biotage SNAP KP-Sil 50 g) at 30 mL / min (Büchi Pure C-815, 50 mL syringe, UV detector as described above). The column was conditioned with ethyl acetate at a flow rate of 15 mL / min for 6.47 minutes and then washed with cyclohexane at 15 mL / min for 22.64 minutes. 30 mL of the 25 wt% squalene solution (permeate 2) was applied to the column using a syringe.

[0079] The fractions obtained between 2.1 and 2.7 minutes and between 2.7 minutes and 3.4 minutes were collected. The second fraction was evaporated under reduced pressure at 40 °C (product: 0.7788 g yellow liquid) and analyzed by GC. (20.5 mg product + 1 mL n-heptane, GC: 99.3 area %)

[0080] Example 3B: Chromatography after diafiltration with a nanofiltration membrane

[0081] 10 g of the permeate containing 15 wt% amaranth oil was chromatographed at 30 mL / min. 66.6 g of cyclohexane was added to 10 g of amaranth oil and stirred with a magnetic stirrer. Approximately 84 mL of a clear yellow solution was obtained.

[0082] Flash chromatography (Büchi C-815, column: Biotage SNAP KP-Sil 50 g, UV detector as described below). The column was passivated with 2 BV (bed volume) of ethyl acetate for 6.47 min (flow rate 30 mL / min) and equilibrated with 6.99 BV of cyclohexane.

[0083] 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 containing 97.1 area% squalene (GC). The product was 0.93 g of a pale yellow oil.

[0084] Example 4A: Chromatography after extraction (chemical enrichment)

[0085] Flash chromatography of 40.8 g of 25 wt% crude amaranth oil on a 340 g column (Biotage SNAP KP-Sil 340 g, Büchi C-815), equilibrated with cyclohexane (3.5 BV) at a flow rate of 80 mL / min. 40.8 g and 122.4 g of cyclohexane were chromatographed as a 25 wt% solution at a flow rate of 80 mL / min in 2.3 min. To obtain a chromatogram with the Büchi apparatus, the flow rate was adjusted to 1 mL / min from 2.2 min to 4.5 min. Before 2.2 min and after 4.5 min, the flow rate was 80 mL / min.

[0086] Three fractions from 8.76 min to 14.71 min were collected and analyzed by GC. Fraction 4 was evaporated on 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.

[0087] Example 4B: Extraction

[0088]

[0089] 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 cooled to room temperature and 50 g of amaranth oil (Stübener )。The mixture was heated to 60 to 65 °C and stirred for 3 hours. Optionally, thin layer chromatography (9.8:0.2 hexane:ethyl acetate in KMnO4). The mixture was cooled to room temperature, and 300 mL of deionized water was added, followed by stirring for 15 minutes. The mixture was heated to 50 to 55 °C and extracted with 500 mL of hexane, optionally analyzed by GC. The hexane phase was separated, and the aqueous phase was extracted with 500 mL of hexane at 50 to 55 °C. The hexane phase was separated and combined with the first hexane phase at 50 to 55 °C and washed with 150 mL of deionized water. Hexane was removed from the separated hexane phase at 45 °C under 300 to 10 mbar in a rotary evaporator (crude product: 3.51 g).

[0090] 250 mL of hexane and celite were added to the obtained crude product. Optionally, GC analysis could be performed before adding celite. A clear yellow solution was obtained before adding celite. The solution was stirred for 45 minutes and filtered through a glass frit, which was washed with 30 mL of hexane. Hexane was removed on a rotary evaporator at 45 °C and 300 to 10 mbar. 3.71 g of the product (orange, oily solid) was obtained, GC analysis: 81.0 area% squalene, 1 1H-NMR (600 MHz, CDCl3): 75.97 wt% squalene. 1 1H-NMR (600 MHz, CDCl3) analysis: 22.43 mg of squalene and 19.49 mg of dimethyl terephthalate.

[0091] Chromatography

[0092] The obtained product (3.71 g of the product) was dissolved in 25 mL of n-hexane and stirred for 30 minutes. The solvent was evaporated at a rotary evaporator, and a milky solid was obtained, which was dissolved in 25 mL of cyclohexane and stirred with 2.5 g of Porocell silica for 30 minutes. The 25 wt% solution was filtered through a coarse filter (No. 4) and washed with cyclohexane. The filtrate was evaporated with a rotary evaporator to obtain a clear yellow solution (oily). A 25 wt% solution was prepared from this oil with 25 mL of n-hexane and applied to a column (Biotage SNAP KP-Sil 340 g). The column was equilibrated with 3.5 BV of cyclohexane for 22.6 minutes. 14 mL was injected into a direct injector in 1:05 minutes. 1 mL of GC sample was analyzed from the obtained fractions. The product was a clear colorless liquid of 1.85 g squalene (GC: 99.8 area%).

[0093] Example 5: Chromatography (30 mL / min) of CO2-extracted enriched amaranth oil (Flavex amaranth seeds, 15 wt%)

[0094] 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 light yellow clear solution was obtained.

[0095] The column was equilibrated with 3.5 BV of cyclohexane for 22.6 minutes. Then, the solution obtained above was injected into the column (24 mL, injection time: 0:48 minutes). 1 mL was taken for gas chromatography.

[0096] GC: 94.8 area% (GC retention time (RT) [minutes]: 19.72 minutes) Description of the Drawings

[0097] Figure 1 Describes an alternative to the method, which discloses the possible enrichment of squalene content in the steps of the method.

[0098] Figure 2 Describes apparatus 1 including percolation device 3.

[0099] Figure 1 Is a schematic overview of the method, starting from plant-based amaranth oil, where the typical squalene content in a total 100 wt% composition is 5 to 10 wt%. After a percolation step carried out as a nanofiltration step, an intermediate composition with a squalene content of 15 to 40 wt% is obtained, where the composition is 100 wt% in total. This composition is subjected to a chromatography step, where a squalene composition with an enriched squalene content is obtained after removal of the mobile phase. As an optionally present step, the composition containing the mobile phase or a portion of the mobile phase can be contacted with an adsorbent such as activated carbon, and this step can be carried out on the activated carbon as a filtration step.

[0100] Figure 2 Describes apparatus 1, which includes percolation device 3 for nanofiltration.

[0101] Example Experiment - Part 1 - Enrichment via Percolation Device 3 as Nanofiltration

[0102] These tests were carried out in continuously operating apparatus 1, see Figure 2 , and were carried out in a closed loop or at total return. In principle, the apparatus 1 includes a feed container 2 and a high-pressure circuit 4, which has a flat channel test unit for testing a portion of the flat membrane in the percolation device 3.

[0103] The feed container 2 filled with amaranth oil (2 L) is overflowed with nitrogen, and a slight overpressure of about 100 mbar is maintained due to this nitrogen. The feed enters the high-pressure circuit 4 from the feed container 2 by means of a piston diaphragm pump for feeding the system. The pressure of this circuit is adjusted to at most 50 bar by an adjustable pre-pressure regulator. There is a flat channel test unit in this circuit, in which a flat membrane to be tested with a plane of 84.5 cm 2 is installed before the start of the test. The built-in membrane (PAN (Polyacrylnitril) coated with silicone Flux, molecular weight cut-off value 280 to 600 g / mol) is overflowed in the said circuit by means of a gear pump, and the temperature (30 to 60 °C) of this circuit is controlled by a heat exchanger connected to an external thermostat and / or cryostat. In addition, the device 1 includes corresponding sensors in the said circuit 4, such as pressure measurement and temperature measurement. It is also possible to take samples in the said feed, retentate and permeate to determine the selectivity of the membrane. The permeate is obtained in the flask 5.

[0104] In normal operation, the permeate (oil enriched with about 10 to 50 wt% squalene), the flow permeating through the membrane, and the retentate (i.e., the excess part of the feed of the piston diaphragm pump minus the said permeate, which is released again via the pre-pressure regulator) are driven back to the said feed container. The said components are conveyed through the system in a closed loop so that constant conditions at the membrane are set for the most feasible measurement. The permeate mass flow rate is indirectly determined selectively by balancing. Thus, the permeance of the membrane can be obtained by dividing the applied transmembrane pressure (which is the difference between the average pressure ((p 进料 +p 渗余物 ) / 2) on the feed side of the membrane and the pressure (p 渗透物 ) on the permeate side of the membrane) by the installed membrane area.

[0105] Example Experiment - Part 2 - Chromatographic Purification of Enriched Oil

[0106] The intermediate composition in the container with a squalene content of about 10 to 50 wt% is diluted with cyclohexane, and a 25 wt% solution is prepared with stirring in a downstream container. A device for chromatography is used for chromatographic purification, and the device includes at least one column (SNAP KP-Sil 50g) and a Büchi Pure C-815 rapid system. The column is pretreated with ethyl acetate (15 ml / min) and washed with cyclohexane (22 minutes, at 15 ml / min). Sample submission is carried out using a 50 ml syringe. A 30 ml sample of the 25% solution is applied to the chromatographic column. The column is used with cyclohexane passing through the packed column at 30 ml / min, and the main fraction is collected in a flask. The main fraction is completely concentrated in a rotary evaporator at a bath temperature of 40 °C, preferably under reduced pressure, and squalene with high drug purity (measured by Agilent GC 7890 as described above) is obtained. Optionally, additional steps may include: contacting the squalene composition with an enriched squalene content with a mobile phase or at least a part of the mobile phase in a container with an adsorbent such as activated carbon, and obtaining the composition with an enriched squalene content, in particular a medicinal squalene composition, in a flask after removing the mobile phase.

Claims

1. A method for preparing a squalene composition having an enriched squalene content from a squalene-containing composition having a lower squalene content, the method comprising the following steps: 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 and obtaining an intermediate composition having a squalene content, and iii) performing a chromatography step on the intermediate composition having a squalene content, and iv) obtaining a squalene composition having an enriched squalene content.

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

3. The method according to claim 1 or 2, characterized in that, The purification step in ii) comprises a percolation 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, wherein the extractant is CO2.

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

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

6. The method according to claim 3, wherein The extraction step comprises a liquid-liquid extraction step, wherein 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, and most preferably is n-hexane, cyclohexane and / or a mixture thereof.

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

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, the alkane is selected from alkanes including pentane, hexane, heptane, octane, nonane, decane, undecane and dodecane, and most preferably is cyclohexane.

9. The method according to any one of claims 1 to 8, characterized in that, After the chromatography step in iii), at least one additional step comprises contacting the composition obtained from the chromatography step with at least one adsorbent.

10. The method according to any one of claims 1 to 9, characterized in that a) the squalene-containing composition having a lower squalene content has a squalene content of 25 wt% or less, preferably 12 wt% or less of squalene, in particular a squalene content of 0.1 wt% to equal or less than 10 wt%, preferably 5 wt% to equal or less than 10 wt%, wherein the composition totals 100 wt%, and / or b) the intermediate composition having a squalene content has a squalene content of at least 14 wt% of squalene, in particular a squalene content of equal or greater than 15 wt% to 90 wt%, preferably equal or greater than 15 wt% to 40 wt% or 25 wt% to 40 wt%, wherein the intermediate composition totals 100 wt%, and / or c) the squalene composition having an enriched squalene content, in particular a squalene content equal to or greater than 91 wt%, wherein the composition totals 100 wt%.

11. The method according to any one of claims 3 to 10, characterized in that, The percolation step is carried out in a reaction system comprising a feed container and at least one percolation device. In particular, the percolation step comprises a membrane filtration step. The reaction system is supplied with a feed, wherein the feed is the squalene-containing composition having a lower squalene content. In particular, the percolation device comprises a membrane, preferably a nanofiltration membrane. And a retentate is obtained from the at least one percolation device, the retentate is recycled to the feed container, and a permeate is obtained from the at least one percolation device. Preferably, the permeate is the intermediate composition having a squalene content.

12. The method according to any one of claims 1 to 11, characterized in that, The squalene-containing composition having a lower squalene content is a plant-based squalene-containing composition. Preferably, it is a plant-based squalene-containing composition having a squalene content of 1 to 14 wt%, preferably 2 to 12 wt%, more preferably 5 to 10 wt%. Most preferably, the squalene-containing composition having a lower squalene content is amaranth oil.

13. A squalene composition having an enriched squalene content, which is prepared by the method according to any one of claims 1 to 12, in particular a pharmaceutical squalene composition.

14. A pharmaceutical composition comprising the squalene composition according to claim 13, in particular a parenteral pharmaceutical composition, more preferably a vaccine.

15. An oil-in-water emulsion comprising the squalene composition according to claim 13.

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