EP4634144A1

EP4634144A1 - Process for obtaining plant-based squalene

Info

Publication number: EP4634144A1
Application number: EP23818465.9A
Authority: EP
Inventors: Andreas Jakob; Jochen Becker; Sebastian DETLEFSEN; Martin GLOBISCH; Marc SCHÄPERTÖNS; Jens Martin DREIMANN
Original assignee: Evonik Operations GmbH
Current assignee: Evonik Operations GmbH
Priority date: 2022-12-12
Filing date: 2023-12-06
Publication date: 2025-10-22
Also published as: IL320137A; AU2023394219A1; MX2025006742A; JP2025538826A; WO2024126194A1; KR20250121571A; CN120359195A

Abstract

Process for the production of a squalene composition having an enriched squalene content, in particular of a pharmaceutical squalene composition, form a squalene comprising composition having a lower squalene content, in particular from a plant-based oil having a lower squalene content, comprising the steps of i) providing a composition comprising squalene having a lower squalene content, ii) subjecting the squalene comprising composition having a lower squalene content to a purification step at a temperature below the boiling point of squalene, and obtaining an intermediate composition with a squalene content, and iii) performing a chromatography step with the intermediate composition with a squalene content, and iv) obtaining a squalene composition having the enriched squalene content. Furthermore, objects of the invention are also a squalene composition having an enriched squalene content, in particular a pharmaceutical squalene composition, in particular a parenteral pharmaceutical composition, more preferred a vaccine and an oil-in water emulsion comprising a squalene composition.

Description

Process for obtaining plant-based squalene

FIELD OF THE INVENTION

Process for the production of a squalene composition having an enriched squalene content, in particular of a pharmaceutical squalene composition, from a squalene comprising composition having a lower squalene content, in particular from a plant-based oil having a lower squalene content, comprising the steps of i) providing a composition comprising squalene having a lower squalene content, ii) subjecting the squalene comprising composition having a lower squalene content to a purification step at a temperature below the boiling point of squalene, and obtaining a composition with an intermediate squalene content, and iii) performing a chromatography step with the composition with an intermediate squalene content, and iv) obtaining a squalene composition having the enriched squalene content. Furthermore, objects of the invention are also a squalene composition having an enriched squalene content, in particular a pharmaceutical squalene composition, in particular a parenteral pharmaceutical composition, more preferred a vaccine and an oil-in water emulsion comprising a squalene composition.

BACKGROUND OF THE INVENTION

Pharmaceutical squalene is used as vaccine adjuvants and pharmaceutical formulations. Currently, squalene-containing oils from animal sources, such as shark liver, are used as the starting raw material for squalene production, as described in WO 2011141819 A1. This squalene is obtained in pharmaceutically pure quality through a distillation process. A thermal distillation process always bears the risk of degradation or isomerisation of components due to thermal distillation and vaporisation of the squalene which is in contact with the internal surface of a distillation system.

The quality required for pharmaceutical applications is for example described in Ph. Eur. 01-2020- 2805. The extraction of technical squalene from plant 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, 13 pages, 2018. https://doi.Org/1 0.1 155/201 8/1829160, and Nisarg Gohil etal, "Engineering Strategies in Microorganisms forthe Enhanced Production of Squalene: Advances, Challenges and Opportunities", Front. Bioeng. BiotechnoL, 201 9, 7. https://d0i.0rg/l 0.3389/fbioe.201 9.00050.

Extractive as well as chromatographic purification techniques are described, but these are not sufficient to produce pharmaceutically pure squalene as it is required according to pharmacopeia’s. Object of the invention was to develop a process that is able to furnish squalene with pharmaceutical purity. Furthermore, it was an object of the invention to reduce the energy input of the process and to reduce the amount of used organic solvents. In addition, object of the invention was to provide a process that is adapted to plant-based squalene sources. Moreover, a process shall be developed with reduced thermal stress applied on squalene.

SUMMARY OF THE INVENTION

Surprisingly a combined process was developed utilising a purification step at a lower temperature, in particular a temperature below the boiling point, which is not a chromatographic purification step and not a distillation step. In particular, the purification step is a filtration step or a chemical extraction step.

The process of the invention is disclosed in claim 1 , a squalene composition is disclosed in claim 13. Furthermore, the invention is disclosed in more detail in the dependent claims and more detailed in the description. A pharmaceutical composition in claim 14, and an oil-in water composition in claim 15.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention a process forthe production of a squalene composition having an enriched squalene content is provided, wherein the process provides in particular a pharmaceutical squalene composition, from a squalene comprising composition having a lower squalene content, and optionally a squalene composition having an enriched squalene content obtainable by the process is provided, wherein the process comprising the steps of i) providing a composition comprising squalene having a lower squalene content, ii) subjecting the squalene comprising composition having a lower squalene content to a purification step at a temperature below the boiling point of squalene, in particular at a temperature below 100 °C optionally at a pressure from 0.01 to 100 bar, in particular below 75 °C, and optionally at a pressure from 0.01 to 10 bar, and obtaining an intermediate composition with a squalene content, and iii) performing a chromatography step with the intermediate composition with a squalene content, and iv) obtaining a squalene composition having the enriched squalene content.

It is preferred that the squalene comprising composition having a lower squalene content is a plantbased oil having a lower squalene content. Below the boiling point means below 275 °C at 20 hPa or at a lower temperature at the corresponding reduced pressure. According to first embodiment of the invention the squalene composition having an enriched squalene content is a pharmaceutical squalene composition. It is particularly preferred that the squalene comprising composition having a lower squalene content is amaranth oil.

Furthermore, it is preferred that, the ii) purification step comprises physical and/or chemical purification step below the boiling point of squalene, such as a filtration step and/or chemical extraction step, in particular a chemical extraction step with recycling of the extraction agent. The filtration step is preferred a filtration step utilizing a membrane filtration. A particularly preferred purification step comprises a diafiltration step and/or an extraction step, in particular the extraction step is a liquid-liquid extraction step and/or the extraction step comprising a fluid-extraction.

In another preferred aspect of the invention the process may comprise i) providing a composition comprising squalene having a lower squalene content having a squalene content of below 12 wt.-% of squalene, wherein the composition sums up to 100 wt.-%, and ii) subjecting the squalene comprising composition having a lower squalene to a purification step at a temperature below the boiling point of squalene, in particular at a temperature below 100 °C, preferred below 75 °C, optionally at a pressure from 0.01 to 100 bar, preferred at 0.01 to 1 bar, and obtaining an intermediate composition with a squalene content of a least 14 wt.- % squalene, wherein the intermediate composition sums up to 100 wt.-%, and iii) performing a chromatography step with the intermediate composition with a squalene content, and iv) obtaining a squalene composition having the enriched squalene content, wherein the squalene content is equal or above 91 wt.-%, wherein the composition sums up to 100 wt.-%.

The newly developed process allows squalene to be obtained on a purely plant-based vegetable basis. Most preferred is amaranth oil with an average squalene content of 1 to 10 wt.% which is first enriched via diafiltration, in particular diananofiltration. This process allows a drastic solvent reduction compared to chemical extraction methods. The resulting squalene composition (intermediate composition) obtained from diafiltration comprises approximately a squalene content of about 20 to 50 wt.-%. This intermediate composition is further enriched via a chromatography step and the isolated composition from a chromatography unit has preferably after the removal of the mobile phase pharmaceutical quality.

Optionally, the composition comprising the mobile phase or part of the mobile phase can be treated with an adsorbent, such as activated carbon or diatomaceous earth, and final removal of the mobile phase, in particular the solvent. This process produces pharmaceutically pure squalene that meets the requirements of Ph. Eur. Monograph 01/2021 :2805.

As an alternative to the combination of diananofiltration and chromatography, the combination of extraction and chromatography can also be used. This can be a fluid-extraction or liquid-liquid- extraction comprising on the one hand a solvent extraction and on the other hand a fluid extraction with CO2 extraction. However, the combination of diafiltration, in particular diananofiltration, and chromatography significantly reduces the use of solvents compared to extraction and chromatography, since diafiltration based on nanofiltration membranes can be performed without the use of additional solvents, in particular without organic solvents. This leads to an environmentally friendly process using significantly less undesired organic solvents. A particular preferred embodiment of the current process is that the solvents used as mobile phase and/or as extracting agent can be reused. The used liquid alkanes, in particular hexane, n-hexane, and cyclohexane, can be reused after they were evaporated form the compositions. It is preferred that more than 90 % of the liquid alkanes used as mobile phase and/or extraction agent can be reused in the process. Due to the reuse of the amount of solvent for a process the amount of solvent per annum can be reduced.

The first purification step is used to separate the fatty acid esters contained in squalene comprising composition having a lower squalene content, and partial separation of phytosterols contained. By the chromatography and optional activated carbon filtration during the second step, the remaining components are separated and squalene with high, pharmaceutical purity is obtained.

The term diafiltration or diananofiltration describes a membrane-based process in which the solvent and, depending on the application, some of the ingredients of a solution or suspension are exchanged. The starting composition, here squalene comprising composition having a lower squalene content, whose further components, especially oily components, are to be exchanged, is circulated from a feed vessel. By regulating the flow after the membrane, in particular after the membrane apparatus, a transmembrane pressure (TMP) is set, which acts as a driving force in the diafiltration. The molecular wheigt cut off (MWCO) of the membrane is chosen so that the molecules, here squalene, to be removed can pass through the membrane. Since the system is operated as a closed system, the discharge of the permeate results in reflux of the exchange squalene comprising composition, which keeps the circulating volume constant during diafiltration. Once the same volume that was initially introduced has left the system as permeate and has been replaced by new squalene comprising composition, a so-called diafiltration volume has been achieved.

Chromatography is a chemical-physical technique for a separation of a mixture into its components. Typically, a mixture comprising the mentioned components is dissolved in a fluid solvent, e.g., in a fluid-extraction, in particular gas, according to the invention in CO2 or in a solvent as liquid-liquid- extraction. According to the invention in particular in a liquid hydrocarbon, preferred in liquid alkanes, the fluid solvent or liquid hydrocarbon is called the mobile phase. The mobile phase carries the components through a system, like a column, a capillary tube, a plate, or a sheet, on which a material called the stationary phase is fixed or comprises the stationary phase. As the different components of the mixture tend to have different affinities to the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites of the stationary phase, the components pass the stationary phase at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

It is a further embodiment of the invention that the ii) purification step may comprise a membrane filtration step, in particular a membrane based diafiltration step. It is further preferred that the membrane is a silicone-based membrane, like polydimethylsiloxane, 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. As there is no standard procedure for the determination of MWCO, this value maybe obtained differently. In this specific case, the rejection R of polystyrenes having different molecular weights (solutes) in toluene (solvent) can be considered. Herein, Ri is the rejection of a polystyrene of a certain molecular weight, c_P,i is the permeate concentration and CR is the retentate concentration of this polystyrene.

The molecular weight cut-off is then described by the molecular weight of the polystyrene which shows a rejection R of 0.9.

According to an alternative embodiment the extraction step may comprise a liquid-liquid extraction step, wherein the extraction agent is at least one liquid hydrocarbon and/or a mixture comprising at least two liquid hydrocarbons, in particular at least a liquid alkane. The alkane or alkanes can be selected from linear alkanes, branched alkanes and/or cyclic alkanes, in particular the alkane is selected from alkanes comprising pentane, hexane, heptane, octane, nonane, decane, undecane and dodecane and as well as all of their isomers, most preferred are n-hexane, n-heptane, cyclohexane, cycloheptane and/or mixtures thereof. In an alternative the extraction step may comprise a fluid-extraction, wherein the extraction agent is CO2. Liquid alkanes mean liquid at a temperature below 100 °C, in particular liquid at room temperature and 20 hPa.

According to a preferred embodiment the purification step, in particularthe extraction step, comprises a saponification step prior to the extraction step, preferred the liquid-liquid extraction step. It is preferred that the saponification step comprises a) that the squalene comprising composition having a lower squalene content is contacted with an alkaline, in particular an alkaline solution in an organic solvent, in particular an alkaline solution of an alcohol, e.g., methanol, ethanol, propanol, and b) a mixture is obtained. And optionally, wherein in step c) the in b) obtained mixture is treated at elevated temperature, in particular the mixture is stirred and/or heated to a temperature in the range of 30 to 80 °C, in particular from 50 to 70 °C, more preferred from 50 °C.

And optionally, in step d) the mixture may be cooled down and in step e) water is added, in particular distilled water. Afterwards, 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 from 50 to 70 °C, more preferred from 50 °C, and the mixture and water are treated with the extraction agent and a resulting mixture is obtained. Optionally, the g) resulting mixture is allowed to separate into a water phase and an organic phase comprising the extraction agent and squalene, the two phases are separated and optionally the water phase is again treated with the extraction agent and optionally step g) is repeated, and in particular organic phases comprising the extraction agent and squalene are obtained. Preferably, h) the obtained organic phases comprising the extraction agent and squalene are combined and optionally contacted with water, in particular with distilled water, i) the resulting mixture is allowed to separate into a water phase and an organic phase comprising the extraction agent and squalene. Optionally, the j) this organic phase comprising the extraction agent and squalene is again contacted with water, in particular with distilled water, k) the resulting mixture is allowed to separate into a water phase and an organic phase comprising the extraction agent and squalene. Further, I) the organic phase comprising the extraction agent and squalene obtained in step i) and/or k) is treated under vacuum and/or elevated temperature to remove the extraction agent and the intermediate composition with a squalene content is obtained.

Optionally, as an alternative in step I) the organic phase comprising the extraction agent, in particular with a reduced content of extraction agent, m) is contacted with an adsorbent, in particular contacted with an adsorbent and stirred, and n) the adsorbent is removed from this organic phase comprising the extraction agent, in particular the adsorbent is filtered off. And following, o) the organic phase comprising the extraction agent is treated under vacuum and/or elevated temperature to remove the extraction agent and the intermediate composition with a squalene content is obtained. A preferred adsorbent is diatomaceous earth or activated charcoal. In a particular preferred embodiment of the invention steps a) to k) are performed consecutively or the steps a) to o) are performed consecutively.

The treatment under vacuum and/or elevated temperature may comprise treatment at a pressure from 0.1 mbar to below 1 bar, preferred in the range from 10 mbar to 600 mbar, more preferred from 100 mbar to 300 bar and in particular at a temperature in the range from room temperature to 60 °C, preferred in the range from 30 to 50 °C.

According to the invention the chromatography step may be performed with a stationary phase comprising silica, alumina, diol functionalised polymers or diol functionalised silica and/or hydroxyl apatite, in particularthe stationary phase is selected from silica, alumina, diol functionalised polymers or diol functionalised silica and/or hydroxyl apatite. In particular, the chromatography step may be performed with the intermediate composition with a squalene content and optionally with the addition of a mobile phase, and after evaporation of the mobile phase a squalene composition having the enriched squalene content may be obtained. It is particular, preferred that in iii) performing a chromatography step with the intermediate composition with a squalene content, the step is performed with a mobile phase, in particular with the intermediate composition and a mobile phase. Therefore, iii) may be performed with the intermediate composition and an added mobile phase.

It is further an object of the invention that the chromatography step may be performed with a mobile phase comprising at least one liquid hydrocarbon, in particular at least one liquid alkane, preferred the at least one or more alkanes are selected from alkanes comprising pentane, hexane, heptane, octane, nonane, decane, undecane and dodecane, most preferred is cyclohexane. Optionally in addition or as an alternative the mobile phase can also be selected from a fluid solvent such as supercritical CO2.

Preferred the liquid phase comprises the composition comprising the intermediate composition with a squalene content and a liquid alkane, in particular as mobile phase, wherein the content of the intermediate composition with a squalene content is from 1 to 80 wt.-%, preferably from 5 to 30 wt.- % and the remaining is a mobile phase, in particular at least one liquid alkane, up to 100 wt.-% of the mobile phase. The liquid alkane is in particular cyclohexane. The liquid alkanes may comprise the above-mentioned liquid alkanes and or mixtures of at least two of them. Most preferred is cyclohexane, n-hexane, n-heptane, cycloheptane and/or mixtures thereof.

It is a further object of the invention that, after the iii) chromatography step at least one additional 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 mixtures of at least two of them.

According to a further embodiment of the process the a) the squalene comprising composition having a lower squalene content may have a squalene content of below 12 wt.-% of squalene, in particular from 0.1 wt.-% to equal or below 10 wt.-%, preferably from 5 wt.-% to equal or below 10 wt.-%, wherein the composition sums up to 100 wt.-%, and/or b) the intermediate composition with a squalene content may have a squalene content of at least 14 wt.-% squalene, in particular equal or above 15 wt.-% to 90 wt.-%, preferably equal or above 15 wt.- % to 40 wt.-%, wherein the intermediate composition sums up to 100 wt.-%, and/or c) the squalene composition having the enriched squalene content, in particular a pharmaceutical squalene composition, may have a squalene content of equal or above 91 wt.-%, in particular equal or above 97 wt.-% to 100 wt.-%, preferably a squalene content equal or above 97 wt.-% to 99.99 wt.- %, wherein the composition sums up to 100 wt.-%.

Another embodiment of the invention may comprise the diafiltration step, in particular the diafiltration step may comprise a membrane filtration step, and the membrane filtration step may be performed in a system comprising a feed vessel and at least one diafiltration apparatus, which is supplied with a feed, wherein the feed is the squalene comprising composition having a lower squalene content, in particular wherein the diafiltration apparatus comprises a membrane, preferred a nanofiltrationmembrane, and wherein from the at least one diafiltration apparatus a retentate, in particular at least one retentate, is obtained which is recycled to the feed vessel and from the at least one diafiltration apparatus a permeate, in particular at least one permeate, is obtained, wherein the permeate is the intermediate composition with a squalene content. The diafiltration step is preferably performed continuously in a closed system. The feed vessel possesses preferably 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, like polydimethylsiloxane, 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.

Preferred is that at least one of the steps of the process of the invention is performed under an inert atmosphere, in particular under nitrogen, argon and/or a mixture of both. Particular preferred the diafiltration step is performed under inert atmosphere.

Wherein the diafiltration apparatus is preferably supplied with a feed having an elevated temperature, in particular the feed, in particular the squalene comprising composition having a lower squalene content, having a temperature from 25 °C to below boiling temperature, in particular from 25 to 90 °C, preferred from 30°C to 70°C, most preferred from 30 to 60 °C or 50°C, and/or the feed is supplied under a pressure equal or above 1 bar to 100 bar, in particular above 20 bar, more preferred above 50 bar. The pressure in the diafiltration apparatus, in particular the pressure lay on the at least one membrane, is in the range from 10 to 100 bar, preferred in the range from 30 to 80 bar, more preferred from 50 to 70 bar, most preferred about 60 bar with +/- 5 bar.

In a preferred embodiment the at least one permeate obtained from the at least one diafiltration apparatus comprises at least three diafiltration apparatuses that are combined after another in a series connection or are combined parallel in a series connection. In case the diafiltration apparatuses are combined after another the first permeate resulting from the first diafiltration apparatus may be fed into the second diafiltration apparatus, wherein the permeate resulting from the second diafiltration apparatus is the intermediate composition with a squalene content. The retentates form the at least two diafiltration apparatuses, in particularthe first and second diafiltration, are fed into the feed vessel. The diafiltration step is preferably performed continuously in a closed system. The apparatus and/or the process may be performed with 1 or 2 to 100 diafiltration apparatuses.

It is a further embodiment of the invention that the squalene comprising composition having a lower squalene content may be in a particular preferred embodiment a plant-based squalene comprising composition, preferred are plant-based squalene comprising compositions with a squalene content from 1 to 14 wt.-%, preferred from 2 to 12 wt.-%, more preferred from 5 to 10 wt.-%. Most preferred as the squalene comprising composition having a lower squalene content are plant-based oils. Plantbased oils that can be used comprise soybean oil, rice bran oil, olive oil, vegetable oils, in particular distillates of vegetable oils, coffee oil, wheat germ oil, corn germ oil, palm oil, andiroba oil, oil from tomato residues and amaranth oil. Most preferred is amaranth oil, in particular amaranth oil with a squalene content from 1 to 10 wt.-%, more preferred from 5 to 9 wt.-%.

In an alternative embodiment of the invention a squalene composition can be used having a squalene content of up to 25 wt.-%. However, in order to obtain squalene compositions having a squalene content of 12 up to 25 wt.-%, the squalene composition needs to be enriched beforehand with an additional process step not claimed in the present application, thus contents of below 12 wt.-% are preferred.

Unless otherwise indicated, any percentages stated herein are percent by weight.

In addition, a squalene composition having an enriched squalene content prepared according to the process of the invention is an embodiment of the invention. The squalene composition is preferably a pharmaceutical squalene composition. Furthermore, an embodiment of the invention is also a pharmaceutical composition comprising a squalene composition, preferred is a pharmaceutical squalene composition, in particular a parenteral composition, more preferred a vaccine. Moreover, it is an embodiment of the invention to furnish an oil-in water emulsion comprising a squalene composition as described above.

The method is preferably performed in an apparatus that may comprise at least one feed vessel and at least one diafiltration apparatus, in particular for performing step ii) of the process, in particular the diafiltration apparatus may comprise a membrane, preferred a nanofiltration-membrane, more preferred a flat nanofiltration-membrane, wherein the at least one diafiltration apparatus is suppliable with a feed, wherein the feed is the squalene comprising composition having a lower squalene content, in particular according to the process of the invention, and wherein from the at least one diafiltration apparatus a retentate is obtainable which is recyclable to the feed vessel and a permeate is obtainable, wherein the permeate is the intermediate composition with a squalene content. The flat membrane may have an area in one plane from 1 cm² to 1 m², preferred from 10 cm² to 100 cm².

The apparatus is preferably a closed system, wherein in the feed vessel the composition having a lower squalene content is supplied and from the at least one diafiltration apparatus a permeate is obtainable, wherein the permeate is the intermediate composition with a squalene content. Wherein the squalene content is preferred from 10 to 80 wt.-%, more preferred from 20 to 80 wt.-%. The feed vessel possesses preferably a pressure of at least 100 mbar at room temperature, which is obtained by using an inert atmosphere, preferably nitrogen.

Examples

Measurement Techniques and Apparatus:

Gas chromatography:

All percentages quoted herein are percentages by area % and may be measured using gas chromatography (GC). A GC technique may be conducted by injecting a sample of squalene in n- heptane or hexane onto a gas chromatograph equipped with a flame ionization detector (FID). The analysis was performed on a 30 m x 0.32 mm x 0.50 mm capillary column maintained at 380°C for 2 minutes and then ramped at 12°C per minute to 310°C, where it is held for 9 minutes. The injection port and the FID are maintained at 300°C and 320°C respectively. Identity of the squalene peak is established using GC/MS (gas chromatography using a mass selective detector). Purity is reported as the area of the squalene peak as a percentage of the sum of the areas of all the peaks in the chromatogram.

Retention time of squalene ca. 19 to 19.80 min, sample: 20.8 mg + 1 mL n-heptane

Type of gas chromatograph: Agilent GC 7890

Type of injector: Split/ split less for capillary columns

Column: capillary column Restek (30 m)

Temp.: 380 °C

Carrier gas: He 4.6

Flash chromatography:

Type of chromatograph: Biichi C-815

Column: Biotage SNAP KP-Sil, Flow rate: 30 mL/min or see examples, Equilibration: 15-25 min (15 mL/min), Run length: 11.0 min, Temp.: RT Detectors: UV1 : 208 nm, UV2 : 220 nm, UV3 : 254 nm, UV4 : 320 nm, UV scan 200 to 800 nm.

Content of squalene according to gas chromatography:

Amaranth-oil (educt, squalene content 5 to 10 wt.-%): GC analysis: 6.1 area%

Amaranth-oil enriched (squalene content 15 to 40 wt.-%): GC analysis: 20.2 area% squalene isolated (squalene content 97 to 99 wt.-%): GC analysis: 99.8 area%

Squalene-molecule: Boiling point: 275 °C at 20 hPa

Example 1 : Flash Chromatography of amaranth-oil

10 g of amaranth oil (15 wt.-%) was chromatographed over a 50 g Silica gel cartridge (Silica 60A, amorphous (irregular), Average Particle Size 50 micron). The cartridge was passivated with ethyl acetate for 6.47 min. Equilibration was performed with cyclohexane for 19.4 min. A 15 wt.-%liquid was prepared from 10 g of amaranth-oil. Flash chromatography was performed with 66.6 mL liquid and the liquid was injected with a pump (flow: 15 mL/min). The pump was previously subjected with the liquid at 50 mL/min. Fractions after ca. 6.7 min to 8.7 min were collected and combined. Yield: 64 %, 273.2 mg, GC: 98.1 area% Example 2: Nanofiltration

The nano-filtration was performed as described below according to figure 2.

Example 3A: Chromatography after diafiltration with a nanofiltration-membrane

6 g of permeate obtained as permeate 2 was prepared as a 25 wt.-% of a squalene solution in cyclohexane and was chromatographed at 30 mL/min (Biichi Pure C-815, 50 mL syringe, UV detectors as mentioned below) over a cartridge (Biotage SNAP KP-Sil 50 g). The cartridge was conditioned with ethyl acetate for 6.47 min at a flow of 15 mL/min, followed by cyclohexane washing for 22.64 min at 15 mL/min. 30 mL of the 25 wt.-% squalene solution (permeate 2) was applied with the syringe onto the cartridge.

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

Example 3B: Chromatography after diafiltration with a nanofiltration-membrane

10 g permeate of amaranth oil 15 wt.-% was chromatographed at 30 mL/min. to 10 g amaranth oil 66.6 g of cyclohexane were added and stirred with a magnetic stir. A clear, yellow solution was obtained of ca. 84 mL.

Flash chromatography (Biichi C-815, cartridge: Biotage SNAP KP-Sil 50 g, UV detectors as mentioned below). Passivation was performed with 2 BV (bedvolume) ethyl acetate for 6.47 min (Flow 30 mL/min) and equilibration was performed with 6.99 BV cyclohexane.

Fractions from 3.4 min to 4.9 min were combined and evaporated at the rotary evaporator (40 °C, 130 mbar) to obtain an oily squalene composition of 97.1 area% squalene (GC). Product 0.93 g slightly yellow oil.

Example 4A: Chromatography after extraction (chem. enriched)

Flash chromatography of 40.8 g of 25 wt.-% crude amaranth oil over 340 g cartridge (Biotage SNAP KP-Sil 340 g, Biichi C-815) was equilibrated with cyclohexane (3.5 BV) at a flow of 80 mL/min. 40.8 g with 122.4 g cyclohexane were chromatographed as 25 wt.-% solution over 2.3 min at a flow of 80 mL/min. To obtain a chromatogram with the Biichi apparatus the flow was adjusted to 1 mL/min from 2.2 min to 4.5 min. Before 2.2 and after 4.5 the flow was 80 mL/min. Three fractions from 8.76 min to 14.71 min were collected and analysed by GC. Fraction 4 was evaporated at 40 °C under reduced pressure at the rotary evaporator. Product: 20.08 g, GC (21.4 mg product + 1 mL n-heptane): 98.2 area% squalene.

Example 4B: Extraction

250.0 mL methanol (techn. grade) was subjected into a 1 litre flask at room temperature and stirred. 12.5 g NaOH were added at room temperature and stirred until NaOH was dissolved. The solution was allowed to cool to room temperature and 50 g amaranth oil (Stubener Krautergarten), was added. 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 demineralized water was added, followed by stirring for 15 min. The mixture was heated to 50 to 55 °C and extracted with 500 mL hexane, optionally GC analytic. The hexane phase was separated and the water phase was extracted at 50 to 55 °C with 500 mL hexane. The hexane phase was separated and combined with the first hexane phase at 50 to 55 °C and washed with demineralized 150 mL water. The hexane from the separated hexane phase was removed in a rotary evaporator at 45 °C at 300 mbar to 10 mbar (Crude product: 3.51 g).

To the obtained crude product 250 mL hexane and celite (diatomaceous earth) were added. Optionally a GC analytic may be performed before the addition of celite. Before the addition of celite a clear yellow solution was obtained. The solution was stirred for 45 min and filtered off with a glass frit which was washed with 30 mL hexane. Hexane was removed on the rotary evaporator at 45 °C and 300 mbar to 10 mbar. 3.71 g product was obtained (orange, greasy solid), GC analysis: 81.0 area% squalene, ¹H-NMR (600 MHz, CDCb): 75.97 wt.-% squalene. ¹H-NMR (600 MHz, CDCb) analysis: 22.43 mg squalene and 19.49 mg dimethylterephtalate)

Chromatography

The obtained product (3.71 g product) was dissolved in 25 mL n-hexane and stirred for 30 min. The solvent was evaporated at the rotary evaporator and a creamy solid obtained which was dissolved in 25 mL cyclohexane and stirred for 30 min with 2.5 g silica (Porocell silica). The 25 wt.-% solution was filtered over a strainer (No. 4) and washed with cyclohexane. The filtrate was evaporated with a rotary evaporator and a clear, yellow solution obtained (oily). From this oil a 25 wt.-% solution was prepared with 25 mL n-hexane and furnished onto the cartridge (Biotage SNAP KP-Sil 340 g. The cartridge was equilibrated with 3.5 BV cyclohexane for 22.6 min. The probe was injected with 14 mL over 1 :05 min. From the obtained fractions a 1 mL GC sample was analysed. The product is a transparent colourless liquid of 1 .85 g squalene (GC: 99.8 area%).

Example 5: Chromatography (30 mL/min) of enriched amaranth oil (Flavex amaranth seed, 15 wt.-%) CO2 extracted

3 g Flavex was mixed with 17 g cylohexane to obtain a yellow solution of 25 wt.-%. The mixture was stirred for 10 minutes with a magnetic stirrer. A slight yellow clear solution was obtained.

The cartridge was equilibrated with 3.5 BV cyclohexane for 22.6 min. Following the above obtained solution was injected to 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)

Description of Figures

Fig. 1 describes one alternative of the process disclosing the possible squalene content enrichment in the steps of the process.

Fig. 2 describes an apparatus 1 comprising the diafiltration apparatus 3.

Fig. 1 is a schematic overview of the process starting form a plant-base amaranth-oil with a typical squalene content from 5 to 10 wt.-% in the composition amounting to 100 wt.-%. Afterthe diafiltration step performed as nanofiltration step an intermediate composition with a squalene content of 15 to 40 wt.-% is obtained, wherein the composition amounts to

100 wt.-%. This composition is subjected to a chromatography step, wherein the squalene composition having an enriched squalene content is obtained after the removal of the mobile phase. As an optional step the composition comprising the mobile phase or a part of the mobile phase may be contacted with an adsorbent, such as activated carbon, the step can be performed as a filtration step over activated carbon.

Fig. 2 describes the apparatus 1 comprising the diafiltration apparatus 3 for nano-filtration.

Example experiment- Part 1 - Enrichment via diafiltration apparatus 3 as nanofiltration

The tests were carried out in a continuously operating apparatus 1 see Fig. 2 in a closed circuit or with 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 a section of a flat membrane in the diafiltration apparatus 3. The feed vessel 2 filled with amaranth oil (2 L) is overflowed with nitrogen and a slight overpressure due to the nitrogen of about 100 mbar is maintained. From the feed vessel 2, the feed enters the high-pressure circuit 4 by means of a piston diaphragm pump, which is used for feeding the system. The pressure of the circuit is regulated by means of an adjustable pre-pressure regulator up to 50 bar. In this circuit there is a flat channel test cell, in which the flat membrane to be tested with a flatness of 84.5 cm² is installed before the start of the test. The built-in membrane (Silicone-coated PAN (Polyacrylnitril) PuraMem® Flux, Molecular Weight Cut off 280 to 600 g/mol) is overflowed in the circuit by means of a gear pump and the temperature of the circuit (30 to 60 °C) is controlled by a heat exchanger connected to an external thermostat and/or cryostat. In addition, the apparatus 1 contains corresponding sensors such as pressure measurement and temperature measurement in the circuit 4. There are also possibilities of sampling in the feed, retentate and permeate to determine the selectivity of the membrane. The permeates are obtained in the flasks 5.

Both the permeate (oil enriched with squalene of about 10 to 50 wt.-%), the current which permeates through the membrane, as well as the retentate, namely the excess portion of the feed of the piston diaphragm pump minus the permeate, which is relaxed again via the pre-pressure regulator, are driven back into the feed vessel in normal operation. The components travel through the system in a closed circuit, so that constant conditions at the membrane are set for the most practicable measurement. The permeate mass flow is determined selectively indirectly by balancing. From this the permeance of the membrane can be obtained by dividing with the applied transmembrane pressure, which is the difference of the average pressure on the feed-side of the membrane ((PFeed+pRetentate)/2) and the pressure on the permeate side of the membrane (ppermeate) and the installed membrane area.

Example Experiment - Part 2 - Chromatographic purification of enriched oil

The intermediate composition with a squalene content of about 10 to 50 wt.-% in vessel is diluted with cyclohexane, wherein a 25 wt.-% solution is prepared in a downstream vessel with stir. An apparatus for chromatography comprising at least one cartridge (SNAP KP-Sil 50 g) and a Biichi Pure C-815 flash system are used for chromatographic purification. The cartridge is preconditioned with ethyl acetate (15 mL/min) and washed with cyclohexane (22 min at 15mL/min). The sample submission is carried out with a 50 ml syringe. 30 ml samples of the 25% solution are applied to the chromatography column. The column is used with 30 mL/min cyclohexane over the filled column and the main fractions are collected in flasks. The main fractions are completely concentrated at a rotary evaporator at 40 °C bath temperature and preferably under reduced pressure and squalene with high pharmaceutical purity is preserved (measured with Agilent GC 7890 as described above). Optionally, an additional step may comprise contacting the squalene composition with enriched squalene content and the mobile phase or at least a part of the mobile phase in a vessel with an adsorbent like activated charcoal and obtaining after removal of the mobile phase the composition with enriched squalene content, in particular a pharmaceutical squalene composition in flask.

Claims

1 . Process for the production of a squalene composition having an enriched squalene content, form a squalene comprising composition having a lower squalene content, comprising the steps of i) providing a composition comprising squalene having a lower squalene content, ii) subjecting the squalene comprising composition having a lower squalene content to a purification step at a temperature below the boiling point of squalene, and obtaining an intermediate composition with a squalene content, and iii) performing a chromatography step with the intermediate composition with a squalene content, and iv) obtaining a squalene composition having the enriched squalene content.

2. Process according to claim 1 , characterised in that, the squalene composition having an enriched squalene content is a pharmaceutical squalene composition.

3. Process according to claim 1 or 2, characterised in that, the ii) purification step comprises a diafiltration step and/or an extraction step, in particular the extraction step is a liquid-liquid extraction step or the extraction step comprising a fluid-extraction, wherein the extraction agent is CO2.

4. Process according to any of claims 1 to 3, characterised in that, the ii) purification step comprises a membrane filtration step, in particular a membrane based diafiltration step.

5. Process according to claim 4, characterised in that, the membrane filtration step is performed with a silicon-based membrane and/or a membrane having a molecular weight cut-off (MWCO) of 200 to 700 g/mol.

6. Process according to claim 3, characterised in that, the extraction step comprising a liquid-liquid extraction step, wherein the extraction agent is a liquid hydrocarbon, in particular a liquid alkane, in particular the alkane is selected from alkanes comprising pentane, hexane, heptane, octane, nonane, decane, undecane and dodecane, most preferred are n-hexane, cyclohexane and/or mixtures thereof.

7. Process according to any of claims 1 to 6, characterised in that, the chromatography step is performed with a stationary phase comprising silica, alumina, diol functionalised polymers or diol functionalised silica and/or hydroxyl apatite, in particular the stationary phase is selected from silica.

8. Process according to any of claims 1 to 7, characterised in that, the chromatography step is performed with a mobile phase comprising a liquid hydrocarbon, in particular a liquid alkane, preferred the alkane is selected from alkanes comprising pentane, hexane, heptane, octane, nonane, decane, undecane and dodecane, most preferred is cyclohexane.

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

10. Process according to any of claims 1 to 9, characterised in that a) the squalene comprising composition having a lower squalene content having a squalene content of below 25 wt.-%, preferably below 12 wt.-% of squalene, in particular from 0.1 wt.-% to equal or below 10 wt.-%, preferably from 5 wt.-% to equal or below 10 wt.-%, wherein the composition sums up to 100 wt.-%, and/or b) the intermediate composition with a squalene content having a squalene content of at least

14 wt.-% squalene, in particular equal or above 15 wt.-% to 90 wt.-%, preferably equal or above

15 wt.-% to 40 wt.-% or 25 wt.-% to 40 wt.-%, wherein the intermediate composition sums up to 100 wt.-%, and/or c) the squalene composition having the enriched squalene content, in particular a squalene content of equal or above 91 wt.-%, wherein the composition sums up to 100 wt.-%.

11. Process according to any of claims 3 to 10, characterised in that, the diafiltration step, in particular the diafiltration step comprising a membrane filtration step, is performed in a reaction system comprising a feed vessel and at least one diafiltration apparatus, which is supplied with a feed, wherein the feed is the squalene comprising composition having a lower squalene content, in particular wherein the diafiltration apparatus comprises a membrane, preferred a nanofiltration-membrane, and wherein from the at least one diafiltration apparatus a retentate is obtained which is recycled to the feed vessel and from the at least one diafiltration apparatus a permeate is obtained, preferably wherein the permeate is the intermediate composition with a squalene content.

12. Process according to any of claims 1 to 11 , characterised in that, the squalene comprising composition having a lower squalene content is a plant-based squalene comprising composition, preferred are plant-based squalene comprising compositions with a squalene content from 1 to 14 wt.-%, preferred from 2 to 12 wt.-%, more preferred from 5 to 10 wt.-%, most preferred the squalene comprising composition having a lower squalene content is amaranth-oil.

13. Squalene composition having an enriched squalene content prepared according to any of the claims 1 to 12, in particular of a pharmaceutical squalene composition.

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

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