Chromatographic method for producing polyunsaturated fatty acids

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 1 CHROMATOGRAPHIC PROCESS FOR THE PRODUCTION OF POLYUNSATURATED FATTY ACIDS FIELD OF THE INVENTION The present invention relates to a chromatographic process for producing polyunsaturated fatty acids, such eicosapentaenoic acid, as well as an installation suitable for carrying out this process.

TECHNICAL BACKGROUND Fatty acids, including polyunsaturated fatty acids (abbreviated as PUFA), are particularly important biological compounds because they are involved in numerous biological processes such as the construction and maintenance of cell membranes, the synthesis of hormones ( e.g. prostaglandins) which play a role in platelet aggregation, inflammatory processes and immunological response, etc.

Most PUFAs can be synthesized by a human body, with the exception of two families of PUFAs which must be provided by food, called essential fatty acids.

The two families of essential fatty acids are:

¨ omega-6, which is particularly abundant in walnut, sunflower, soy, grapeseed or corn oils and fatty poultry (such as duck);

¨ omega-3 which are mainly present in nut oils, in vegetables such as rapeseed and flax and in fatty fish (such as salmon, tuna, sardines, mackerel or herring).

Processes for the production of omega-3 using cultures of microalgae, transgenic yeasts or krill have recently been developed.

Omega-3s are particularly interesting PUFAs for their antioxidant properties.

Among these omega-3s, purified EPA (eicosapentaenoic acid, CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 2 C20-5w3) and DHA (docosahexaenoic acid, C22-6w3) and their enriched combinations are the most used as food supplements or drugs to reduce triglyceride levels, cardiovascular risks, improve cognition or vision, etc.

Recent clinical studies have shown that treatment of patients with triglyceride levels greater than 500 ml/dL with 4 grams per day of 96% EPA ethyl ester without DHA lowers triglyceride levels without causing increase LDL (bad cholesterol) levels, whereas treatment with 4 grams per day of a mixture of ethyl esters of EPA and DHA, approximately 50% and 35% respectively, led to an increase in LDL levels concomitant with the decrease in triglycerides.

Until now, the PUFA food supplements used, in particular omega-3s, are essentially based on mixtures containing 30 to 60% of the mixture of EPA and DHA.

In the separation methods used to date, the mixture is obtained by transesterification of the triglycerides into ethyl esters then by an enrichment of the omega-3s by molecular distillation and/or co-crystallization of the saturated and monounsaturated fatty acids with urea.

The enriched ethyl esters are optionally reconverted into triglycerides chemically or preferably enzymatically.

However, these separation processes are not satisfactory for the production of an omega-3 such as EPA, DHA or even stearidonic acid (SDA, C18-Sw3) at more than 80%, or even at more of 96%, in particular in esterified form.

However, the purification of omega-3s is delicate because these compounds contain several carbon-carbon double bonds which make them sensitive to oxidation or degradation.

In the presence of oxygen and when they are heated, these PUFAs notably undergo isomerization, oxidation, peroxidation and oligomerization reactions.

Thus, the separation techniques stated above make it possible to obtain a mixture of PUFAs with a good yield and an acceptable degree of purity; but they cannot be used for the individual separation of PUFAs.

They therefore do not make it possible to separate omega 3s from each other.

Indeed, molecular distillation, for example, cannot economically remove DHA from EPA or SDA; it does not allow an efficient separation of long-chain omega-3s of the C20 and C22 type.

The combination of urea clathration and molecular distillation allows CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 3 to obtain blends of omega-3s of higher purity, at the cost of 'a generally low yield and a high operating cost, but cannot be used for the separation of long-chain omega-3s from each other, and from EPA and DHA in particular.

There is therefore a need to provide an industrial purification process for omega-3 in esterified form with very high purity.

Chromatography is a fine separation technique allowing the efficient purification or enrichment of molecules under mild conditions and away from light and air.

This technology is based on the separation of molecules which are brought into contact with a stationary phase with which they have different interactions. The use of one or more fluids, called mobile phases or eluents, allows the percolation of different molecules at different speeds.

These different speeds make it possible to physically separate the molecules and to collect them in purified form at the end of chromatographic processes with one or more columns.

The purified fractions are generally concentrated, under mild conditions at ambient or moderate temperature, by means such as vacuum evaporation or membrane processes.

In some cases, the starting product for chromatographic purification is an oil composed of fatty acid esters already enriched by molecular distillation, preferably comprising more than 30% of the omega-3 of interest, which has undergone treatment for eliminating the oxidized compounds, either by the last molecular distillation, or by adsorption, preferably on silica derivatives (silica gel, bentonite, diatomaceous earth) or on activated carbon for example.

Numerous documents describe the elimination of oxidized compounds in compositions derived from oils.

By way of example, document EP 0682006 describes a treatment of oils containing omega-3s by dilution in hexane and addition of 10 to 40% by weight of activated carbon.

Document US Pat. No. 4,874,629 describes a treatment of oils containing omega-3s by steam distillation followed by adsorption of the polar compounds on silica.

Document WO 2005/049772 also describes the treatment of an oil rich in omega-3 by dissolution in an aprotic solvent and contact with a silicon derivative.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 4 Document EP 0773283 describes the treatment of an oil containing a PUFA of at least 18 carbons by contact for at least 10 minutes with at least 0.1% in weight of diatomaceous earth previously treated in an acid medium at a temperature of 5 to 80 C, followed or not by steam distillation.

A certain number of chromatographic processes have moreover been described, for obtaining omega-3 with high purity.

Thus, document US 5,719,302 describes a process in which the PUFAs are separated in particular using a supercritical eluent (carbon dioxide under pressure), and in particular on a simulated moving bed (SMB for Simulated Moving Bed according to the English terminology). Saxon).

Document US 2011/0091947 describes another omega-3 purification process using the technique of simulated moving bed chromatography.

The document describes in particular the succession of an enzymatic transesterification step, two molecular distillation steps, and a step of the SMB type, these last three steps make it possible to separate the products into two fractions in order of retention time.

Document WO 2011/080503 describes the purification of omega-3 from a device comprising two SMB chromatographic devices arranged in series and a washing zone, each SMB chromatographic device defining a separation zone and consisting of several columns.

The feed to be treated is injected into a first separation zone to obtain an extract stream and a raffinate stream, said raffinate stream comprising the compounds of interest then being injected into a column of the second separation zone not adjacent to a column from the first area.

Document WO 2013/005051 describes the purification of omega-3 by two chromatographic separations by SMB or AMB (Actual Moving Bed, that is to say real moving bed) in reversed phase with a hydroorganic eluent, in which the two Separations by SMB or AMB are carried out sequentially on the same chromatographic device, or on two different devices, the intermediate purified by the first device being introduced into the second.

Document WO 2013/005048 describes the purification of EPA to more than 90% purity by a first chromatographic separation followed by two chromatographic separations by SMB or AMB in reversed phase with a hydro-organic eluent at each stage, the purified intermediate by the first chromatographic separation being introduced into the second chromatographic separation, and the intermediate purified by the second chromatographic separation being introduced into the third chromatographic separation.

In the two preceding documents, the purified EPA is preferably obtained in the extract of the last separation; thus the last separation 5 separates the EPA from the less retained impurities collected in the raffinate (as is well known to those skilled in the art of reversed-phase chromatography).

Such less retained impurities than EPA can be, for example, shorter chain PUFAs such as SDA, or oxidation compounds such as peroxides or aldehydes.

There is still a need to provide a PUFA (or a derivative, in particular an ester thereof) with high purity, allowing its use in drug compositions, from a multi-compound charge comprising said PUFA.

In particular, there is still a need to provide a PUFA (or a derivative, in particular an ester thereof) essentially devoid of oxygenated contaminants of the peroxide or aldehyde type.

SUMMARY OF THE INVENTION The invention relates firstly to a method for purifying a first polyunsaturated fatty acid from an initial mixture, the initial mixture comprising at least one second fatty acid in addition to the first polyunsaturated fatty acid, the process comprising:

¨ at least one stage of chromatographic separation in liquid phase of the first polyunsaturated fatty acid and the second fatty acid, making it possible to recover on the one hand a flow enriched in first polyunsaturated fatty acid and on the other hand a flow enriched in second fatty acid;

¨ a step of processing the stream enriched in the first polyunsaturated fatty acid, leading to a reduction in the peroxide index and/or the anisidine index of this stream.

According to one embodiment, the treatment step is a molecular distillation step.

According to one embodiment, the treatment step is a step of bringing into contact with an adsorption substrate, which is preferably chosen from silica, alumina, activated carbon, diatomaceous earth, clays and in particular bentonite, and derivatives thereof; said bringing into contact being preferably carried out without diluting the first polyunsaturated fatty acid and without adding a solvent.

CA 02932928 2016-06-03 WO 2015/086672 PCT / EP2014/077209 6 According to one embodiment, the flux enriched in first unsaturated fatty acid has, after the treatment step, a peroxide number less than or equal to 10, preferably less than or equal to 5, or less than or equal to 2, or less than or equal to 1; and/or an anisidine number less than or equal to 20, preferably less than or equal to 10, or less than or equal to 5, or less than or equal to 3; and/or the stream enriched in first unsaturated fatty acid has, before the treatment step, a peroxide number greater than or equal to 1, preferably greater than or equal to 2, or greater than or equal to 4, or greater than or equal to 6, and/or an anisidine number greater than or equal to 1, preferably greater than or equal to 2, or greater than or equal to 4, or greater than or equal to 6.

According to one embodiment:

¨ the first polyunsaturated fatty acid is eicosapentaenoic acid, and is preferably recovered at the end of the process with a purity greater than or equal to 80%, or 90%, or 96%; or - the first polyunsaturated fatty acid is docosahexaenoic acid, and is preferably recovered at the end of the process with a purity greater than or equal to 70%, or 80%, or 90%, or 95%; or ¨ the first polyunsaturated fatty acid is arachidonic acid, and is preferably recovered at the end of the process with a purity greater than or equal to 70%, or 80%, or 90%, or 95%; or ¨ the first polyunsaturated fatty acid is docosapentaenoic acid, and is preferably recovered at the end of the process with a purity greater than or equal to 70%, or 80%, or 90%, or 95%.

According to one embodiment, the stage of chromatographic separation of the first polyunsaturated fatty acid and of the second fatty acid is carried out with a hydro-organic eluent.

According to one embodiment, the second fatty acid is a polyunsaturated fatty acid, preferably chosen from docosahexaenoic acid, eicosapentaenoic acid, arachidonic acid, docosapentaenoic acid and stearidonic acid.

According to one embodiment, the initial mixture further comprises a third fatty acid, and the method comprises a liquid phase chromatographic separation step of the first polyunsaturated fatty acid and of the third fatty acid, making it possible to recover on the one hand an enriched flow in the first polyunsaturated fatty acid and on the other hand a flow enriched in third fatty acid; this chromatographic separation step preferably being carried out with a hydro-organic eluent; and this chromatographic separation step preferably being carried out upstream of the step of liquid phase chromatographic separation of the first polyunsaturated fatty acid and the second fatty acid.

According to one embodiment, the initial mixture further comprises a fourth fatty acid, and the method comprises a liquid phase chromatographic separation step of the first polyunsaturated fatty acid and of the fourth fatty acid, making it possible to recover on the one hand an enriched flow first polyunsaturated fatty acid and secondly a flow enriched fourth fatty acid; this chromatographic separation step preferably being carried out with a hydro-organic eluent; and this step of chromatographic separation being preferably carried out upstream of the step of chromatographic separation in the liquid phase of the first polyunsaturated fatty acid and of the second fatty acid.

According to one embodiment:

¨ the first polyunsaturated fatty acid is retained less than the second fatty acid during the stage of chromatographic separation of the first polyunsaturated fatty acid and the second fatty acid; and/or the first polyunsaturated fatty acid is retained less than the third fatty acid during the stage of chromatographic separation of the first polyunsaturated fatty acid and of the third fatty acid; and/or the first polyunsaturated fatty acid is less retained than the fourth fatty acid during the stage of chromatographic separation of the first polyunsaturated fatty acid and of the fourth fatty acid;

¨ preferably, the first polyunsaturated fatty acid is less retained than two fatty acids chosen from the second fatty acid, the third fatty acid and the fourth fatty acid, during the respective chromatographic separation steps of the first polyunsaturated fatty acid and the second, the third and fourth fatty acid;

- more particularly preferably, the method comprises successively (i) the liquid phase chromatographic separation step of the first polyunsaturated fatty acid and the fourth fatty acid, the first polyunsaturated fatty acid being less retained than the fourth fatty acid, then ( ii) the liquid phase chromatographic separation step of the first polyunsaturated fatty acid and the third fatty acid, the first polyunsaturated fatty acid being retained less than the third fatty acid, then (iii) the liquid phase chromatographic separation step of the first polyunsaturated fatty acid and the second fatty acid, the first polyunsaturated fatty acid being retained more than the second fatty acid.

According to one embodiment, the method comprises a preliminary treatment step leading to a reduction in the peroxide index and/or the anisidine index compared to the initial mixture, upstream of the chromatographic separation step(s). in the liquid phase, said preliminary treatment step preferably being a step of molecular distillation, or a step of bringing into contact with an adsorption substrate, chosen in particular from silica, alumina, activated carbon, diatomaceous earth, clays and in particular bentonite, and derivatives thereof.

The invention also relates to an installation for purifying a first polyunsaturated fatty acid from an initial mixture, the installation comprising:

- at least one liquid phase chromatographic separation unit for the first polyunsaturated fatty acid and a second fatty acid, to which are connected at the output, on the one hand, a flow line enriched with the first polyunsaturated fatty acid and, on the other hand, a conduct of flow enriched in second fatty acid;

- at least one processing unit supplied by the flow line enriched with the first polyunsaturated fatty acid, the processing unit being adapted to effect a reduction in the peroxide index and/or the anisidine index.

According to one embodiment, the processing unit is a molecular distillation unit; or is a unit for contacting an adsorption substrate, preferably chosen from silica, alumina, activated carbon, diatomaceous earth, clays and in particular bentonite, and derivatives thereof; said contacting unit preferably comprising no solvent supply.

According to one embodiment, the installation comprises a liquid phase chromatographic separation unit for the first polyunsaturated fatty acid and a third fatty acid, and optionally a liquid phase chromatographic separation unit for the first polyunsaturated fatty acid and a fourth fatty acid, said liquid phase chromatographic separation units being preferably located upstream of the liquid phase chromatographic separation unit of the first polyunsaturated fatty acid and a second fatty acid.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 9 According to one embodiment, at least one liquid phase chromatographic separation unit, preferably all of the liquid phase chromatographic separation units, are chromatographic separation comprising a plurality of chromatographic separation columns, and preferably are simulated moving bed and/or real moving bed chromatographic separation units.

In addition, the invention also relates to the following objects:

1.

A process for purifying a first polyunsaturated fatty acid from an initial mixture of fatty acids, the process comprising lo successively:

¨ the supply of a flux from the initial mixture;

¨ the enrichment of the flow in first polyunsaturated fatty acid in at least one liquid phase chromatographic separation step, said flow having a higher peroxide number and/or an anisidine number after said chromatographic separation step than before said chromatographic separation step;

¨ the treatment of the flux so as to reduce the peroxide index and/or the anisidine index of said flux.

2.

The method according to object 1, in which the treatment step is chosen from among a step of molecular distillation and a step of bringing into contact with an adsorption substrate.

3.

The method according to object 1 or 2, in which the chromatographic separation step is carried out with a hydro-organic eluent.

4.

The method according to one of objects 1 to 3, comprising an enrichment of the flow in the first polyunsaturated fatty acid in at least two liquid phase chromatographic separation steps.

5.

The method according to one of objects 1 to 4, comprising an enrichment of the flow in the first polyunsaturated fatty acid in at least three liquid phase chromatographic separation steps.

6.

The method according to one of objects 1 to 5, successively comprising:

¨ the supply of the initial mixing flow;

¨ the enrichment of the flow in first polyunsaturated fatty acid in a first stage of chromatographic separation in liquid phase, said flow having a peroxide index and/or a higher anisidine index after the first stage of chromatographic separation than before the first stage of chromatographic separation;

CA 02932928 2016-06-03 WO 2015/086672 PCT / EP2014/077209 ¨ the enrichment of the flow in first polyunsaturated fatty acid in a second step of liquid phase chromatographic separation, said flow having a peroxide index and / or an index higher anisidine after the second chromatographic separation step than before the second chromatographic separation step;

¨ the enrichment of the flow in first polyunsaturated fatty acid in a third stage of chromatographic separation in liquid phase, said flow having a peroxide index and/or an anisidine index 10 lower after the third stage of chromatographic separation than before the third stage of chromatographic separation;

¨ the treatment of the flux so as to reduce the peroxide index and/or the anisidine index of the flux.

7.

A process for purifying a first polyunsaturated fatty acid from an oil, the oil having a peroxide index lower than 10 and an anisidine index lower than 15, the method successively comprising:

¨ the supply of an oil flow;

¨ the enrichment of the flow in the first polyunsaturated fatty acid in at least one liquid phase chromatographic separation step;

¨ the treatment of the flux so as to reduce the peroxide index and/or the anisidine index of this flux.

8.

The method according to object 7, in which the treatment step is chosen from among a step of molecular distillation and a step of bringing into contact with an adsorption substrate.

9.

The process according to object 7 or 8, in which the process comprises enriching the stream with the first polyunsaturated fatty acid in a sequence of at least two stages of liquid chromatographic separation, said sequence comprising at least a first stage of chromatographic separation and a final chromatographic separation step.

10.

The process according to one of objects 7 to 9, in which the peroxide index of the stream before the first stage of chromatographic separation in liquid phase is lower than the peroxide index of the stream after the last stage of chromatographic separation in liquid phase liquid.

11.

The method according to one of objects 7 to 10, in which the anisidine number of the flow before the first stage of chromatographic separation CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 11 in the liquid phase is lower than the anisidine number of the stream after the last step of liquid chromatographic separation.

12.

A purified polyunsaturated fatty acid product obtained according to the process of any one of the preceding objects, or according to any other process in accordance with the invention described above, this product containing at least 96% by weight of eicosapentaenoic acid.

The present invention makes it possible to overcome the drawbacks of the state of the art.

It more particularly provides a process for obtaining a PUFA (or derivative, in particular an ester thereof) of high purity and capable of being used in drug compositions, from a multi-component charge comprising said PUFA.

In particular, the PUFA thus obtained has a low level, or is essentially free, of oxygenated contaminants of the peroxide or aldehyde type.

The invention is based on the observation by the inventors that the separation of an omega-3 by one or more stages of liquid phase chromatography, carried out in the absence of light and oxygen, leads surprisingly to a increase in the peroxide index and/or the anisidine index, so that an additional treatment to eliminate the oxidation compounds is necessary, and this even if the last stage of chromatography separates the desired omega-3 from the less retained compounds.

More precisely, each time the compound of interest is less retained than the compounds to be eliminated (sampling with raffinate, within the framework of a continuous process), the peroxide index and/or the anisidine index increase , necessitating an additional treatment to remove the oxidation compounds.

The presence of one or more steps in which the compound of interest is retained more than the compounds to be eliminated (sampling at the extract, within the framework of a continuous process) can reduce the peroxide index and/or the anisidine index during the process.

However, it has been observed that this reduction is not necessarily sufficient, necessitating an additional treatment for the elimination of the oxidation compounds.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 schematically represents an embodiment of an installation for implementing the invention.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION The invention is now described in more detail and in a non-limiting manner in the following description.

In general, the proportions expressed are mass proportions, unless otherwise stated.

General Principle of the Process The process of the invention makes it possible to obtain a first PUFA in purified form, from an initial mixture.

The initial mixture comprises at least one second undesired fatty acid, which is preferably a second undesired PUFA, and preferably a certain number of other undesired fatty acids, such as saturated or monounsaturated fatty acids and other PUFAs, as well as other possible impurities.

The initial mixture can be a mixture of fatty acids derived from fish, plants, algae and/or yeast, and preferably from fish.

H can be a raw material, for example fish oil or algae oil or yeast oil.

It can also be a product derived from the raw materials above, and for example derived from fish oil, algal oil and/or yeast oil. The oil can for example be extracted from plants, algae or natural or genetically modified yeasts.

By product derived from a raw material, we mean a raw material that has been subjected to one or more processing steps.

These processing steps may include one or more cell disruption, milling, separation or purification (e.g. fractionation) steps and/or a hydrolysis step to convert triglycerides to free fatty acids and/or a esterification to convert fatty acids into alkyl esters and/or a transesterification step to convert fatty triglycerides into alkyl esters, and preferably into ethyl esters, and/or a step of reducing the peroxide index and/or the anisidine index (cf.

below), and/or a molecular distillation step, and/or one or more chromatographic separation steps, etc.

According to a preferred embodiment, the initial mixture has a peroxide number less than or equal to 15, or less than or equal to 10, or less than or equal to 8, or less than or equal to 6, or less than or equal to 5.

In addition, according to a preferred embodiment, the initial mixture has an anisidine number less than or equal to 25, or less than or equal to 20, or less than or equal to 15, or less than or equal to 12, or less than or equal 10, or less than or equal to 8, or less than or equal to 6, or less than or equal to 5.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 13 Generally, this is possible when the initial mixture is an oil which has been subjected to a stage of reduction of the peroxide and/or anisidine number before the method of the invention is implemented.

This step can be carried out in a manner similar to what is described below in connection with the reduction of the peroxide and/or anisidine number according to the method of the invention.

According to a preferred embodiment, the peroxide number and the anisidine number of the initial mixture, upstream of the liquid phase chromatographic separation step or steps, comply with the specifications of the peroxide number and of the anisidine value required for the first purified polyunsaturated fatty acid.

According to an advantageous embodiment, the initial mixture is an esterified or transesterified product, such as a fish oil, a vegetable oil, an algae oil or a transesterified yeast oil.

Thus, each fatty acid (and in particular each PUFA) obtained or used in the process of the invention can be a fatty acid derivative, in particular in the form of a monoglyceride, diglyceride or triglyceride, of an ester, of a phospholipid, an amide, a lactone or a salt.

The free fatty acid and ester forms are preferred, especially the esters.

The esters are typically alkyl esters, for example C1-C6, in particular C1-C4, alkyl esters, for example methyl esters and ethyl esters.

Ethyl esters are preferred.

Thus, the first PUFA, the second fatty acid, the third fatty acid and the fourth fatty acid mentioned in the present application can be, for example, in free fatty acid or ester form, and preferably are in the form of ethyl ester compounds.

Referring to Figure 1, the method according to the invention can be implemented in an installation comprising a first chromatographic unit 10.

The first chromatographic unit 10 ensures the separation between the first PUFA and the second fatty acid.

Whenever mention is made in the present application of a separation between the first PUFA and a given fatty acid, it is understood that other fatty acids can also be separated from the first PUFA simultaneously with the separation with respect to -vis of the given fatty acid.

Generally, each chromatographic separation separates the first PUFA from a set of compounds that are more polar than it or less polar than it.

The separation can also be carried out according to size, aliphatic chain length and number of unsaturation criteria.

More CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 14 generally, since the effects may be dependent on the eluents used, the separation is carried out according to retention time criteria which are different depending on the case, thus allowing to separate the first PUFA from impurities which are more or less retained than it.

The first chromatographic unit 10 is supplied by a fatty acid mixture supply line 9 as well as by an eluent supply line 11.

At the output of the first chromatographic unit 10 are connected on the one hand a flow line enriched in first PUFA 12 and on the other hand a flow line enriched in second fatty acid 13.

In the context of the present application, the term enriched has a relative meaning: a separation between a species A and a species B from an initial flow, making it possible to recover a flow enriched in species A, thus means that the flow thus recovered has a higher NB mass ratio than that of the initial flow.

The PUFA-first enriched flow line 12 feeds a processing unit 14, which is adapted to carry out a reduction in the peroxide index and/or the anisidine index.

At the output of the latter is connected a first purified PUFA collection pipe 15.

If the method of the invention comprises a single chromatographic step, the other parts of the installation shown in the figure are omitted.

In this case, the fatty acid mixture feed line 9 feeds the first chromatographic unit 10 with the initial mixture (it being understood that this initial mixture may have undergone preliminary treatment steps as described above, in which case the corresponding processing units, not shown, can be included in the installation).

Alternatively, and as shown in the figure, a second chromatographic unit 6 can be provided, to ensure separation between the first PUFA and a third fatty acid.

This second chromatographic unit 6 is supplied by a fatty acid mixture supply line 5 as well as by an eluent supply line 7.

At the output of the second chromatographic unit 6 are connected on the one hand a flow line enriched in first PUFA 9 and on the other hand a flow line enriched in third fatty acid 8.

The first PUFA-enriched flow line 9 constitutes the fatty acid mixture feed line 9 for the first chromatographic unit 10.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 If the method of the invention comprises only two chromatographic steps, the other parts of the installation shown in the figure are omitted.

In this case, the fatty acid mixture feed line 5 feeds the second chromatographic unit 6 with the initial mixture (it being understood that this initial mixture may have undergone preliminary treatment steps as described above, to which case the corresponding processing units, not shown, can be included in the installation).

Alternatively, and as shown in the figure, a third chromatographic unit 3 can be provided, to ensure separation between the first PUFA and a fourth fatty acid.

This third chromatographic unit 3 is fed by a fatty acid mixture feed line 1 as well as by an eluent feed line 2.

At the outlet of the third chromatographic unit 3 are connected on the one hand a flow line enriched in first PUFA 5 and on the other hand a flow line enriched in fourth fatty acid 4.

The flow line enriched in the first PUFA 5 constitutes the fatty acid mixture feed line 5 for the second chromatographic unit 6.

If the process of the invention comprises three chromatographic steps, the fatty acid mixture feed pipe 1 feeds the third chromatographic unit 3 with the initial mixture (it being understood that this initial mixture may have undergone preliminary treatment steps such as as described above, in which case the corresponding processing units, not shown, can be included in the installation).

Alternatively, further analogous chromatographic separation steps may be provided.

Thus, in the illustrated embodiment, the initial mixture undergoes three successive stages of liquid phase chromatography:

¨ a step making it possible to separate the first PUFA from the fourth fatty acid (in the third chromatographic unit 3);

¨ a step making it possible to separate the first PUFA from the third fatty acid (in the second chromatographic unit 6);

¨ a step making it possible to separate the first PUFA from the second fatty acid (in the first chromatographic unit 10).

Alternatively, the initial mixture only undergoes two successive stages of liquid phase chromatography:

¨ a step making it possible to separate the first PUFA from the third fatty acid (in the second chromatographic unit 6);

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 16 ¨ a step making it possible to separate the first PUFA from the second fatty acid (in the first chromatographic unit 10).

Alternatively, the initial mixture undergoes a single liquid phase chromatography step, namely the separation of the first PUFA and the second fatty acid (in the first chromatographic unit 10).

Alternatively, the initial mixture undergoes four successive stages of liquid phase chromatography:

¨ a step for separating the first PUFA from a fifth fatty acid (in a chromatographic unit not shown);

- a step making it possible to separate the first PUFA from the fourth fatty acid (in the third chromatographic unit 3);

¨ a step making it possible to separate the first PUFA from the third fatty acid (in the second chromatographic unit 6);

¨ a step making it possible to separate the first PUFA from the second fatty acid (in the first chromatographic unit 10).

Nature of Chromatographic Separations The term chromatographic unit designates either a single-column chromatographic system or a multi-column chromatographic system.

Examples of single-column chromatography systems are HPLC (high performance liquid chromatography) or CYCLOJETTM (system with steady-state recycle) systems.

Examples of multi-column chromatography systems are SMB, iSMB, AMB, VARICOLTM, MODICONTM, POWERFEEDTM, DCC, MCSGP or GSSR (multi-column gradient chromatography) systems.

The CYCLOJET™ system is as described in US 6,063,284, to which express reference is made.

It is a discontinuous single-column chromatographic separation system, in which the (i) most retained then (ii) the least retained species are collected separately at the outlet of the column, an unseparated portion of the chromatogram being recycled by a main pump.

The mixture to be separated is periodically injected by means of an injection loop into the recycled portion of the chromatogram.

The injection loop is preferably connected between the main pump and the column.

After several chromatographic cycles, the process reaches a periodic stationary state in which the quantity of products injected is equal to the quantity of products collected separately at the outlet of the column.

A SMB system comprises a plurality of individual columns containing an adsorbent, which are connected in series.

A flow of eluent passes through the columns in a first direction.

The injection points of the feed stream and the eluent, as well as the collection points of the separated compounds, are staggered periodically and simultaneously by means of a set of valves. The overall effect is to simulate the operation of a single column containing a moving bed of solid adsorbent, with the solid adsorbent moving in a direction countercurrent to the flow of eluent.

Thus, an SMB system is composed of columns that contain stationary beds of solid adsorbent through which the eluent passes, but the operation is such that a continuous countercurrent moving bed is simulated.

The most conventional form of an SMB system is the four-zone SMB system. Other possible forms are three-zone SMB systems and two-zone SMB systems (as described in the article Two Section Simulated Moving Bed Process by Kwangnam Lee, in Separation Science and Technology 35(4):519-534 , 2000, to which express reference is made).

An iSMB system is as described in EP 0342629 and US 5,064,539, to which express reference is made.

An SSMB system divides the introductions and collections of flows into sub-sequences applied periodically.

In iSMB and SSMB systems, there is at least one step in which the system operates in a closed loop, with no product inflow or outflow.

Other variants of SMB systems are: the time-varying SMB system and the POWERFEEDTM system, as described in US 5,102,553 and the article PowerFeed operation of simulated moving bed units: changing flow-rates during the switching interval , from Zhang et al. in Journal of Chromatography A, 1006:87-99, 2003, to which express reference is made; the MODICONTM system, as described in document US Pat. No. 7,479,228, to which express reference is made; and the SMB system with internal recirculation, as described in US 8,282,831, to which express reference is made.

An AMB system operates similarly to an SMB system.

However, instead of moving the feed stream and eluent injection points, as well as collection points, by means of a valve system, a set of adsorption units (columns) are physically displaced from supply and collection points. Again, the operation makes it possible to simulate a continuous counter-current moving bed.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 18 A VARICOLTM chromatography system is as described in the documents US 6,136,198, US 6,375,839 US 6,413,419 and US 6,712,973, to which reference is expressly made.

A VARICOLTM system comprises a plurality of individual columns containing an adsorbent which are connected in series.

An eluent is passed through the columns in a first direction.

Unlike the SMB system, the injection points for the mixture to be separated and for the eluent and the collection points for the separated compounds in the system are moved periodically but asynchronously, by means of a set of valves. The overall effect is to create separation zones of varying length over time, thereby dynamically allocating the stationary phase to the areas where it is most useful, and allowing similar separation power with fewer chromatographic units and increased productivity.

Unlike an SMB system, a VARICOLTM system does not simulate the operation of a single column containing a moving bed of solid adsorbent, the solid adsorbent moving in a direction countercurrent to the flow of eluent, and thus the VARICOLTM operating principle cannot be implemented in an equivalent AMB system.

A DCC chromatography system is as described in document FR 2889077, to which express reference is made.

A DCC system is a sequential process with periodic displacement of the mobile phase injection points and of the mixture to be separated, having the characteristic of being constantly in open loop.

It uses two or more columns.

According to one embodiment, the method of the invention comprises two successive chromatographic separation steps (and two only), which can be AMB, SMB or VARICOL™ separation steps.

According to one embodiment, the method of the invention comprises three successive chromatographic separation steps (and only three), which can be AMB, SMB or VARICOLTM separation steps.

According to one embodiment, the method of the invention comprises three successive chromatographic separation steps (and only three), with first a separation step of the VARICOL™ type (to separate the first PUFA from the fourth fatty acid), then a separation step of the CYCLOJETTM or HPLC type (to separate the first PUFA from the third fatty acid), then a separation step of the VARICOLTM type (to separate the first PUFA from the second fatty acid).

According to one embodiment, the method of the invention comprises three successive chromatographic separation steps (and only three), CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 19 with first a separation step of the VARICOLTM type (to separate the first PUFA from the fourth fatty acid), then a separation step of the CYCLOJETTm or HPLC type (to separate the first PUFA from the third fatty acid), then a separation step of the CYCLOJET™ or HPLC type (to separate the first PUFA from the second fatty acid).

When the process comprises two or more chromatographic separation steps, these steps may be carried out simultaneously in physically separate units (of the same type or of different types and/or sizes), or may be carried out sequentially, in physically separate units or in the same units.

Furthermore, when two chromatographic separation steps are carried out in a system of the SMB or AMB type, it is possible to implement them simultaneously on the same SMB or AMB system.

An example of simultaneous implementation on the same device is described in document WO 2011/080503, or document WO 2013/005048, or document WO 2013/005051, to which reference is expressly made.

Thus, some of the separation units may be the same.

For example, the first chromatographic unit 10 and the second chromatographic unit 6 can be the same unit; or the third chromatographic unit 3 and the second chromatographic unit 6 can be the same unit; or the first chromatographic unit 10 and the third chromatographic unit 3 can be the same unit; or the first chromatographic unit 10, the second chromatographic unit 6 and the third chromatographic unit 3 can be the same unit.

Alternatively, all of the chromatographic units can be separate.

Each chromatographic separation step can be carried out on a reversed phase, as an adsorbent (stationary phase).

For example, it is possible to use adsorbents based on weakly polar resins or stationary phases based on silica chemically modified with organic groups such as alkyl groups (in particular C4, C8, C18, C24, C30), phenyls, or others.

Each chromatographic separation step can be carried out using a hydro-organic eluent, that is to say a mixture of one or more organic solvents with water.

Preferably, all the chromatographic separation steps are carried out using hydroorganic eluents.

Alternatively, it is possible to implement certain chromatographic separation steps with purely organic eluents.

The organic solvents that can be used in the context of the invention (in particular to form the hydro-organic eluents) are, for example, alcohols such as ethanol, propanol, isopropanol and more preferably methanol; ketones such as acetone or methyl ethyl ketone; nitriles such as acetonitrile; esters such as methyl acetate or ethyl acetate; furans such as tetrahydrofuran; ethers such as diethyl ether or methyl ethyl ether; and combinations of two or more of these solvents.

Methanol and acetone are preferred organic solvents.

Each hydro-organic eluent is characterized by a water/organic ratio, which is the volume ratio of water relative to the organic solvent(s) in the eluent.

15 The water/organic ratio of each hydro-organic eluent can preferably vary from 0.01:99.99 to 30:70, and preferably from 5:95 to 25:75.

When the process comprises at least two chromatographic separation steps, these can be carried out with eluents having the same composition or different compositions.

20 It is preferred to use eluents having different compositions, and in particular having different water/organic ratios, this making it possible to adjust the eluent strength of the eluent at each separation step and therefore to obtain the separation of different compounds at each step.

It may also be desired to use eluents composed of different organic solvents in the different stages, in order to adjust the chromatographic selectivity between certain species that have to be separated at each separation stage and thus obtain the separation of different compounds at each stage.

Preferably, the mass concentration of the first eluent in organic solvent(s) is controlled to within 2%, preferably within 1%, or within 0.5%, or within 0.2%, or within close to 0.1%; if necessary, preferably the mass concentration of the second eluent in organic solvent(s) is controlled to within 2%, preferably within 1%, or within 0.5%, or within 0.2%, or to within 0.1%; if necessary, preferably the mass concentration of the third eluent in organic solvent(s) is controlled to within 2%, preferably within 1%, or within 0.5%, or within 0.2%, or within 0.1%.

The control of the composition of the eluents is carried out ensuring the addition of water and/or organic solvent(s) in order to make the necessary adjustments.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 21 At the output of the first chromatographic unit 10, the stream enriched in first PUFA can be the raffinate, and the stream enriched in second fatty acid can be the extract ; or conversely, the stream enriched in the first PUFA can be the extract, and the stream enriched in the second fatty acid can be the raffinate.

Thus, the second fatty acid may be less polar than the first PUFA, or conversely the second fatty acid may be more polar than the first PUFA.

At the outlet of the second chromatographic unit 6, the stream enriched in the first PUFA may be the raffinate, and the stream enriched in the third fatty acid may be the extract; or conversely, the stream enriched in the first PUFA can be the extract, and the stream enriched in the third fatty acid can be the raffinate.

Thus, the third fatty acid may be less polar than the first PUFA, or conversely the third fatty acid may be more polar than the first PUFA.

At the outlet of the third chromatographic unit 3, the stream enriched in the first PUFA may be the raffinate, and the stream enriched in the fourth fatty acid may be the extract; or conversely, the stream enriched in the first PUFA may be the extract, and the stream enriched in the fourth fatty acid may be the raffinate.

Thus, the fourth fatty acid may be less polar than the first PUFA, or conversely the fourth fatty acid may be more polar than the first PUFA.

According to a particular embodiment, the stream enriched in the first PUFA at the outlet of the third chromatographic unit 3 is the raffinate, the stream enriched in the first PUFA at the outlet of the second chromatographic unit 6 is the raffinate and the stream enriched in the first PUFA in output of the first chromatographic unit 10 is the extract.

Thus, the second fatty acid is more polar than the first PUFA, while the third fatty acid and the fourth fatty acid are less polar than the first PUFA.

Each stream (raffinate or extract) resulting from a chromatographic separation step is generally concentrated so as to eliminate the eluent (organic solvents and water) or to reduce the mass content of the stream in organic solvents and water to a level of less 10%, or less than 5%, or less than 2%, or less than 1%, or less than 0.5%, or less than 0.1 A).

Thus, in a preferred embodiment, at least one concentration unit (not shown in the figure) is associated with each chromatographic unit 3, 6, 10.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 22 In particular, preferably, the stream enriched in first PUFA collected at the end of the chromatographic separation between the first PUFA and the second fatty acid is a stream concentrated (depleted in eluent or devoid or essentially devoid of organic solvents and water); likewise, where appropriate, the stream enriched in the first PUFA collected at the end of the chromatographic separation between the first PUFA and the third fatty acid, and the stream enriched in the first PUFA collected at the end of the chromatographic separation between the first PUFA and fourth fatty acid are concentrated fluxes (elutant depleted or devoid or substantially devoid of organic solvents and water); optionally, the flux enriched in second fatty acid, and where appropriate the flux enriched in third fatty acid and the flux enriched in fourth fatty acid are concentrated fluxes (depleted in eluent or devoid or essentially devoid of organic solvents and water) .

In each concentration unit, the eluent can be evaporated and condensed, so as to separate it from the mixture of fatty acids.

One can use for example a recirculating falling film evaporator, a rising flow evaporator, a wiped film evaporator, a thin film evaporator, a thermosiphon evaporator, a rotary evaporator, a distillation column, a rectification column or any other evaporator or combination of evaporators allowing the evaporation of the eluent and the concentration of the concentrated fatty acids at the bottom of the apparatus. The evaporation is preferably carried out at a pressure below atmospheric pressure, in particular at a pressure less than or equal to 750 mbar, or less than or equal to 500 mbar, or less than or equal to 300 mbar.

Alternatively, a membrane separation device can be used, with one or more separation stages, or a combination of evaporation and membrane separation means.

The evaporated and condensed, or otherwise separated, eluent can be recycled to one or more process steps, including one or more of the chromatographic separation steps.

According to one embodiment, each concentration step (and in particular each concentration step of flow enriched in the first PUFA) can be carried out at a temperature less than or equal to 120 C, preferably less than or equal to 100 C, preferably less than or equal to equal to 90 C, preferably less than or equal to 80 C, less than or equal to 75 C.

According to one embodiment, each concentration step (and in particular each concentration step of flow enriched in the first PUFA) has a duration of less than or equal to 6 hours, preferably less CA 02932928 2016-06-03 WO 2015/086672 PCT/ EP2014/077209 23 or equal to 4 hours, preferably less than or equal to 3 hours, preferably less than or equal to 2 hours, preferably less than or equal to 1 hour 30 minutes, preferably less than or equal to 1 hour.

According to particular embodiments, the eluent separated from the first stream enriched in the first PUFA is recycled to more than 50%, preferably to more than 60%, preferably to more than 70%, preferably to more than 80 %, preferably more than 90%, preferably more than 95%, preferably more than 98%, preferably more than 99%.

According to particular embodiments, the eluent separated from the second stream enriched in the first PUFA is recycled to more than 50%, preferably to more than 60%, preferably to more than 70%, preferably to more than 80 %, preferably more than 90%, preferably more than 95%, preferably more than 98%, preferably more than 99%.

According to particular embodiments, the eluent separated from the third stream enriched in the first PUFA is recycled to more than 50%, preferably to more than 60%, preferably to more than 70%, preferably to more than 80 %, preferably more than 90%, preferably more than 95%, preferably more than 98%, preferably more than 99%.

According to particular embodiments, the eluent separated from the stream enriched in second fatty acid is recycled at more than 50%, preferably at more than 60%, preferably at more than 70%, preferably at more than 80 %, preferably more than 90%, preferably more than 95%, preferably more than 98%, preferably more than 99%.

According to particular embodiments, the eluent separated from the stream enriched in third fatty acid is recycled at more than 50%, preferably at more than 60%, preferably at more than 70%, preferably at more than 80 %, preferably more than 90%, preferably more than 95%, preferably more than 98%, preferably more than 99%.

According to particular embodiments, the eluent separated from the stream enriched in fourth fatty acid is recycled at more than 50%, preferably at more than 60%, preferably at more than 70%, preferably at more than 80 %, preferably more than 90%, preferably more than 95%, preferably more than 98%, preferably more than 99%.

Preferably, the feed product which is supplied at the input of each chromatographic separation unit and which is intended to be separated is as free of solvents as possible.

So :

CA 02932928 2016-06-03 WO 2015/086672 PCT / EP2014/077209 24 ¨ the initial mixture comprises less than 80% organic solvents, preferably less than 60% or less than 40% or less than 20% or less than 10 % or less than 5% or less than 2% or less than 1% of organic solvents, and more particularly preferably is a mixture of fatty acids essentially devoid of organic solvents; and/or where appropriate, the first stream enriched in first PUFA which feeds the second chromatographic separation unit 6 comprises less than 80% of organic solvents, preferably less than 60% or less than 40% or less than 20% or less than 10% or less than 5% or less than 2% or less than 1% of organic solvents, and more particularly preferably is a mixture of fatty acids essentially devoid of organic solvents;

and/or the second stream enriched in first PUFA which feeds the third chromatographic separation unit 10 comprises less than 80% organic solvents, preferably less than 60% or less than 40% or less than 20% or less than 10% or less than 5% or less than 2% or less than 1% of organic solvents, and more particularly preferably is a mixture of fatty acids essentially free of organic solvents.

Elimination of the oxygenated compounds The method according to the invention provides for a stage of elimination (or reduction of the quantity) of the oxygenated compounds after the chromatographic separation, or after the chromatographic separations.

Preferably, this step is not a chromatographic separation step, and is not implemented in a chromatographic unit.

Preferably, this step does not separate the first PUFA from other fatty acids present in the stream (with the exception of oxygenated compounds of the aldehyde and peroxide type).

The stage of elimination of the oxygenated compounds is implemented in the processing unit 14.

This processing unit 14 can in particular be a molecular distillation unit or a short-path evaporator.

A short path evaporator is equipped with an internal condenser and can produce evaporations with a residence time of preferably less than 1000 s, preferably less than 100 s, preferably less than 10 s, under pressure CA 02932928 2016- 06-03 WO 2015/086672 PCT/EP2014/077209 preferably less than 10 mbar, preferably less than 1 mbar, preferably less than 0.1 mbar, preferably less than 0.01 mbar, preferably less than 0.001 mbar, at a temperature less than or equal to 200 C, preferably less than or equal to 150 C, preferably less than or equal to 120 C, preferably less than or equal to 100 C, preferably less than or equal to 80 C.

Alternatively, the processing unit 14 can be a unit for bringing into contact with an adsorption substrate.

The adsorption substrate is any substrate capable of adsorbing oxygenated compounds such as peroxides and aldehyde compounds.

It can be chosen for example from silica, alumina, activated carbon and derivatives thereof, in particular silica gels, silicates, aluminates and aluminosilicates.

A clay such as bentonite is an example of a suitable substrate, as is diatomaceous earth.

15 The adsorption can be carried out discontinuously, that is to say in batch, or continuously, by percolation through a bed of adsorbent.

Preferably, the fatty acids are not diluted during this step, and in particular no solvent is added.

The bringing into contact can last, for example, from 5 minutes to 24 hours, and in particular from 10 minutes to 10 hours, from 20 minutes to 20 hours, from 30 minutes to 2 hours, and from 45 minutes to 1 hour 30 minutes.

The amount of adsorbent used depends on the nature of the adsorbent and its ability to capture oxygenated compounds.

It may for example be from 1 to 1000 g of adsorbent per kg of stream to be treated (mixture of fatty acids), in particular from 10 to 500 g/kg, and more particularly from 25 to 200 g/kg.

25 At the end of the contacting, the adsorbent is separated from the mixture of fatty acids, and the latter can be filtered in order to avoid any contamination by residual adsorbent.

Preferably, the binding of the oxygenated compounds to the adsorbent is essentially irreversible, that is to say the adsorbent is not regenerated.

It is however possible to regenerate the adsorbent, by heat treatment for example, in order to limit the volume and/or the cost of waste treatment.

The treatment step in the treatment unit 14 can make it possible to reduce the peroxide number of the treated stream by at least 25%, preferably by at least 50%, preferably by at least 75%, by preferably at least 80% or at least 90% or at least 95% or at least 98%.

The treatment step in the treatment unit 14 can make it possible to reduce the anisidine index of the treated stream by at least 25%, preferably by at CA 02932928 2016-06-03 WO 2015/086672 PCT/ EP2014/077209 26 at least 50%, preferably at least 75%, preferably at least 80 'Vo or at least 90 'Vo or at least 95 'Vo or at least 98%.

The peroxide number measures the amount of peroxide compounds in a mixture of fatty acids.

The analysis method used is preferably Ph Eur 2.5.5 met A.

The anisidine index measures the amount of aldehyde compounds in a mixture of fatty acids.

The analysis method used is preferably Ph Eur 2.5.36.

According to one embodiment, the peroxide index of the stream resulting from the treatment step (product collected in the first purified PUFA collection line 15) is less than or equal to 10, or less than or equal to 9, or less or equal to 8, or less than or equal to 7, or less than or equal to 6, or less than or equal to 5, or less than or equal to 4, or less than or equal to 3, or less than or equal to 2, or less than or equal to 1.5, or less than or equal to 1.

According to one embodiment, the anisidine index of the stream resulting from the treatment step (product harvested in the first purified PUFA collection line 15) is less than or equal to 20, or less than or equal to 18, or less than or equal to 16, or less than or equal to 14, or less than or equal to 12, or less than or equal to 10, or less than or equal to 9, or less than or equal to 8, or less than or equal to 7, or less or equal to 6, or less than or equal to 5, or less than or equal to 4, or less than or equal to 3, or less than or equal to 2.

Another treatment step analogous to that described above, and more particularly a molecular distillation step, can also be provided upstream, in particular before any chromatographic separation step.

Thus, according to one embodiment, the peroxide index of the initial mixture which is subjected to the chromatographic separation(s) is less than or equal to 10, or less than or equal to 9, or less than or equal to 8, or less than or equal 7, or less than or equal to 6, or less than or equal to 5, or less than or equal to 4, or less than or equal to 3, or less than or equal to 2, or less than or equal to 1.5, or less than or equal to 1.

According to one embodiment, the anisidine number of the initial mixture which is subjected to the chromatographic separation(s) is less than or equal to 20, or less than or equal to 18, or less than or equal to 16, or less than or equal to 14, or less than or equal to 12, or less than or equal to 10, or less than or equal to 9, or less than or equal to 8, or less than or equal to 7, or less than or equal to 6, or less than or equal to 5 , or less than or equal to 4, or less than or equal to 3, or less than or equal to 2.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 27 The inventors have observed that the peroxide index and/or the anisidine index increase during the chromatographic separation steps described above, when the first PUFA is separated from compounds more apolar than it, and this although the chromatographic separation conditions are mild conditions ¨ the chromatographic separations being carried out in particular in the absence of oxygen and away from light, and/or at a moderate temperature.

Without wishing to be bound by theory, the inventors believe that this increase may be due in particular to the use of hydro-organic eluents.

According to one embodiment, the peroxide index of the stream containing the first PUFA increases during the chromatographic separation step between the first PUFA and the second fatty acid (that is to say between the fatty acid mixture feed 9 and first PUFA enriched flow line 12), preferably at least 30%, or at least 50%, or at least 75%, or at least 100%, or at least 150%, or at least 200%, or at least 300%, or at least 500%.

According to one embodiment, the anisidine index of the stream containing the first PUFA increases during the step of chromatographic separation between the first PUFA and the second fatty acid (that is to say between the pipe line d fatty acid mixture feed 9 and first PUFA enriched flow line 12), preferably at least 30%, or at least 50%, or at least 75%, or at least 100%, or at least 150%, or at least 200%, or at least 300%, or at least 500%.

According to one embodiment, the peroxide index of the stream containing the first PUFA increases during the chromatographic separation step between the first PUFA and the third fatty acid (that is to say between the fatty acid mixture feed 5 and first PUFA enriched flow line 9), preferably at least 30%, or at least 50%, or at least 75%, or at least 100%, or at least 150%, or at least 200%, or at least 300%, or at least 500%.

According to one embodiment, the anisidine number of the stream containing the first PUFA increases during the step of chromatographic separation between the first PUFA and the third fatty acid (that is to say between the pipe line d fatty acid mixture feed 5 and first PUFA enriched flow line 9), preferably at least 30%, or at least 50%, or at least 75%, or at least 100%, or at least 150%, or at least 200%, or at least 300%, or at least 500%.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 28 According to one embodiment, the peroxide index of the stream containing the first PUFA increases during the step of chromatographic separation between the first PUFA and the fourth fatty acid (i.e. between the fatty acid mixture feed line 1 and the first PUFA enriched flow line 5), preferably by at least 30%, or d at least 50%, or at least 75%, or at least 100%, or at least 150%, or at least 200%, or at least 300%, or at least 500%.

According to one embodiment, the anisidine index of the stream containing the first PUFA increases during the step of chromatographic separation between the first PUFA and the fourth fatty acid (that is to say between the pipe line d fatty acid mixture feed 1 and first PUFA enriched flow line 5), preferably at least 30%, or at least 50%, or at least 75%, or at least 100%, or at least 150%, or at least 200%, or at least 300%, or at least 500 A.

According to one embodiment, the peroxide index of the stream enriched in first PUFA 13 from the chromatographic separation of the first PUFA and the second fatty acid (that is to say from the first chromatographic unit 10) is greater than the peroxide index of the initial mixture (that is to say the optionally pretreated mixture which is intended to be subjected to the stage or stages of chromatographic separation), preferably of at least 30%, or of at least 50%, or at least 75%, or at least 100%, or at least 150%, or at least 200%, or at least 300%, or at least 500%.

According to one embodiment, the anisidine number of the stream enriched in first PUFA 13 resulting from the chromatographic separation of the first PUFA and the second fatty acid (that is to say resulting from the first chromatographic unit 10) is higher to the anisidine index of the initial mixture (that is to say possibly pretreated mixture which is intended to be subjected to the stage or stages of chromatographic separation), preferably of at least 30%, or of at least least 50%, or at least 75%, or at least 100%, or at least 150%, or at least 200%, or at least 300%, or at least 500 In the event that one of the chromatographic separations carried out does not have the consequence of significantly increasing the peroxide index or the anisidine index of the flux of interest, it is possible to reverse the order of the steps, i.e. to carry out this or these chromatographic separation steps after the peroxide value/anisidine value reduction step.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 29 Thus, in this case, one can consider for example:

¨ a chromatographic separation step of the first PUFA and the second fatty acid, followed by a treatment step comprising the reduction of the peroxide index and / or the anisidine index of the stream enriched in the first PUFA, followed by a stage of chromatographic separation of the first PUFA and of the third fatty acid; or ¨ a stage of chromatographic separation of the first PUFA and of the third fatty acid, followed by a stage of chromatographic separation of the first PUFA and of the second fatty acid, followed by a treatment stage comprising the reduction of the peroxide index and / or the anisidine index of the stream enriched in the first PUFA, followed by a stage of chromatographic separation of the first PUFA and of the fourth fatty acid; or - a stage of chromatographic separation of the first PUFA and the second fatty acid, followed by a treatment stage comprising the reduction of the peroxide index and/or the anisidine index of the stream enriched in the first PUFA, followed a stage of chromatographic separation of the first PUFA and of the fourth fatty acid, followed by a stage of chromatographic separation of the first PUFA and of the third fatty acid.

The method may include a step of adding a stabilizer (antioxidant), such as tocopherol, ascorbic acid or any other compound or mixture of compounds known to those skilled in the art, in order to avoid degradation of the product and a further increase in the peroxide or anisidine value.

This stabilizer addition step is preferably carried out at the end of the process, after all the chromatographic separation steps and after the treatment step.

Product Obtained According to one embodiment, the first PUFA is an omega-3 fatty acid.

According to different embodiments, the first PUFA can be EPA, or DHA, or ARA, or DPA, or SDA.

According to one embodiment, the first PUFA is EPA and the second fatty acid is DHA.

According to one embodiment, the first PUFA is DHA and the second fatty acid is EPA.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 According to one embodiment, the first PUFA is EPA and the second fatty acid is SDA.

According to one embodiment, the first PUFA is SDA and the second fatty acid is EPA.

According to one embodiment, the first PUFA is DPA and the second fatty acid is DHA.

According to one embodiment, the first PUFA is DHA and the second fatty acid is DPA.

According to one embodiment, the first PUFA is ARA and the second fatty acid is DHA.

According to one embodiment, the first PUFA is DHA and the second fatty acid is ARA.

According to one embodiment, the first PUFA is EPA, the second fatty acid is DHA or SDA, and the third fatty acid and the fourth fatty acid are chosen from that of DHA and SDA which is not the second fatty acid, and among saturated fatty acids and monounsaturated fatty acids.

The concentration of first PUFA in the final product obtained at the end of the process (harvested in the purified first PUFA collection line 15) may be greater than or equal to approximately 80%, preferably greater than or equal to approximately 90%, or about 95%, or about 97%, or about 98%, or about 99% (based on total fatty acids).

The second fatty acid concentration in this final product may be less than or equal to about 1%, or about 0.1%, or about 0.05%, or about 0.03%, or about 0, 01`)/0 (relative to total fatty acids).

The third fatty acid concentration in this final product may be less than or equal to about 1%, or about 0.1%, or about 0.05%, or about 0.03%, or about 0.01 `)/0 (relative to total fatty acids).

The fourth fatty acid concentration in this final product may be less than or equal to about 1%, or about 0.1%, or about 0.05%, or about 0.03%, or about 0. 01`)/0 (relative to total fatty acids).

For example, the final product obtained at the end of the process (harvested in the first purified PUFA collection pipe 15), may contain EPA in a proportion greater than or equal to approximately 80%, or greater than or equal to approximately 95%, or greater than or equal to about 97%, or greater than or equal to about 98%, or greater than or equal to about 99% (based on total fatty acids); as well as DHA in a proportion less than or equal to about 1%, or less than or equal to about 0.1%, or less or CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 31 equal to about 0 0.05%, or less than or equal to about 0.03%, or less than or equal to about 0.01%.

The oil enriched in the first PUFA is preferably stored in the absence of air and light before its packaging and/or its use, comprising for example the final formulation and/or the encapsulation.

According to one embodiment, the final product is combined with a pharmaceutically and/or dietetically acceptable vehicle and/or excipients and/or diluents.

This product can thus be formulated for example in the form of gelatin capsules, capsules or tablets (or in any other form suitable for oral or topical or parenteral administration).

Each individual dosage form (eg capsule or capsule) may contain, for example, 250 to 1500 mg, preferably 300 to 1000 mg of the above product.

The product can thus be used for the preparation of a pharmaceutical composition for the prevention and/or the treatment and/or the prophylaxis of risk factors for cardiovascular diseases, such as hypertriglyceridemia, hypercholesterolemia and hypertension; and cardiovascular diseases such as arrhythmia, atrial and/or ventricular fibrillation, decompensation and heart failure; for the primary and secondary prevention of infarction and re-infarction; for the treatment of any other pathology which can be treated by the aforementioned PUFAs, such as for example autoimmune diseases, ulcerative cholic, tumor pathologies, diseases of the nervous system, cell aging, cerebral infarction, ischemic diseases , psoriasis.

Alternatively, the product can be used in parapharmaceutical uses, and in particular dietetic uses, in particular in infant nutrition.

EXAMPLES The following example illustrates the invention without limiting it.

In this example, an ethyl ester of EPA with a purity of more than 96% is obtained by three successive chromatography steps, from a mixture of ethyl esters containing more than 50% of EPA, and having a peroxide number of 4.5 and an anisidine number of 11.

All of the chromatography operations are carried out under an inert atmosphere and away from light.

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 32 All the chromatography operations are carried out on a VARICOLTM type chromatographic system, comprising five columns 20 cm in diameter, filled with a stationary phase of reverse phase silica in 018.

The chromatography system is coupled to two recirculating falling film evaporators, for the concentration of the extract and the raffinate, respectively.

The chromatography and evaporation conditions are as follows:

Step 1 :

¨ eluent: acetone/water in a 90/10 v/v ratio;

- Complete evaporation of the raffinate eluent (target product) at 250 mbar and 75°C;

¨ complete evaporation of the extract eluent at 250 mbar and 75 C.

The concentrated raffinate contains about 70% EPA (area measurement by gas chromatography) and less than 1% residual solvents. The average peroxide index obtained is 8.0 and the average anisidine index obtained is 13.8.

2nd step:

¨ eluent: methanol/water in a 93/7 v/v ratio;

¨ complete evaporation of the raffinate eluent (target product) at 250 mbar and 75°C;

¨ partial evaporation of the extract eluent at 1000 mbar and 75 C.

The concentrated raffinate contains approximately 92% EPA (measured by area by gas chromatography) and less than 1% residual solvents. The average peroxide index obtained is 7.4 and the average anisidine index obtained is 22.6.

Step 3:

¨ eluent: acetone/water in a 79/21 v/v ratio;

¨ complete evaporation of the raffinate eluent at 250 mbar and 75°C;

¨ complete evaporation of the extract eluent (target product) at 250 mbar and 75 C.

The concentrated extract contains approximately 97% EPA (measured by area by gas chromatography) and less than 1% residual solvents. The average peroxide index obtained is 6.5 and the average anisidine index obtained is 6.1.

A final stage of stripping with nitrogen (stripping) and elimination of the oxidation compounds is carried out discontinuously (batch) under the following conditions:

¨ stripping by nitrogen bubbling at 75 C for 1 hour;

CA 02932928 2016-06-03 WO 2015/086672 PCT/EP2014/077209 33 ¨ addition of 100 g of bentonite per kg of oil, stirring under vacuum at 50 mbar for 1 hour;

¨ filtration.

An antioxidant (0.2% tocopherol) is added. The peroxide number and the anisidine number in the purified EPA are measured between 0.6 and 0.7 and between 1.0 and 2.6 respectively.