CHROMATOGRAPHIC SEPARATION PROCESS TO RECOVER A POLY-INSATURATED FATTY ACID (PUFA) PRODUCT AND COMPO

专利摘要:
"chromatographic separation process to recover a fatty acid product, composition, and computer program". the present invention provides a chromatographic separation process to recover a polyunsaturated fatty acid product (pufa) from a feed mixture, the process of which introduces the feed mixture to a simulated or real moving bed chromatography apparatus having a plurality of columns linked chromatographs containing, as an eluent, an aqueous alcohol, in which the apparatus has a plurality of zones comprising at least a first zone and a second zone, each zone having an extract stream and a raffinate stream from which the liquid can be collected of said plurality of linked chromatographic columns, and in which (a) a stream of raffinate containing the pufa product together with more polar components is collected from a column in the first zone and introduced into a non-adjacent column in the second zone, and / or ( b) an extract stream containing the pufa product together with less polar components is collected from a column in the second zone ei Introduced in a non-adjacent column in the first zone, said pufa product being separated from different components of the feed mixture in each zone.
公开号:BR112012016308B1
申请号:R112012016308-6
申请日:2010-12-24
公开日:2020-03-31
发明作者:Adam Kelliher；Angus Morrison；Anil Oroskar；Rakesh Vikraman Nair Rema；Abhilesh Agarwal
申请人:Basf Pharma (Callanish) Limited；
IPC主号:

专利说明:

“CHROMATOGRAPHIC SEPARATION PROCESS TO RECOVER A POLY-INSATURATED FATTY ACID (PUFA) PRODUCT AND COMPOSITION”
The present invention relates to an improved chromatographic fractionation process to purify polyunsaturated fatty acids (PUFAs) and their derivatives. In particular, the present invention relates to a better chromatographic separation process from simulated or real moving bed to purify PUFAs and their derivatives. .
Fatty acids, in particular PUFAs, and their derivatives are precursors to biologically important molecules, which play an important role in regulating biological functions such as platelet aggregation, inflammation and immune responses. Thus, PUFAs and their derivatives can be therapeutically useful in the treatment of a wide range of pathological conditions including CNS conditions, neuropathies, including diabetic neuropathy, cardiovascular diseases, general immune system and inflammatory conditions, including inflammatory skin diseases.
It was observed that PUFAs are natural raw materials, such as vegetable oils and marine oils. Such PUFAs are, however, often present in such oils in admixture with saturated fatty acids and numerous other impurities. PUFAs should therefore be desirably purified before nutritional or pharmaceutical uses.
Unfortunately, PUFAs are extremely fragile. Thus, when heated in the presence of oxygen, they are prone to isomerization, peroxidation and oligomerization. The fractionation and purification of PUFA products to prepare pure fatty acids is therefore difficult. Distillation, even under vacuum, can lead to unacceptable product degradation.
Simulated and real moving bed chromatography are known techniques, familiar to those skilled in the art. The principle of operation
Petition 870190102397, of 11/10/2019, p. 12/176 involves countercurrent movement of a liquid eluting phase and a solid adsorbent phase. This operation allows minimal use of solvent making the process economically viable. Such separation technology has found several applications in several areas, including hydrocarbons, industrial chemicals, oils, sugars and APIs. Such separation technology has also been applied to purify PUFAs and their derivatives.
As is well known, in a conventional stationary bed chromatographic system, a mixture whose components have to be separated percolates through a container. The container is generally cylindrical, and is typically referred to as the column. The column contains a packaging of a porous material (usually called the stationary phase) that exhibits a high permeability to fluids. The percolation speed of each component of the mixture depends on the physical properties of this component, so that the components leave the column successively and selectively. Thus, some of the components tend to fix strongly in the stationary phase and thus will seep slowly, while others tend to fix weakly and leave the spine more quickly. Many different stationary bed chromatographic systems have been proposed and are used for both analytical and industrial production purposes.
In contrast, a simulated moving bed system consists of numerous individual columns containing adsorbent that are connected to each other in series. Eluent passes through the columns in a first direction. The injection points of the feed stock and the eluent, and the collection points of the separate components in the system, are periodically displaced by means of a series of valves. The general effect is to simulate the operation of a single column containing a moving bed of the solid adsorbent. Thus, a simulated moving bed system consists of columns that, as in a conventional stationary bed system, contain solid adsorbent stationary beds through which the eluent passes, but in a
Petition 870190102397, of 11/10/2019, p. 13/176 simulated moving bed the operation is just like simulating a continuous countercurrent moving bed.
Processes and equipment for simulated moving bed chromatography are described in several patents, including US 2,985,589, US 3,696,107, US 3,706,812, US 3,761,533, FR-A-2103302, FR-A-2651148 and FR- A-2651149, which are incorporated in their entirety here by reference. The topic is also dealt with in detail in “Preparative and Production Scale Chromatography”, edited by Ganetsos and Barker, Marcel Dekker Inc, New York, 1993, which is incorporated in its entirety here by reference.
A real moving bed system is similar in operation to a simulated moving bed system. However, instead of displacing the injection points of the feed mixture and the eluent, and the component collection points separated by means of a valve system, instead of a series of adsorption units (ie, columns) it is physically moved in relation to the feeding and withdrawal points. Again, the operation is like simulating a continuous countercurrent moving bed.
Processes and equipment for real moving bed chromatography are described in several patents; including US 6,979,402, US 5,069,883 and US 4,764,276, which are incorporated herein in full by reference.
Simulated and real moving bed technology is generally suitable for separating binary mixtures. Thus, a more polar component will move with the eluent, and will be collected as a raffinate stream and a less polar component will move with the adsorbent, and will be collected as an extract stream. It is therefore difficult to use simulated or real moving bed technology to separate a desired product from a crude mixture containing both polar and non-polar impurities. This limits the applicability of such techniques in the purification of PUFA products from
Petition 870190102397, of 11/10/2019, p. 14/176 fish, for example.
In this way, when simulated or real moving bed technology was used to separate PUFAs from natural oils in the past, it was generally necessary to first subject the natural oil to a preliminary separation step (for example, fixed column chromatography) before product purification intermediate obtained using simulated or real moving bed technology (see, for example, EP-A-0697034). Typically, the initial purification step removes polar or non-polar components, thus creating an essentially binary mixture that is then subjected to moving bed chromatography.
This process of separating a binary mixture is illustrated with reference to figure 1. The concept of a simulated or real continuous countercurrent chromatographic separation process is explained considering a vertical chromatographic column containing stationary S phase divided into sections, more precisely in four I sub-areas, II, III and IV overlapping from the base to the top of the column. The eluent is introduced into the base in IE by means of a P pump. The mixture of components A and B that have to be separated is introduced in IA + B between sub-area II and sub-area III. An extract containing mainly B is collected in SB between subarea I and subarea II and a raffinate containing mainly A is collected in SA between subarea III and subareaIV.
In the case of a simulated moving bed system, a simulated downward movement of the stationary S phase is caused by movement of the introduction and collection points relative to the solid phase. In the case of a real moving bed system, downward movement of the stationary S phase is caused by movement of the various chromatographic columns relative to the points of introduction and collection. In figure 1, the eluent flows upwards and the A + B mixture is injected between sub-area II and sub-area III. The components will move according to their chromatographic interactions with the
Petition 870190102397, of 11/10/2019, p. 15/176 stationary phase, for example, adsorption in a porous medium. Component B that exhibits the strongest affinity in the stationary phase (the component that runs the slowest) will be dragged more slowly by the eluent and will follow it with delay. The component A that exhibits the weakest affinity in the stationary phase (the component that runs the fastest) will be dragged easily by the eluent. If the correct set of parameters, especially the flow in each zone, is correctly estimated and controlled, component A that exhibits the weakest affinity in the stationary phase will be collected between sub-area III and sub-area IV as a raffinate and component B that displays the strongest affinity in the stationary phase will be collected between subarea I and subarea II as an extract.
Therefore, it should be noted that the conventional mobile bed system schematically illustrated in Figure 1 is limited to binary fractionation.
Thus, there is a need for a single simulated or real moving bed chromatographic separation process that can separate PUFAs or their derivatives from components that run both slower and faster (ie, more polar and less polar impurities), to produce an essentially pure PUFA or derivative thereof. It is additionally desired that the process involves cheap eluents that operate under standard temperature and pressure conditions.
Surprisingly, it has now been observed that a PUFA product can be effectively purified with a single simulated or real moving bed apparatus using an aqueous alcohol eluent. The present invention therefore provides a chromatographic separation process to recover a polyunsaturated fatty acid (PUFA) product from a feed mixture, the process of which introduces the feed mixture into a simulated moving bed chromatography apparatus or with a plurality of linked chromatography columns containing, as an eluent, an aqueous alcohol, in which the apparatus has a plurality of
Petition 870190102397, of 11/10/2019, p. 16/176 zones comprising at least a first zone and a second zone, each zone with an extract stream and raffinate stream from which the liquid can be collected from said plurality of linked chromatography columns, and in which (a) a stream of raffinate containing the PUFA product together with more polar components is collected from a column in the first zone and introduced into a non-adjacent column in the second zone and / or (b) an extract stream containing PUFA product together with less polar components is collected from a column in the second zone and introduced into the non-adjacent column in the first zone, said PUFA product being separated from the different components of the feed mixture in each zone.
A PUFA product obtainable by the process of the present invention is also provided.
The PUFA products produced by the process of the present invention are produced in high yield, and are of high purity. In addition, the content of the different impurities that typically increase from the distillation of PUFAs is very low. As used herein, the terms "isomeric impurities" are used to denote those impurities typically produced during the distillation of PUFA containing natural oils. These include PUFA isomers, peroxidation products and oligomerization.
Figure 1 illustrates the basic principles of a simulated or real moving bed process to separate the binary mixture.
Figure 2 illustrates a first preferred embodiment of the invention that is suitable for separating EPA from components that run faster and slower (i.e., more polar and less polar impurities).
Figure 3 illustrates a second preferred embodiment of the invention that is suitable for separating DHA from components that run faster and slower (i.e., more polar and less polar impurities).
Figure 4 illustrates in more detail the first modality
Petition 870190102397, of 11/10/2019, p. 17/176 preferred invention that is suitable for separating EPA from components that run faster and slower (i.e., more polar and less polar impurities).
Figure 5 illustrates in more detail the second preferred embodiment of the invention that is suitable for separating DHA from components that run faster and slower (i.e., more polar and less polar impurities).
Figure 6 illustrates in more detail an alternative method for the first preferred embodiment of the invention that is suitable for separating EPA from components that run faster and slower (i.e., more polar and less polar impurities).
Figure 7 illustrates in more detail an alternative method for the second preferred embodiment of the invention that is suitable for separating DHA from components that run faster and slower (i.e., more polar and less polar impurities).
Figure 8 illustrates a particularly preferred embodiment of the invention for purifying EPA from components that run faster and slower (i.e., more polar and less polar impurities).
Figure 9 illustrates an alternative method for a particularly preferred embodiment of the invention for purifying EPA from components that run faster and slower (i.e., more polar and less polar impurities).
Figure 10 illustrates a particularly preferred embodiment of the invention for purifying EPA from components that run faster and slower (i.e., more polar and less polar impurities).
Figure 11 shows a GC analysis of an EPA product produced according to the invention.
Figure 12 shows FAMES GC traces of the first extract and raffinate streams obtained according to the invention.
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Figure 13 shows FAMES GC traces of the second extract and raffinate streams obtained according to the invention.
Figure 14 shows a FAMES GC trace of a DHA product produced according to the invention.
Figure 15 shows a FAMES GC trace of a DHA product produced by distillation.
The terms "polyunsaturated fatty acid" (PUFA) refer to fatty acids that contain more than one double bond. Such PUFAs are well known to those skilled in the art. As used herein, a PUFA derivative is a PUFA in the form of a mono, di or triglyceride, ester, phospholipid, amide, lactone or salt. Triglycerides and esters are preferred. Esters are more preferred. Esters are typically alkyl esters, preferably C1-C6 alkyl esters, more preferably C1-C4 alkyl esters. Examples of esters include methyl and ethyl esters. Ethyl esters are more preferred.
As used herein, the terms "PUFA product" refer to a product comprising one or more polyunsaturated fatty acids (PUFAs) and / or their derivatives, typically of nutritional or pharmaceutical significance. Typically, the PUFA product is a single PUFA or derivative thereof. Alternatively, the PUFA product is a mixture of two or more PUFAs or their derivatives, for example, two.
As used herein, the term "zone" refers to a plurality of linked chromatography columns containing, as an eluent, an aqueous alcohol, and with one or more injection points for a feed mixture stream, one or more points injection for water and / or alcohol, a raffinate removal stream from which the liquid can be collected from said plurality of linked chromatography columns, and an extract removal stream from which the liquid can be collected from said plurality of columns chromatography linked. Typically, each zone has only one
Petition 870190102397, of 11/10/2019, p. 19/176 injection point for a mixture of food in one mode, each zone has only one injection point for the aqueous alcohol eluent. In another mode, each zone has two or more injection points for water and / or alcohol.
The term "raffinate" is well known to those skilled in the art. In the context of real and simulated moving bed chromatography it refers to the stream of components that move faster with the liquid eluting phase compared to the solid adsorbent phase. Thus, a raffinate stream is typically enriched with more polar components, and depleted of less polar components compared to a feed stream.
The term "extract" is well known to those skilled in the art. In the context of real and simulated moving bed chromatography it refers to the stream of components that move faster with the solid adsorbent phase compared to the liquid eluent phase. Thus, an extract stream is typically enriched with less polar components, and depleted of more polar components compared to a feed stream.
As used herein, the terms "non-adjacent" when applied to columns in the same apparatus refer to columns separated by one or more columns, preferably 3, or more columns, more preferably 5 or more columns, above all preferably about 5 columns.
Thus, where (a) a stream of raffinate containing the PUFA product together with more polar components is collected from a column in the first zone and introduced into a non-adjacent column in the second zone, the stream of raffinage collected from the first zone is the mixture of supply to the second zone. Where (b) an extract stream containing the PUFA product together with less polar components is collected from a column in the
Petition 870190102397, of 11/10/2019, p. 20/176 second zone and introduced in a non-adjacent column in the first zone, the extract stream collected from the second zone is the feed mixture in the first zone.
Typically, the PUFA product comprises at least one PUFA ω-3 or ω-6, preferably at least one PUFA ω-3. Examples of ω-3 PUFAs include alpha-linolenic acid (ALA), stearidonic acid SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosaexaenoic acid DHA). SDA, EPA, DPA and DHA are preferred. EPA and DHA are more preferred. Examples of ω-6 PUFAs include linoleic acid (LA), gamma-linolenic acid (GLA), eicosadienic acid, diomogamalinolenic acid (DGLA), arachidonic acid (ARA), docosadienic acid, adrenic acid and docosapentaenoic acid (ω-6 ). LA, ARA, GLA and DGLA are preferred.
In one embodiment, the PUFA product is ethyl ester EPA and / or EPA (EE).
In another embodiment, the PUFA product is ethyl ester DHA and / or EPA (EE).
In an additional modality, the PUFA product is a mixture of EPA and DHA and / or EPA EE and DHA EE.
Feed mixtures suitable for fractionation by the process of the present invention can be obtained from natural sources including vegetable and animal oils and fats, and from synthetic sources including oils obtained from genetically modified plants, animals, and microorganisms including yeast. Examples include fish oils, algae and microalgae oils and plant oils, for example, borage oil, Echium oil and evening primrose oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is an algae oil. Seaweed oils are particularly suitable when the PUFA product
Petition 870190102397, of 11/10/2019, p. Desired 21/176 is EPA and / or DHA. Genetically modified sunflower oil is particularly suitable when the desired PUFA product is GLA. Genetically modified yeast is particularly suitable when the desired PUFA product is EPA.
The feed mixture can undergo chemical treatment before fractionation by the process of the invention. For example, it may undergo glyceride transesterification or glyceride hydrolysis followed in certain cases by selective processes such as crystallization, molecular distillation, urea fractionation, extraction with silver nitrate or other metal salt solutions, iodolactonization or fluid fractionation supercritical.
Feed mixtures typically contain the PUFA product and at least one more polar component and at least one less polar component. The less polar components have a stronger adherence to the adsorbent used in the process of the present invention than the PUFA product. During operation, such less polar components typically move with the solid adsorbent phase in preference to the liquid eluent phase. The more polar components have weaker adhesion to the adsorbent used in the process of the present invention than the PUFA product. During operation, such more polar components typically move with the liquid eluting phase in preference to the solid adsorbent phase. In general, more polar components will be separated in a raffinate stream, and less polar components will be separated in an extract stream.
Examples of the more and less polar components include (1) other compounds that occur in natural oils (for example, marine oils or vegetable oils), (2) by-products formed during the stages of storage, refining and prior concentration, and (3) contaminants from solvents or reagents that are used during refining or prior purification steps.
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Examples of (1) include other undesirable PUPAs; saturated fatty acids; sterols, for example, cholesterol; vitamins, and environmental pollutants, such as polychlorinated biphenyl (PCB), polyaromatic hydrocarbon (PAH) pesticides, chlorinated pesticides, dioxins and heavy metals. PCBs, PAHs, dioxins and chlorinated pesticides are all highly non-polar components.
Examples of (2) include isomers and oxidation or decomposition products of the PUFA product, for example, polymeric products of the autooxidation of fatty acids or their derivatives.
Examples of (3) include urea that can be added to remove saturated or monounsaturated fatty acids from the feed mixture.
Preferably, the feed mixture is a marine oil containing PUFA, more preferably a marine oil comprising EPA and / or DHA.
A typical feed mixture for preparing concentrated EPA by the process of the present invention comprises 50-75% EPA, 0 to 10% DHA, and other components including other essential ω-3 and ω-6 fatty acids.
A preferred feed mixture for preparing concentrated EPA by the process of the present invention comprises 55% EPA, 5% DHA, and other components including other essential ω-3 and ω-6 fatty acids. DHA is less polar than EPA.
A typical feed mixture for preparing concentrated DHA by the process of the present invention comprises 50-75% DHA, 0 to 10% EPA, and other components including other essential ω-3 and ω-6 fatty acids.
A preferred feed mixture for preparing concentrated DHA by the process of the present invention comprises 75% DHA, 7% EPA and other components including other ω-3 and ω-6 fatty acids
Petition 870190102397, of 11/10/2019, p. 23/176 essential, EPA is more polar than DHA.
A typical feed mixture for preparing a concentrated mixture of EPA and DHA by the process of the present invention comprises more than 33% EPA and more than 22% DHA.
The process of the invention requires a plurality of zones in said chromatography apparatus. Typically, two or more zones are used. The number of zones is not particularly limited, but in general there are 2 to 5 zones. Preferably, there are two or three zones, more preferably there are two zones.
Typically, the separate components in each zone of the apparatus used in the process of the present invention have different polarities.
Typically, a) the aqueous alcohol eluent present in each zone has a different water: alcohol ratio; and / or (b) the rate at which liquid collected through the extract and raffinate streams in each zone is recycled to the same zone so that the PUFA product can be separated from different components of the feed mixture in each zone.
When the apparatus used in the process of the present invention has two zones, the present invention typically provides a chromatographic separation process to recover a polyunsaturated fatty acid (PUFA) product from a feed mixture, the process of which comprises introducing the feed mixture into a simulated or real moving bed chromatography apparatus with a plurality of connected chromatography columns containing, as an eluent, an aqueous alcohol, in which the apparatus has a first zone and a second zone, each zone with an extract stream and a stream of raffinate from which the liquid can be collected from said plurality of linked chromatography columns, and in which (a) a stream of raffinate containing the PUFA product together with more polar components is collected from a column in the first zone and
Petition 870190102397, of 11/10/2019, p. 24/176 introduced in the non-adjacent column in the second zone, and / or (b) an extract stream containing the PUFA product together with less polar components is collected from a column in the second zone and introduced in a non-adjacent column in the first zone, said PUFA product being separated from less polar components of the feed mixture in the first zone, and said PUFA product being separated from the more polar components of the feed mixture in the second zone.
Typically, when the apparatus used in the process of the present invention contains two zones, the eluent in the first zone contains more alcohol than the eluent in the second zone, and the second zone is downstream of the first zone with respect to the flow of eluent in the system. Thus, the eluent in the system typically moves from the first zone to the second zone. In contrast, the solid adsorbent phase typically moves from the second zone to the first zone. Typically, the two zones do not overlap, that is, there is no chromatographic column that is in both zones.
In a further embodiment of the invention, the apparatus has a first zone, a second zone and a third zone. The water: alcohol ratios of the aqueous alcohol eluent present in the first second and third zones are typically different. As will become evident to those skilled in the art; this has the consequence that impurities with different polarities can be removed in each zone.
Preferably, when the apparatus has three zones, the eluent in the first zone contains more alcohol than the eluent in the second zone and the third zone and the first zone is upstream of the second and third zones with respect to the flow of eluent in the system. Typically, the eluent in the second zone contains more alcohol than the eluent in the third zone and the second zone is upstream of the third zone with respect to the flow of eluent in the system. Typically, in the first zone, said PUFA product is separated from the components of the feed mixture that are less polar than the
Petition 870190102397, of 11/10/2019, p. 25/176 PUFA product. Typically, in the second zone, said PUFA product is separated from the components of the feed mixture, which are less polar than the PUFA product, but more polar than the components separated in the first zone. Typically, in the third zone, said PUFA product is separated from components of the feed mixture that are more polar than the PUFA product.
In an additional embodiment, in the first zone, said PUFA product is separated from the components of the feed mixture that are less polar than the PUFA product, in the second zone, said PUFA product is separated from the components of the feed mixture that are more polar. polar than the PUFA product and, in the third zone, said PUFA product is separated from the components of the feed mixture which are more polar than the PUFA product and also more polar than the separate components in the second zone.
Such a configuration with three zones would be suitable for separating EPA and DHA from a mixture containing impurities that are less polar than DHA and EPA and also containing impurities that are more polar than EPA. In the first zone, components that are less polar than DHA and EPA are removed as an extract stream and raffinate stream comprising DHA, EPA and components that are more polar than EPA is collected and introduced into the second zone. In the second zone, DNA is removed as an extract stream and a raffinate stream comprising EPA and components that are more polar than EPA is collected and introduced into the third zone. In the third zone, components that are more polar than EPA are removed as a streak of raffinate and purified EPA is collected as an extract stream. In this embodiment, the purified EPA is the purified PUFA product. Such a configuration has an advantage that a secondary PUFA can also be recovered. In this case, the secondary PUFA is DNA
Petition 870190102397, of 11/10/2019, p. 26/176 collected as the extract stream from the second zone.
Typically, in addition to said PUFA product, an additional secondary PUFA product is collected in the chromatographic separation process of the invention. Preferably, the PUFA product is EPA and the additional secondary PUFA product is DHA.
In a further embodiment of the invention, the apparatus is configured to collect a PUFA product which is a concentrated mixture of EPA and DHA. Thus, a feed mixture that contains EPA, DNA, components that are more polar than EPA and DHA, and components that are less polar than EPA and DNA is used. In the first zone, less polar material than EPA and DHA is removed. In the second zone, material that is more polar than EPA and DHA is removed, and a concentrated mixture of EPA and DHA is collected as the PUFA product.
Any known simulated or real moving bed chromatography apparatus can be used for the purposes of the method of the present invention, provided that the apparatus is configured with the multiple zones, in particular two, that characterize the process of the present invention, whose apparatus described in US 2,985,589, US 3,696,107, US 3,706,812, US 3,761,533, FR-A-2103302, FR-A-2651148, FR-A-2651149, US 6,979,402, US 5,069,883 and US 4,764 .276 can all be used if configured according to the process of the present invention.
The number of speakers used in the device is not particularly limited. Those skilled in the art can easily determine an appropriate number of columns to use. The number of columns is typically 8 or more, preferably 15 or more. In a more preferred embodiment, 15 or 16 columns are used. In another more preferred embodiment, 19 or 20 columns are used. In other more preferred embodiments, 30 or more columns are used. Typically, there are no more than 50 columns, preferably no more than 40.
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Each zone typically consists of an approximately equal portion of the total number of columns. Thus, in the case of a device configured with two zones, each zone typically consists of approximately half the total number of chromatographic columns in the system. Thus, the first zone typically comprises 4 or more, preferably 8 or more, more preferably about 8 columns. The second zone typically comprises 4 or more, preferably 7 or more preferably 7 or 8 columns.
The dimensions of the columns in the manner used in the apparatus are not particularly limited, and will depend on the volume of feed mixture to be purified. Those skilled in the art will be able to easily determine appropriately sized columns to use. The diameter of each column is typically between 10 and 500 mm, preferably between 25 and 250 mm, more preferably between 50 and 100 mm, and above all preferably between 70 and 80 mm. The length of each column is typically between 10 and 200 cm, preferably between 25 and 150 cm, more preferably between 70 and 110 cm, and above all preferably between 80 and 100 cm.
The columns in each zone typically have identical dimensions, but may, for certain applications, have different dimensions.
Flow rates for the column are limited by maximum pressures across the series of columns and will depend on the column dimensions and particle size of the solid phases. Experienced in the technique, they can easily establish the flow required for each column dimension to ensure efficient desorption. Larger diameter columns will generally need greater flows to maintain linear flow through the columns.
For typical column sizes outlined above, and for a two-zone apparatus, the eluent flow rate for the first zone is typically 1 to 4.5 L / min, preferably 1.5 to 3.5 L / min. Typically, the extract flow rate for the first zone is 0.1 to 2.5 L / min,
Petition 870190102397, of 11/10/2019, p. 28/176 preferably from 0.5 to 2.25 L / min. In modalities where part of the extract from the first zone is recycled to the first zone, the recycling rate is typically 0.7 to 1.4 L / min, preferably about 1 L / min. Typically, the flow rate of the rafinado in the first zone is 0.2 to 2.5 L / min, preferably 0.3 to 2.0 L / min. In modalities where part of the raffinate from the first zone is recycled to the first zone, the recycling rate is typically 0.3 to 1.0 L / min, preferably about 0.5 L / min. Typically, the flow rate of introducing the feed mixture into the first zone is from 5 to 150 ml / min, preferably from 10 to 100 L / min, more preferably from 20 to 60 ml / min.
For typical column sizes outlined above, and for a two-zone apparatus, the eluent flow rate for the second zone is typically 1 to 4 L / min, preferably 1.5 to 3.5 L / min. Typically, the flow rate of the extract from the second zone is 0.5 to 2 L / min, preferably 0.7 to 19 L / min. In modalities where part of the extract from the second zone is recycled to the second zone, the recycling rate is typically 0.6 to 1.4 L / min, preferably 0.7 to 1.8 mL / min, more preferably about 0.9 L / min. Typically, the flow rate of the raffinate in the second zone is 0.5 to 2.5 L / min, preferably 0.7 to 1.8 L / min, more preferably about 1.4 L / min.
As skilled in the art, you will see references to the rates at which the liquid is collected or removed through the various streams of extract and raffinate refer to the volumes of liquid removed in a period of time, typically L / minute. Similarly, references to the rates at which the liquid is recycled to the same zone, typically to an adjacent column in the same zone, refer to the volumes of liquid recycled over a period of time, typically L / minute.
Typically, part of one or more of the extract streams from the first zone, the streak stream from the first zone, the
Petition 870190102397, of 11/10/2019, p. 29/176 extract from the second zone and the raffinate stream from the second zone is recycled to the same zone, typically to an adjacent column in the same zone.
This recycling is different from feeding an extract or raffinate stream in a non-adjacent column in another zone. Instead, recycling involves feeding part of the extract or raffinate stream outside a zone back to the same zone, typically to an adjacent column in the same zone.
The rate at which liquid collected via the extract or raffinate stream from the first or second zones is recycled to the same zone is the rate at which liquid collected via this stream is fed back to the same zone, typically to an adjacent column. in the same zone. This can be seen with reference to figure 9. The extract recycling rate in the first zone is the rate at which the extract collected from the bottom of column 2 is fed to the top of column 3, that is, the flow of liquid at the top of the column 3. The extract recycling rate in the second zone is the rate at which the extract collected at the bottom of column 10 is fed at the top of column 11, that is, the flow of liquid to the top of column 11.
The recycling of the extract and / or raffinate streams is typically carried out by feeding the liquid collected through this stream into a container, and then pumping a quantity of this liquid from the container back to the same zone. In this case, the rate of recycling of liquid collected through a particular extract or raffinate stream, typically back to an adjacent column in the same zone, is the rate at which liquid is pumped out of the container back into the same zone , typically for an adjacent column.
As those skilled in the art will realize, the amount of liquid being introduced into a zone by means of the eluent and feed stock currents is balanced with the amount of liquid removed from a zone and recycled to the same zone. So, with reference to figure 9,
Petition 870190102397, of 11/10/2019, p. 30/176 for the extract stream, the flow of eluent (desorbent) for the first or second zone (D) is equal to the rate at which liquid collected through the extract stream of this zone accumulates in a container (E1 / E2) added with the rate at which the extract is recycled to the same zone (D-E1 / DE2). For the raffinate stream in a zone, the rate at which the extract is recycled to a zone (D-E1 / D-E2) plus the rate at which the feed stock is introduced into a zone (F / R1) is equal to the rate at which liquid collected through the raffinate stream from this zone accumulates in a container (R1 / R2) plus the rate at which raffinate is recycled to the same zone (D + F-E1-R1 / D + R1 = E2-R2).
The rate at which liquid collected from a particular streak or extract stream from a zone accumulates in a container can also be considered the net rate of removal of this streak or extract stream from that zone.
Typically, the rate at which liquid collected via the extract stream out of the first zone is recycled to the first zone differs from the rate at which the liquid collected via the extract stream out of the second zone is recycled to the second zone, and / or the rate at which liquid collected via this raffinate stream out of the first zone differs from the rate at which liquid collected via the raffinate stream out of the second zone is recycled to the second zone .
The variation in the rate at which liquid collected through the extract and / or raffinate streams in each zone is recycled to the same zone has the effect of varying the amount of more polar and less polar components present in the other extract and raffinate streams. Thus, for example, a lower rate of recycling of the extract causes some of the less polar components in this zone to be carried through the raffinate stream in this zone. A higher rate of recycling of the extract makes
Petition 870190102397, of 11/10/2019, p. 31/176 more of the less polar components in this zone are loaded through the raffinate chain in this zone. This can be seen, for example, in the specific embodiment of the invention shown in figure 6. The rate at which liquid collected via the extract stream in the first zone is recycled to the same zone (D-E1) will affect the extent to which any component A is loaded through the raffinate current in the first zone (R1).
Typically, the rate at which liquid collected via the extract stream from the first zone is recycled to the first zone is greater than the rate at which liquid collected via the extract stream from the second zone is recycled to the second zone. Preferably, a stream of raffinate containing the PUFA product together with more polar components is collected from a column in the first zone and introduced into a non-adjacent column in the second zone, and the rate at which liquid is collected via the extract stream from the first zone is recycled to the first zone is greater than the rate at which liquid collected through the extract stream from the second zone is recycled to the second zone.
Alternatively, the rate at which liquid collected via the extract stream from the first zone is recycled to the first zone is less than the rate at which liquid collected via the extract stream from the second zone is recycled to the second zone.
Typically, the rate at which liquid collected via the streak from the second zone is recycled to the second zone is greater than the rate at which liquid collected via the streak from the first zone is recycled to the first zone. Preferably, an extract stream containing the PUFA product together with less polar components is collected from a column in the second zone and introduced into a non-adjacent column in the first zone, and the rate at which liquid is collected via the raffinate stream from the second zone. is recycled to the second zone is greater than the rate at which liquid collected through the
Petition 870190102397, of 11/10/2019, p. 32/176 first zone is recycled to the first zone.
Alternatively, the rate at which liquid collected via the streak from the second zone is recycled to the second zone is less than the rate at which liquid collected via the streak from the first zone is recycled to the first zone.
The stage time, that is, the time between the displacement of the injection points of the feed and eluent mixture, and the various points of withdrawal of the collected fractions, is not particularly limited, and will depend on the number and dimensions of the columns used, and flow through the device. Those skilled in the art will be able to easily determine the appropriate time of steps to use in the process of the present invention. The step time is typically from 100 to 1,000 seconds, preferably from 200 to 800 seconds, more preferably from about 250 to about 750 seconds. In some embodiments, a step time of 100 to 400 seconds, preferably 200 to 300 seconds, more preferably about 250 seconds is appropriate. In other embodiments, a step time of 600 to 900 seconds, preferably 700 to 800 seconds, more preferably about 750 seconds is appropriate.
In the process of the present invention, real moving bed chromatography is preferred.
Conventional adsorbents known in the art for real and simulated moving bed systems can be used in the process of the present invention. Each chromatographic column can contain the same adsorbent, or a different adsorbent. Typically, each column contains the same adsorbent. Examples of such commonly used materials are polymeric microspheres, preferably cross-linked polystyrene with DVB (divinylbenzene), and silica gel, preferably silica gel bound in the reverse phase with C8 or C18 alkanes; especially C18. C18 reverse phase bonded silica gel is preferred. The adsorbent used in the process of the present invention is
Petition 870190102397, of 11/10/2019, p. 33/176 preferably non-polar.
The shape of the material of the stationary phase of the adsorbent can be, for example, spherical or non-spherical granules, preferably substantially spherical granules. Such microspheres typically have a diameter of 40 to 500 microns; preferably 100 to 500 microns, more preferably 250 to 500 microns, even more preferably 250 to 400 microns, above all preferably 250 to 350 microns. These preferred particle sizes are slightly larger than the microsphere particle sizes used in the past in simulated and real moving bed processes. Use of large particles allows a lower eluent pressure to be used in the system. This, in turn, has advantages in terms of cost reductions, efficiency and device life. It has surprisingly been found that adsorbent microspheres of large particle size can be used in the process of the present invention (with its associated advantages) without any loss in resolution.
The adsorbent typically has a pore size of 10 to 50 nm, preferably 1.5 to 45 nm, more preferably 20 to 40 nm, above all preferably 25 to 35 nm.
The eluent used in the process of the present invention is an aqueous alcohol. Aqueous alcohol typically comprises water and one or more small chain alcohols. Small chain alcohol typically has 1 to 6 carbon atoms. Examples of suitable alcohols include methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol and t-butanol. Methanol and ethanol are preferred. Methanol is more preferred.
Typically, the eluent is not in a supercritical state. Typically, the eluent is a liquid.
Typically, the average water: alcohol ratio of the eluent throughout the apparatus is 0.1: 99.9 to 9:91 parts by volume, preferably 0.25: 99.75 to 7:93 parts by volume, more preferably 0.5: 99.5 to 6:94
Petition 870190102397, of 11/10/2019, p. 34/176 parts by volume.
The elution strength of the eluent in each of the zones is typically different. Preferably, the eluting power of the eluent in the first zone is greater than that of the eluent in the second zone and in the subsequent zones. In practice, this is achieved by varying the relative amounts of water and alcohol in each zone. Alcohols are generally more powerful desorbents than water. Thus, the amount of alcohol in the eluent in the first zone is typically greater than the amount of alcohol in the eluent in the second zone and subsequent zones.
In modalities where the aqueous alcohol present in each zone has a different content of water and alcohol, the water: alcohol ratio of the eluent in the first zone is typically 0: 100 to 5:95 parts by volume, preferably 0.1: 99 , 9 to 2.5: 97.5 parts by volume, more preferably from 0.25: 99.75 to 2:98 parts by volume, and above all preferably from 0.5: 99.5 to 1.5: 98.5 parts by volume. In these embodiments, the water: alcohol ratio of the eluent in the second zone is typically from 3:97 to 7:93 parts by volume, preferably from 4:96 to 6:94 parts by volume, more preferably from 4.5: 95.5 to 5.5: 94.5 parts by volume.
In a particularly preferred modality where the aqueous alcohol present in each zone has a different water and alcohol content, the water: alcohol ratio of the eluent in the first zone is 0.5: 99.5 to 1.5: 99.5 parts by volume, and the water: alcohol ratio of the eluent in the second zone is 4: 5 95: 5 to 5.5: 94.5 parts by volume.
In modalities where the rate at which liquid collected by means of extract and raffinate streams in each zone is recycled to the same zone is adjusted in such a way that the PUFA product can be separated from different components of the feed mixture in each zone , the water: alcohol ratio of the eluents in each zone can be the same or different. Typically, water: alcohol ratio of the eluent in each zone is
Petition 870190102397, of 11/10/2019, p. 35/176
0.5: 99.5 to 5.5: 94.5 parts by volume. In one embodiment, the water: alcohol ratio of the eluent in the first zone is less than the water: alcohol ratio of the eluent in the second zone. In another embodiment, the water: alcohol ratio of the eluent in the first zone is greater than the water: alcohol ratio of the eluent in the second zone. In an additional modality, the water: alcohol ratio of the eluent in the first zone is the same as the water: alcohol ratio in the second zone.
It is noticed that the water and alcohol ratios in each zone mentioned above are average ratios in the entire zone.
Typically, the water: alcohol ratio of the eluent in each zone is controlled by introducing water and / or alcohol into one or more columns in the zones. Thus, for example, to obtain a water: lower alcohol ratio in the first zone than in the second zone, water is typically introduced more slowly in the first zone than in the second zone. In some modalities, essentially pure alcohol and essentially pure water can be introduced at different points in each zone. The relative flow rates of these two streams will determine the general solvent profile across the zone. In other modalities, different alcohol / water mixtures can be introduced at different points in each zone. This will involve introducing two or more different alcohol / water mixtures into the area, each alcohol / water mix with a different alcohol: water ratio. The relative flow rates and relative concentrations of alcohol / water mixtures in this mode will determine the profile of the general solvent across the zone. In other modalities where the water: alcohol ratio of the eluent in each zone is the same, the same mixture of alcohol / water is introduced in each zone.
Typically, the process of the present invention is conducted at 15 to 55 ° C, preferably 20 to 40 ° C; more preferably at about 30 ° C. Thus, the process is typically carried out at room temperature, but can be conducted at elevated temperatures.
The process of the present invention involves introducing a
Petition 870190102397, of 11/10/2019, p. 36/176 supply current in one zone (for example, the first zone), collect a first intermediate stream enriched with the PUFA product and introduce the first intermediate stream in another zone (for example, the second zone). Thus, when the device has two zones, the process involves both (a) collecting a first intermediate stream from the first zone and introducing it into the second zone, and (b) collecting a first intermediate stream from the second zone and introducing it into the first zone zone. In this way, the PUFA product can be separated from both more and less polar components in a single process.
Both (a) a stream of raffinate containing the PUFA product together with more polar components is collected from a column in the first zone and introduced into a non-adjacent column in the second zone, and (b) an extract stream containing the PUFA product together with less polar components are collected from a column in the second zone and introduced into a non-adjacent column in the first zone.
In a particularly preferred embodiment, the apparatus has two zones and the process of the present invention comprises:
(i) introduce the feed mixture into the first zone, and remove a first stream of raffinate enriched with the PUFA product and a first stream of depleted extract from the PUFA product, and (ii) introduce the first stream of raffinate into the second zone, move a second stream of raffinate depleted of the PUFA product, and collect a second stream of extract to obtain the PUFA product.
This particularly preferred embodiment is suitable for purifying EPA from a feed mixture.
This particularly preferred embodiment is illustrated in figure 2. A feed mixture F comprising the product PUFA (B)
Petition 870190102397, of 11/10/2019, p. 37/176 and more polar (C) and less polar (A) components are introduced in the first zone. In the first zone, the less polar components (A) are removed as a stream of extract E1. The PUFA product (B) and more polar components (C) are removed as stranded R1. The raffinate stream R1 is then introduced into the second zone. In the second zone, the more polar components (C) are removed as a raffinate chain R2. The PUFA product (B) is collected as an E2 extract stream.
This modality is illustrated in more detail in figure 4. Figure 4 is identical to figure 2, except that the points of introduction of the alcohol (D) and water (W) desorbent in each zone are shown. The alcohol (D) and water (W) desorbent together constitute the eluent. Phase (D) is typically essentially pure alcohol, but may, in certain embodiments, be an alcohol / water mixture comprising mainly alcohol. The (W) phase is typically essentially pure water, but can, in certain embodiments, be an alcohol / water mixture comprising mainly water, for example, a mixture of 98% water / 2% methanol.
A further illustration of this particularly preferred embodiment is shown in figure 6. There is no separate water injection point here and instead a water-alcoholic desorbent is injected into (D).
The separation in the stream of raffinate and extract can be helped by varying the desorption potency of the eluent in each zone. This can be achieved by introducing the alcohol (or rich in alcohol) component of the eluent and the water (or rich in water) component at different points in each zone. Thus, typically, alcohol is introduced upstream of the extract withdrawal point and water is introduced between the extract withdrawal point and the feed introduction point in the zone, with respect to the eluent flow in the system. This is shown in figure 4.
Alternatively, separation can be helped by varying the
Petition 870190102397, of 11/10/2019, p. 38/176 rates at which the liquid collected through the extract and raffinate streams from the two zones is recycled to the same zone.
Typically, in this particularly preferred embodiment, the rate at which liquid collected via the extract stream from the first zone is recycled to the first zone is greater than the rate at which liquid collected via the extract stream from the second zone is recycled to the second zone, or the water: alcohol ratio of the eluent in the first zone is less than that in the second zone.
In this particularly preferred embodiment, the first streak of streak in the first zone is typically removed downstream of the point of introduction of the feed mixture in the first zone, with respect to the flow of eluent in the first zone.
In this particularly preferred embodiment, the first extract stream in the first zone is typically removed upstream of the point of introduction of the feed mixture in the first zone, with respect to the flow of eluent in the first zone.
In this particularly preferred embodiment, the second streak in the second zone is typically removed downstream of the point of introduction of the first streak in the second zone, with respect to the eluent flow in the second zone.
In this particularly preferred embodiment, the second extract stream in the second zone is typically collected upstream of the point of introduction of the first raffinate stream in the second zone, with respect to the eluent flow in the second zone.
Typically, in this particularly preferred embodiment, alcohol or aqueous alcohol is introduced into the first zone upstream of the point of removal of the first extract stream, with respect to the flow of eluent in the first zone.
Typically, in this particularly preferred embodiment,
Petition 870190102397, of 11/10/2019, p. 39/176 when water is introduced in the first zone, water is introduced in the first zone upstream of the point of introduction of the feed mixture, but downstream of the removal point of the first extract stream, with respect to the flow of eluent in the first zone.
Typically, in this particularly preferred embodiment, alcohol or aqueous alcohol is introduced into the second zone upstream of the point of removal of the second extract stream with respect to the flow of eluent into the second zone.
Typically, in this particularly preferred embodiment, when water is introduced into the second zone, water is introduced into the second zone upstream of the point of introduction of the first streak of raffinate, but downstream of the point of removal of the second extract stream, with respect to the eluent flow in the second zone.
In another particularly preferred embodiment, the apparatus has two zones, and the process comprises:
(i) introducing the feed mixture into the second zone, and removing a first stream of depleted raffinate from the PUFA product and a first extract stream enriched in the PUFA product, and (ii) introducing the first extract stream into the first zone, removing a second stream of depleted extract of the PUFA product, and collect a second stream of raffinate to obtain the PUFA product.
This particularly preferred embodiment is suitable for purifying DHA from a feed mixture.
This modality is illustrated in figure 3. A feed mixture F comprising the product PUFA (B) and more polar (C) and less polar (A) components is introduced in the second zone in the second zone, the more polar components (C) are removed as stranded chain R1. The PUFA product (B) and less polar components (A) are
Petition 870190102397, of 11/10/2019, p. 40/176 collected as extract chain E1. The extract stream E1 is then introduced into the first zone. In the first zone, the less polar components (A) are removed as a stream of extract E2. The PUFA product (B) is collected as a raffinate stream R2.
This modality is illustrated in more detail in figure 5. Figure 5 is identical to figure 3, except that the points of introduction of the small chain alcohol (D) and water (W) desorbent in each zone are shown. As previously, phase (D) is typically essentially pure alcohol, but in certain embodiments it may be an alcohol / water mixture comprising mainly alcohol. The (W) phase is typically essentially pure water, but may, in certain embodiments, be an alcohol / water mixture comprising mainly water, for example, a mixture of 98% water / 2% methanol.
A further illustration of this particularly preferred embodiment is shown in figure 7. Here, there is no separate water injection point, and instead a water alcohol desorbent is injected into (D).
Typically, in this embodiment, the rate at which liquid collected via the streak from the second zone is reintroduced into the second zone is greater than the rate at which liquid collected via the streak from the first zone is reintroduced into the first zone or the water: alcohol ratio of the eluent in the first zone is less than that in the second zone.
In this particularly preferred second embodiment, the first raffinate stream in the second zone is typically removed downstream from the point of introduction of the feed mixture in the second zone, with respect to the flow of eluent in the second zone.
In this particularly preferred second embodiment, the first extract stream in the second zone is typically collected upstream of the feed mixture introduction point in the second zone,
Petition 870190102397, of 11/10/2019, p. 41/176 with respect to the flow of eluent in the second zone.
In this particularly preferred second embodiment, the second stream of raffinate in the first zone is typically collected downstream from the point of introduction of the first extract stream in the first zone, with respect to the flow of eluent in the first zone.
In this particularly preferred second embodiment, the second extract stream in the first zone is typically removed upstream of the point of introduction of the first extract stream in the first zone, with respect to the eluent flow in the first zone.
Typically, in this particularly preferred second embodiment, alcohol or aqueous alcohol is introduced into the second zone upstream of the point of removal of the first extract stream, with respect to the flow of eluent into the second zone.
Typically, in this particularly preferred second embodiment, when water is introduced into the second zone, water is introduced into the second zone upstream of the feed mixture introduction point, but downstream of the removal point of the first extract stream, with respect to the eluent flow in the second zone.
Typically, in this particularly preferred second embodiment, alcohol or aqueous alcohol is introduced into the first zone upstream of the point of removal of the second extract stream, with respect to the flow of eluent in the first zone.
Typically, in this particularly preferred second mode, when water is introduced into the first zone, water is introduced into the first zone upstream of the point of introduction of the first stream of raffinate, but downstream of the point of removal of the second stream of extract, with respect to to the eluent flow in the first zone.
In a preferred embodiment of the invention, the simulated or real moving bed chromatography apparatus consists of fifteen columns
Petition 870190102397, of 11/10/2019, p. 42/176 chromatographic. These are referred to as columns 1 through 15. The fifteen columns are arranged in series so that the base of column 1 is attached at the top of column 2, the base of column 2 is attached at the top of column 3, etc. This can optionally be by means of a containment container, with a recycling stream in the next column. The flow of eluent through the system is from column 1 to column 2 to column 3 etc. The adsorbent flow through the system is from column 15 to column 14 to column 13, etc.
In an above all preferred embodiment, the first zone typically consists of eight adjacent columns, columns 1 to 8, which are connected as discussed above. In this most preferred mode, the second zone typically consists of seven columns, columns 9 to 15, which are connected as discussed above. To avoid duplication, the base of column 8 in the first zone is connected at the top of column 9 in the second zone.
An above all preferred embodiment is illustrated in figure 8. A feed mixture F comprising the product PUFA, (B) and more polar (C) and less polar (A) components is introduced at the top of column 5 in the first zone. Alcohol desorbent is introduced at the top of column 1 in the first zone. Water is introduced at the top of column 4 in the first zone. In the first zone, the less polar components (A) are removed as an extract stream E1 from the base of column 2. The product PUFA (B) and more polar components (C) are removed as a raffinate stream R1 from the base of column 7. The raffinate stream R1 is then introduced in the second zone at the top of column 13. Alcohol desorbent is introduced at the top of column 9 in the second zone. Water is introduced at the top of column 12 in the second zone. In the second zone, the most polar components (C) are removed as raffinate stream R2 at the base of column 15. The PUFA product (B) is collected as extract stream E2 at the base of column 10.
In this most preferred mode, alcohol is
Petition 870190102397, of 11/10/2019, p. 43/176 typically introduced at the top of column 1 in the first zone.
In this most preferred embodiment, water is typically introduced at the top of column 4 in the first zone.
In this most preferred embodiment, alcohol is typically introduced at the top of column 9 in the second zone.
In this most preferred embodiment, alcohol is typically introduced at the top of column 12 in the second zone.
In this above all preferred embodiment, the supply current is typically introduced at the top of column 5 in the first zone.
In this above all preferred embodiment, a first streak of streak is typically collected from the base of column 7 in the first zone and introduced at the top of column 13 in the second zone. The first stream of raffinate can optionally be collected in a container before being introduced in column 13.
In this most preferred embodiment, a first extract stream is typically removed from the base of column 2 in the first zone. The first extract stream can optionally be collected in a container and reintroduced at the top of column 3 in the first zone.
In this most preferred embodiment, a second stream of raffinate is typically removed from the base of the column 15 in the second zone.
In this most preferred mode, a second extract stream is typically collected from the base of the column in the second zone. This second extract stream typically contains the purified PUFA product. The second extract stream can optionally be collected in a container and reintroduced at the top of column 11 in the second zone.
Typically, in this most preferred mode, the water: alcohol ratio in the first zone is less than the water: alcohol ratio in the second zone.
Petition 870190102397, of 11/10/2019, p. 44/176
An additional, above all preferred embodiment is illustrated in figure 9. A feed mixture F comprising the product PUFA (B) and more polar (C) and less polar (A) components is introduced at the top of column 5 in the first zone. Aqueous alcohol desorbent is introduced at the top of column 1 in the first zone. In the first zone, the less polar components (A) are removed as an extract stream E1 from the base of column 2. The product PUFA (B) and more polar components (C) are removed as a raffinate stream R1 from the base of column 7. The raffinate stream R1 is then introduced in the second zone at the top of column 12. Aqueous alcohol desorbent is introduced at the top of column 9 in the second zone. In the second zone, the more polar components (C) are removed as raffinate stream R2 at the base of column 14. The PUFA product (B) is collected as extract stream E2 at the base of column 10.
In this most preferred embodiment, aqueous alcohol is typically introduced at the top of column 1 in the first zone.
In this most preferred embodiment, aqueous alcohol is typically introduced at the top of column 9 in the second zone.
In this above all preferred embodiment, the supply current is typically introduced at the top of column 5 in the first zone.
In this above all preferred embodiment, a first streak of streak is typically collected from the base of column 7 in the first zone and introduced at the top of column 12 in the second zone. The first streak of raffinate can optionally be collected in a container before being introduced in column 12.
In this most preferred embodiment, a first extract stream is typically removed from the base of column 2 in the first zone. The first extract stream can optionally be collected in a container and a portion reintroduced at the top of column 3 in the first zone. The rate of recycling of liquid collected through the extract stream of the
Petition 870190102397, of 11/10/2019, p. 45/176 first zone back to the first zone is the rate at which liquid is pumped from this container to the top of column 3.
In this most preferred embodiment, a second strand of streak is typically removed from the base of the column 14 in the second zone.
In this most preferred mode, a second extract stream is typically collected from the base of column 10 in the second zone. This second extract stream typically contains the purified PUFA product. The second extract stream can optionally be collected in a container and a portion reintroduced at the top of column 11 in the second zone. The rate of recycling of liquid collected through the extract stream from the second zone back into the second zone is the rate at which liquid is pumped from this container at the top of column 11.
In this most preferred mode, the rate at which liquid collected via the extract stream from the first zone is typically recycled to the first zone is typically greater than the rate at which liquid collected via the extract stream from the second zone is recycled to the second zone.
In this most preferred embodiment, the aqueous alcohol eluent is substantially the same in each zone.
In a further preferred embodiment of the invention, the simulated or real moving bed chromatography apparatus consists of nineteen chromatographic columns. These are referred to as columns 1 through 19. The fifteen columns are arranged in series so that the base of column 1 is linked at the top of column 2, the base of column 2 is linked at the top of column 3, etc. The eluent flow through the system is from column 11 to column 2 to column 3 etc. The adsorbent flow through the system is from column 19 to column 18 to column 17, etc.
In this modality, the first zone typically consists of
Petition 870190102397, of 11/10/2019, p. 46/176 ten adjacent columns, columns 1 to 10, which are connected as discussed earlier. The second zone typically consists of eight columns, columns 11 to 19 that are connected as discussed earlier.
This additional preferred embodiment is illustrated in figure 10. A feed mixture F comprising the product PUFA (B) and more polar (C) and less polar (A and A ') components is introduced at the top of column 7 in the first zone. A first desorbent (D1) comprising 100% alcohol is introduced at the top of column 1 in the first zone. A second desorbent (D2) comprising a water / alcohol mixture (preferably 2% methanol and 98% water) is introduced at the top of column 5 in the first zone. In the first zone, less polar components (A ') and (A) are removed as extract streams E1' and E1 from the bases of columns 1 and 4, respectively. The PUFA product (B) and more polar components (C) are removed as raffinate chain R1 at the base of the column
10. The raffinate stream R1 is then introduced into the second zone at the top of the column 17. A second desorbent (D2) comprising a water / alcohol mixture (preferably 2% methanol and 98% water) is introduced at the top of the column 11 in the second zone. In the second zone, the more polar components (C) are removed as raffinate stream R2 at the base of column 19. The product PUFA (B) is collected as extract stream E2 at the base of column, 14.
In this preferred embodiment, alcohol is typically introduced at the top of column 1 in the first zone.
In this preferred embodiment, a mixture of 2% MeOH / 98% water is typically introduced at the top of column 5 in the first zone.
In this preferred embodiment, a mixture of 2% MeOH / 98% water is typically introduced at the top of column 11 in the second zone.
In this preferred embodiment, the supply current is
Petition 870190102397, of 11/10/2019, p. 47/176 typically introduced at the top of column 7 in the first zone.
In this preferred embodiment, a first streak of streak is typically collected from the base of the column 10 in the first zone and introduced at the top of the column 17 in the second zone. The first streak of raffinate can optionally be collected in a container before being introduced in column 17.
In this preferred embodiment, extract streams are typically removed from the bases of columns 1 and 4 in the first zone. The extract stream collected from the base or column 4 can optionally be collected in a container and reintroduced at the top of column 5 in the first zone.
In this preferred embodiment, a second streak of streak is typically removed from the base of the column 19 in the second zone.
In this preferred embodiment, a second extract stream is typically collected from the base of column 14 in the second zone. This second extract stream typically contains the purified PUFA product. The second extract stream can optionally be collected in a container and reintroduced at the top of column 15 in the second zone.
Typically, in this most preferred mode, the water: alcohol ratio in the first zone is less than the water: alcohol ratio in the second zone.
The process of the invention allows much higher purities of PUFA product to be obtained than was possible with conventional chromatographic techniques. The PUFA products produced by the process of the invention also have particularly advantageous impurity profiles, which are very different from those observed in oils prepared by known techniques. The present invention therefore also relates to compositions comprising a PUFA product, for example, one obtainable by the process of the present invention.
Petition 870190102397, of 11/10/2019, p. 48/176
Thus, in one embodiment, the present invention also relates to a composition comprising a PUFA product, in which the PUFA product is EPA, the PUFA product is present in an amount greater than 93% by weight, and the total fatty acid content polyunsaturated ω-6 is up to 0.40% by weight.
As used herein,% by weight of a component is relative to the total weight of the composition.
The product PUFA and PUFAs ω-6 are optionally in the form of their alkyl esters, typically ethyl esters. Preferably, the PUFA EPA product is in the form of its ethyl ester.
Typically, in this embodiment, the PUFA EPA product is present in an amount greater than 94% by weight, preferably greater than 95% by weight, greater preferably greater than 96% by weight, even greater preferably greater than 97% by weight, and above preferably everything greater than 98% by weight.
The total content of ω-6 polyunsaturated fatty acids in this modality is up to 0.40% by weight. Thus, the composition typically comprises an amount of ω-6 polyunsaturated fatty acids even in this amount. Typically, the total content of polyunsaturated fatty acids ω-6 is up to 0.35% by weight, preferably up to 03% by weight, more preferably up to 0.25% by weight, and above all preferably up to 0.22% in weight. Typically, the total content of ω-6 polyunsaturated fatty acids is 0.05% by weight or more, preferably 0.1% by weight or more.
Typically, in this embodiment, the content of arachidonic acid is up to 025% by weight, preferably up to 0.24% by weight, more preferably up to 0.23% by weight and above all preferably up to 0.22% by weight. Thus, the composition typically comprises an amount of arachidonic acid up to those amounts. Typically, the total arachidonic acid content is 0.05% by weight or more, preferably 0.1% by weight
Petition 870190102397, of 11/10/2019, p. 49/176 or more.
Typically, in this embodiment, the total content of polyunsaturated fatty acids ω-3 is greater than 97% by weight, preferably greater than 97.5% by weight, more preferably greater than 97.9% by weight, in certain embodiments, the total content of ω-3 polyunsaturated fatty acids is greater than 99% by weight.
Typically, in this embodiment, the total DHA content is up to 1% by weight, preferably up to 0.6% by weight, more preferably up to 0.3% by weight, most preferably up to 0.2% by weight. Thus, the composition typically comprises an amount of DHA up to those amounts. Typically, the total DHA content is 0.05% by weight or more, preferably 0.1% by weight or more.
Typically, in this embodiment, the total content of DHA is up to 0.2% by weight, preferably up to 0.175% by weight, more preferably up to 0.16% by weight. Thus, the composition typically comprises an amount of DHA up to those amounts. Typically, the total DHA content is 0.05% by weight or more, preferably 0.1% by weight or more.
Typically, in this embodiment, the total content of αlinolenic acid is up to 1% by weight, preferably up to 0.6% by weight, more preferably up to 0.3% by weight. Thus, the composition typically comprises an amount of α-linolenic acid up to those amounts. Typically, the total content of α-linolenic acid is 0.05% by weight or more, preferably 0.1% by weight or more.
Typically, in this embodiment, the total content of αlinolenic acid is up to 0.35% by weight, preferably up to 0.3% by weight, more preferably up to 0.29% by weight. Thus, the composition typically comprises an amount of α-linolenic acid in these amounts. Typically, the total content of α-linolenic acid is 0.05% by weight or more, preferably 0.1% by weight or more.
Petition 870190102397, of 11/10/2019, p. 50/176
Typically, in this embodiment, the total stearidonic acid content is up to 1% by weight, preferably up to 0.6% by weight, more preferably up to 0.3% by weight. Thus, the composition typically comprises an amount of stearidonic acid up to those amounts. Typically, the total stearidonic acid content is 0.05% by weight or more, preferably 0.1% by weight or more.
Typically, in this embodiment, the total stearidonic acid content is up to 0.4% by weight, preferably up to 0.35% by weight, more preferably up to 0.34% by weight. Thus, the composition typically comprises an amount of stearidonic acid up to those amounts. Typically, the total stearidonic acid content is 0.05% by weight or more, preferably 0 1% by weight or more.
Typically, in this embodiment, the total eicosatetraenoic acid content is up to 1% by weight, preferably up to 0.75% by weight, more preferably up to 0.5% by weight. Thus, the composition typically comprises an amount of eicosatetraenoic acid up to those amounts. Typically, the total content of stearidonic acid is 0.5% by weight or more preferably 0.1% by weight or more.
Typically, in this embodiment, the total eicosatetraenoic acid content is up to 1% by weight, preferably up to 0.75% by weight, more preferably up to 0.5% by weight. Thus, the composition typically comprises an amount of eicosatetraenoic acid up to those amounts. Typically, the total eicosatetraenoic acid content is 0.05% by weight or more, preferably 0.1% by weight or more.
Typically, in this embodiment, the total eicosatetraenoic acid content is up to 0.5% by weight, preferably up to 0.475% by weight, more preferably up to 0.46% by weight. Thus, the composition typically comprises an amount of eicosatetraenoic acid up to those amounts. Typically, the total eicosatetraenoic acid content is 0.05% by weight or
Petition 870190102397, of 11/10/2019, p. 51/176 more, preferably 0.1% by weight or more.
Typically, in this embodiment, the total content of docosapentaenoic acid is up to 1% by weight, preferably up to 0.6% by weight, more preferably up to 0.3% by weight. Thus, the composition typically comprises an amount of docosapentaenoic acid up to those amounts. Typically, the total content of docosapentaenoic acid is 0.5% by weight or more, preferably 0.1% by weight or more.
Typically, in this embodiment, the total content of docosapentaenoic acid is up to 0.4% by weight, preferably up to 0.35% by weight, more preferably up to 0.33% by weight. Thus, typically, the composition comprises an amount of docosapentaenoic acid. up to those quantities. Typically, the total content of docosapentaenoic acid is 0.05% by weight or more, preferably 0.1% by weight or more.
In this embodiment, the composition preferably comprises more than 96.5% by weight of EPA, up to 1% by weight of DHA up to 1% by weight of α-linolenic acid, up to 1% by weight of stearidonic acid, up to 1% by weight eicosatetraenoic acid, up to 1% by weight of docosapentaenoic acid, and up to 0.25% by weight of arachidonic acid.
In this embodiment, the composition preferably comprises more than 96.5% by weight of EPA, up to 0.2% by weight of DHA, up to 0.3% by weight of α-linolenic acid, up to 0.4% by weight of acid stearidonic, up to 0.5% by weight of eicosatetraenoic acid, up to 0.35% by weight of docosapentaenoic acid, and up to 0.25% by weight of arachidonic acid.
In this embodiment, the composition most preferably comprises from 96.5 to 99% by weight of EPA, up to 0.6% by weight of DHA, up to 0.6% by weight, α-linolenic acid from 0.15 to 0.6 % by weight of stearidonic acid from 0.1 to 0.75% by weight of eicosatetraenoic acid, up to 0.6% by weight of docosapentaenoic acid, and up to 0.6% by weight of arachidonic acid.
Petition 870190102397, of 11/10/2019, p. 52/176
In this embodiment, the composition most preferably comprises from 96.5 to 99% by weight of EPA, up to 0.2% by weight of DHA, up to 0.3% by weight of α-linolenic acid, from 0.15 to 0, 4% by weight of stearidonic acid, from 0.1 to 0.5% by weight of eicosatetraenoic acid, up to 0.35% by weight of docosapentaenoic acid and up to 0.25% by weight of arachidonic acid.
In this embodiment, the composition above all preferably comprises from 98 to 99% by weight of EPA, from 0.1 to 0.3% by weight of DHA, from 0.3 to 035% by weight of stearidonic acid, from 0, 1 to 0.3% by weight of eicosatetraenoic acid, and from 0.3 to 0.35% by weight of docosapentaenoic acid.
In this embodiment, the composition above all preferably comprises from 96.5 to 99% by weight of EPA, from 0.1 to 0.5% by weight of DHA, from 0.1 to 05% by weight of stearidonic acid, of 0.1 to 0.5% by weight of eicosatetraenoic acid, and from 0.1 to 0.5% by weight of docosapentaenoic acid, and from 0.1 to 0.3% by weight of arachidonic acid.
In this embodiment, the composition above all preferably comprises from 98 to 99% by weight of EPA, from 0.1 to 0.2% by weight of DHA, from 0.3 to 0.35% by weight of stearidonic acid, of 0.1 to, 0.2% by weight of eicosatetraenoic acid and from 0.3 to 0.35,% by weight of docosapentaenoic acid.
In this embodiment, the composition above all preferably comprises from 96.5 to 97.5% by weight of EPA, from 0.25 to 0.35% by weight of α-linolenic acid, from 0.18 to 0.24% by weight of stearidonic acid, from 0.4 to 0.46% by weight of eicosatetraenoic acid and from 0.15 to 0.25% by weight of arachidonic acid.
Typically, in this embodiment, the content of isomeric impurities is up to 1.5% by weight. Typically, the content of isomeric impurities is up to 1% by weight, preferably up to 0.5% by weight, more preferably
Petition 870190102397, of 11/10/2019, p. 53/176 up to 0.25% by weight, even more preferably up to 0.25% by weight, and above all preferably up to 0.1% by weight.
In a further embodiment, the present invention also relates to a composition comprising a PUFA product, in which the PUFA product is a mixture of EPA and DHA, in which (i) the total content of EPA and DHA is 80% by weight o or more, (ii) the EPA content is 41 to 60% by weight and the DHA content is 16 to 48% by weight, and (iii) the total content of ω-3 polyunsaturated fatty acids is 94 % by weight or more and / or the total content of polyunsaturated fatty acids ω-3 is up to 4% by weight.
The PUFA product, PUFAs ω-3 and ω-6 are optionally in the form of their alkyl esters, typically ethyl esters. Preferably, the PUFA EPA / DHA product is in the form of its ethyl esters.
Thus, in this additional embodiment the composition is typically a composition comprising a PUFA product, in which the PUFA product is a mixture of EPA and DHA, in which (i) the total content of EPA and DHA is 80% by weight or more, ( ii) the EPA content is 41 to 60% by weight and the DHA content is 16 to 48% by weight, and (iii) the total content of ω-3 polyunsaturated fatty acids is 94% by weight or more .
Alternatively, in this additional embodiment the composition is a composition comprising a PUFA product, in which the PUFA product is a mixture of EPA and DHA, in which (i) the total content of EPA and DHA is 80% by weight or more, (ii ) the EPA content is 41 to 60% by weight and the DHA content is 16 to 4.8% by weight and (iii) the total content of ω-6 polyunsaturated fatty acids is up to 4% by weight.
Typically, in this additional embodiment the total content of EPA and DHA is 82% by weight or more, preferably 83% by weight or more, more preferably 84% by weight or more, even more preferably 85% by weight or more, and above all preferably 86% by weight or more.
Typically, in this additional modality the EPA content is
Petition 870190102397, of 11/10/2019, p. 54/176 to 60% by weight, preferably from 45 to 60% by weight, more preferably from 47 to 60% by weight, even more preferably from 47 to 57% by weight, and above all preferably from 50 to 55% by weight .
Typically, in this additional embodiment the DHA content is 16 to 48% by weight, preferably 20 to 45% by weight, more preferably 25 to 42% by weight, even more, preferably 28 to 38% by weight, and above all preferably from 30 to 35% by weight.
Typically, in this additional embodiment the total content of polyunsaturated fatty acids ω-3 is 94% by weight or more, preferably 95% by weight or more, more preferably 96% by weight or more, and above all preferably 97% by weight. weight or more.
Typically, in this additional embodiment the total α-linolenic acid content is up to 0.4% by weight, preferably up to 0.35% by weight, more preferably up to 0.31% by weight. Thus, the composition typically comprises an amount of α-linolenic acid up to those amounts. Typically, the total content of α-linolenic acid is 0.05% by weight or more, preferably 0.1% by weight or more, more preferably 0.2% by weight or more, and still preferably 0.2 to 0 , 4% by weight.
Typically, in this additional embodiment the total stearidonic acid content is up to 1.9% by weight, preferably up to 1.5% by weight, more preferably up to 1.25% by weight. Thus, the composition typically comprises an amount of stearidonic acid up to those amounts. Typically, the total stearidonic acid content is 0.05% by weight or more, preferably 0.1% by weight or more.
Typically, in this additional embodiment the total eicosatetraenoic acid content is up to 2.0% by weight, preferably up to 1.9% by weight. Thus, the composition typically comprises an amount of eicosatetraenoic acid up to those amounts. Typically, the total eicosatetraenoic acid content is 0.05% by weight or more, preferably 0.1% by weight
Petition 870190102397, of 11/10/2019, p. 55/176 or more, more preferably 1.0% by weight or more, and even more preferably from 1.0 to 1.9% by weight.
Typically, in this additional embodiment the total eicosapentaenoic acid content is up to 3.0% by weight, preferably up to 2.75% by weight. Thus, the composition typically comprises an amount of eicosapentaenoic acid up to those amounts. Typically, the total eicosapentaenoic acid content is 0.05% by weight or more, preferably 0.1% by weight or more, more preferably 2% by weight or more, acid even more preferably from 2 to 2.75% by weight .
Typically, in this additional embodiment the total content of docosapentaenoic acid is up to 6% by weight, preferably up to 5.5% by weight, more preferably up to 5.25% by weight. Thus, the composition typically comprises an amount of docosapentaenoic acid up to those amounts. Typically, the total content of docosapentaenoic acid is 0.05% by weight or more, preferably 0.1% by weight or more, more preferably 4% by weight or more, and even more preferably from 4 to 5.25% by weight .
The total content of ω-6 polyunsaturated fatty acids in this additional embodiment is typically up to 4% by weight. Thus, the composition typically comprises an amount of ω6 polyunsaturated fatty acids up to those amounts. Typically, the total content of polyunsaturated fatty acids ω-6 is up to 3.75% by weight, preferably up to 3.5% by weight, more preferably up to 3.25% by weight, even more preferably up to 3% by weight, above all preferably up to 2.85% by weight. Typically, the total content of polyunsaturated fatty acids ω-6 is 0.05% by weight or more, preferably 0.1% by weight or more.
Typically, in this additional embodiment the total linoleic acid content is up to 0.5% by weight, preferably up to 0.4% by weight, more preferably up to 0.25% by weight. So, typically, the composition
Petition 870190102397, of 11/10/2019, p. 56/176 comprises an amount of linoleic acid up to those amounts. Typically, the total linoleic acid content is 0.05% by weight or more, preferably 0.1% by weight or more, more preferably 0.15% by weight or more, and even more preferably from 0.15 to 0, 25% by weight.
Typically, in this additional embodiment the total gamma-linolenic acid content is up to 0.19% by weight, preferably up to 0.15% by weight, more preferably up to 0.1% by weight. Thus, the composition typically comprises an amount of gamma-linolenic acid up to those amounts. Typically, the total gamma-linolenic acid content is 6.05% by weight or more, preferably 0.1% by weight or more.
Typically, in this additional embodiment the total content of diomo-gamma-linolenic acid is up to 0.1% by weight. Thus, the composition typically comprises an amount of diomo-gamma-linolenic acid up to those amounts. Typically, the total content of diomo-gamma-linolenic acid is 0.05% by weight or more.
Typically, in this additional embodiment the total arachidonic acid content is up to 2.5% by weight, preferably up to 2.25% by weight, more preferably up to 2.1% by weight. Thus, the composition typically comprises an amount of arachidonic acid up to those amounts. Typically, the total arachidonic acid content is 0.05% by weight or more, preferably 0.1% by weight or more.
Typically, in this additional embodiment, the total content of adrenic acid is up to 0.1% by weight. Thus, the composition typically comprises an amount of adrenic acid even in this amount. Typically, the total content of adrenic acid is 0.05% by weight or more.
Typically, in this additional embodiment the total content of docosapentaenoic acid (ω-6) is up to 0.9% by weight, preferably up to 0.75% by weight. More preferably up to 0.65% by weight. Thus, the composition typically comprises an amount of docosapentaenoic acid (ω-6)
Petition 870190102397, of 11/10/2019, p. 57/176 up to those quantities. Typically, the total content of docosapentaenoic acid (ω-6) is 0.05% by weight or more, preferably 0.1% by weight or more.
In this additional embodiment, the composition preferably comprises from 50 to 55% by weight of EPA, from 30 to 35% by weight of DHA, up to 0.4% by weight of α-linolenic acid, up to 1.25% by weight of acid stearidonic, up to 1.9% by weight of eicosatetraenoic acid, up to 2.75% by weight of eicosapentaenoic acid, up to 5.25% by weight of docosatetraenoic acid, up to 0.25% by weight of linoleic acid, up to 0.1 % by weight of gamma-linolenic acid, up to 0.1% by weight of diomo-gammalinolenic acid, up to 2.1% by weight of arachidonic acid, up to 0.1% by weight of adrenic acid, and up to 0.75% by weight of docosapentaenoic acid (ω-6).
In this additional embodiment, the composition most preferably comprises from 50 to 55% by weight of EPA, from 30 to 35% by weight of DHA, from 0.2 to 0.4% by weight of α-linolenic acid, up to 1.25 % by weight of stearidonic acid, from 1.0 to 1.9% by weight of eicosatetraenoic acid, from 2 to 2.75% by weight of eicosapentaenoic acid, from 4 to 5.25% by weight of docosapentaenoic acid, from 0 , 15 to 0.25% by weight of linoleic acid, up to 0.1% by weight of gamma-linolenic acid, up to 0.1% by weight of diomo-gamma-linolenic acid, up to 2.1% by weight of acid arachidonic, up to 0.1% by weight of adrenic acid, and up to 0.75% by weight of docosapentaenoic acid (ω-6).
Typically, in this additional embodiment, the content of isomeric impurities is up to 1.5% by weight. Typically, the content of isomeric impurities is up to 1% by weight, preferably up to 0.5% by weight, more preferably up to 0.25% by weight, even more preferably up to 0.25% by weight, and above all preferably up to 0.1% by weight.
The inventors have also surprisingly observed that oils can be produced with a small amount of environmental pollutants, compared to known oils. So, still in a
Petition 870190102397, of 11/10/2019, p. In a further embodiment, the present invention also relates to a composition comprising a PUFA product, as defined herein, in which (a) the total amount of polyaromatic hydrocarbons without the composition is up to 0.89 pg / kg (b) a total amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans is up to 0.35 pg / g (c) the total amount of polychlorinated biphenyls is up to 0.0035 mg / kg, and / or (d) the total amount of dioxins, furans, dibenzene-to-dioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls is up to 1 pg / g.
Typically, the present invention relates to a composition comprising a PUFA product as defined herein, in which (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.89 pg / kg (b) the total amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans is up to 0.35 pg / g and / or (d) the total amount of dioxins, furans and dioxin-like polychlorinated biphenyls is up to 1 pg / g.
The total amount of polyaromatic hydrocarbons still in this additional form in the composition is up to 0.89 pg / kg. Thus, typically, the composition comprises an amount of polyaromatic hydrocarbons up to this amount. Typically, the total amount of polyaromatic hydrocarbons in the composition is up to 0.85 pg / kg, preferably up to 0.9 pg / kg, more preferably up to 0.7 pg / kg, even more preferably up to 0.5 pg / kg, even more preferably up to 0.5 pg / kg, even more preferably up to 0.4 pg / kg, even more preferably up to 0.3 pg / kg, even more preferably up to 0.2 pg / kg, even more preferably up to 0.1 pg / kg and above all preferably up to 0.05 pg / kg.
Typical polyaromatic hydrocarbons are well known to those skilled in the art and include acenaphene, acenaphthene, anthracene, benzo [a] anthracene, benzo [a] pyrene, benzo [e] pyrene, benzo [b] fluoranthene, benzo [ghi] perylene, benzo [ j] fluoranthene, benzo [k] fluoranthene, chrysene,
Petition 870190102397, of 11/10/2019, p. 59/176 dibenz (ah) anthracene, fluoranthene, fluorine, indene (1,2,3-cd) pyrene, phenanthrene, pyrene, coronene, coranulene, tetracene, naphthalene, pentacene, triphenylene, and ovalene. Typically, the amounts mentioned above refer to the content of benzo [a] pyrene.
The total amount of dioxins, furans, dibenzene-paradioxins and polychlorinated dibenzofurans still in this additional modality is up to 0.35 pg / g. Thus, the composition typically comprises an amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans up to this amount. Typically, the total amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans is up to 0.325 pg / g, preferably up to 0.3 pg / g, more preferably up to 0.275 pg / g, even more preferably up to 0.25 pg / g, even more preferably up to 0.225 pg / g, even more preferably up to 0.2 pg / g, and most of all preferably up to 0.185 pg / g. These amounts are expressed in the World Health Organization (WHO) of toxic equivalence using toxic equivalent WHO factors (TEFs). Toxic equivalent WHO factors are well known to those skilled in the art.
Dioxins, furans, dibenzene-to-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are well known to those skilled in the art. Typically, these are as defined in Community regulations (EC) No. 1881/2006 and 1883/2006 whose full text is incorporated here by reference.
PCDDs, PCDFs and dioxin-like PCBs defined in
Community regulations (EC) No. 1881/2006 and 1883/2006 together with their TEF value areas below.
_________ Branch ______
Dibenzo-p-dioxins (PCDDs)
2.3.7.8-TCDD
1,2,3,7, S-PeCDD
1.2.3.4.7.8-HxCDD
1.2.3.6.7.8-HxCDD
1.2.3.7.8.9-HxCDD
1,2,3,4,6,7,8-HpCDD
TEF V alue
0.1
0.1
0.1
0.01 ____________ Branch _______
Dioxin-like PCBs: non-ortho PCBs + mono ortho PCBs
PCBs do not ortho
PCB 77
PCB 81
PCB 126
TEF value
0.0001
0.0001
0.1
Petition 870190102397, of 11/10/2019, p. 60/176
Branch TEF V alue Branch TEF value OCDD 0.0001 KB 169 0.01 Dibenzofurans (PCDFs)Mono ortho PCBs2,3,7,8-TCDF 0.1 PCB 105 0.0001 1,2,3,7,8-PeCDF 0.05 PCB 114 0.0005 2,3,4,7,8-PeCDF 0.5 1,2,3,4,7,8-HxCDF 0.1 PCB 118 0.0001 1,2,3,6,7,8-HxCDF 0.1 KB 123 0.0001 1,2,3,7, S, 9-HxCDF 0.1 PCB 156 0.0005 2,3,4,6,7,8-HxCDF 0.1 PCB 157 0.0005 1,2,3,4,6,7, S-HpCDF 0.01 1,2,3,4,7,8,9-HpCDF 0.01 KB 167 0.00001 OCDF 0.0001 PCB 189 0.0001
Abbreviations used: T = tetra, Pe = penta, Hx = hexa, Cx = octa, CDD = chlorodibenzodioxin, CDF = chlorodibenzofuran, CB = chlorobiphenyl
Typically, the amount of PCDDs, PCDFs dioxin-like PCBs is determined according to the method demonstrated in Community regulations (EC) No: 1881/2006 and 1883/2006.
The total amount of polychlorinated biphenyls still in this additional modality is up to 0.0035 mg / kg. Thus, the composition typically comprises an amount of polychlorinated biphenyls up to these amounts. Typically, the total amount of polychlorinated biphenyls is up to 0.03 mg / kg, preferably up to 0.0025 mg / kg, more preferably up to 0.002 mg / kg, even more preferably up to 0.0015 mg / kg, even more preferably up to 0.001 mg / kg, even more preferably up to 0.00075 mg / kg, and most of all preferably up to 0.0007 mg / kg.
Polychlorinated biphenyls (PCBs) are well known in the art and include biphenyl, morioclorobiphenyl, dichlorobiphenyl trichlorobiphenyl, tetrachlorobiphenyl, pentachlorobiphenyl, hexachlorobiphenyl, heptachlorobiphenyl, octachlorobiphenyl, nonachlorobiphenyl, decachlorobiphenyl, decachlorobiphenyl, decachlorphenyl.
The total amount of dioxins, furans, dibenzene-paradioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls in this additional embodiment is up to 1 pg / g. Thus, the composition typically comprises an amount of dioxins, furans, dibenzene-dioxins, polychlorinated dibenzofurans, and polychlorinated biphenyls even in this amount. Typically, the total amount of dioxins, furans, dibenzene-dioxins, polychlorinated dibenzofurans and polychlorinated dioxin-like biphenyls is up to 0.75 pg / g, preferably up to 0.5 pg / g, more preferably up to
Petition 870190102397, of 11/10/2019, p. 61/176
0.45 pg / g, even more preferably up to 0.4 pg / g, even more preferably up to 0.35 pg / g, and most of all preferably up to 0.3 pg / g.
Dioxins, furans, dibenzene-to-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated dioxin-like biphenyls are well known to those skilled in the art. Typically, these are as defined in Community regulation (EC) No. 1881/2006 and 1883/2006 whose full text is incorporated herein by reference.
The dioxin-like PCDDs, PCDFs and PCBs defined in Community regulation (EC) No. 1881/2006 and 1883/2006 together with their TEF values are as previously defined.
Still in this additional embodiment, preferably (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.05 pg / kg, (b) the total amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans is up to 0, 2 pg / g (c) the total amount of polychlorinated biphenyls is up to 0.0015 mg / kg, and / or (d) the total amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls is up to 0.3 pg / g.
Still in this additional embodiment, preferably (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.05 pg / kg, (b) the total amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans is up to 0, 2 pg / g and / or (d) the total amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls is up to 0.3 pg / g.
Still in this additional embodiment, more preferably (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.05 pg / kg, (b) the total amount of dioxins, furans, dibenzene-to-dioxins, and polychlorinated dibenzofurans is up to 0.2 pg / g (c) the total amount of polychlorinated biphenyls is up to 0.0015 mg / kg, and (d) the total amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans and biphenyls
Petition 870190102397, of 11/10/2019, p. 62/176 polychlorinated dioxin type is up to 0.3 pg / g.
Still in this additional embodiment, more preferably (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.05 gg / kg, (b) the total amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans is up to 0 , 2 pg / g and (d) the total amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls is up to 0.3 pg / g.
Typically, even in this additional embodiment, the content of isomeric impurities is up to 1.5% by weight. Typically, the content of isomeric impurities is up to 1% by weight, preferably up to 0.5% by weight, more preferably up to 0.25% by weight, even more preferably up to 0.25% by weight, and above all preferably up to 0.1% by weight.
The inventors also noted that high purity oils can be produced that avoid the isomerization, peroxidation and oligomerization problems associated with distilled oils. The amount of isomeric impurities present in a PUFA product of the present invention will depend on the amount of isomeric impurities present in the feed mixture. Crucially, however, the amount of isomeric impurities is greater by the process of the present invention, as opposed to distillation. Thus, the limit of the isomer content in the PUFA product is the isomeric content of the starting material. If the starting material has no isomers present, then the resulting PUFA product will also be substantially free of isomers. This advantage is not seen in distillation.
Thus, in one embodiment, the chromatographic separation process of the present invention does not substantially increase the amount of isomeric impurities in the PUFA product with respect to the amount of isomeric impurities present in the feed mixture. "Increasing substantially typically means increasing by 10% by weight or less, preferably 5% by weight or less, more preferably
Petition 870190102397, of 11/10/2019, p. 63/176% by weight or less, even more preferably 1% by weight or less, even more preferably 0.5,% by weight or less and above all preferably, 0.1% by weight or less.
Thus, even in a further embodiment, the present invention also provides a composition comprising a PUFA product, in which the content of isomeric impurities is up to 1.5% by weight. Typically, the composition contains an amount of isomeric impurities up to this amount. Typically, the content of isomeric impurities is up to 1% by weight, preferably up to 0.5% by weight, more preferably up to 0.25% by weight, and above all preferably up to 0.1% by weight. Isomerization is particularly problematic in the preparation of high purity DHA by distillation, due to the higher temperatures required for separation. Typically, the PUFA product is DHA, optionally in the form of its ethyl ester. Typically the composition comprises more than 85% by weight of the PUFA product, preferably more than 90% by weight, more preferably more than 92.5% by weight, above all preferably more than 95% by weight. Preferably, the composition comprises more than 85% by weight of DHA, optionally in the form of its ethyl ester, preferably more than 90% by weight more preferably more than 92.5% by weight, above all preferably more than 95% by weight . In this embodiment, the composition typically comprises the PUFA DHA product optionally, in the form of its ethyl ester, in an amount greater than 95% by weight, where the content of isomeric impurities is up to 1% by weight, preferably up to 0.5% by weight, more preferably up to 0.25% by weight, and above all preferably up to 0.1% by weight.
The improved process of the invention allows much higher purities of PUFA product to be obtained efficiently, since both more and less polar impurities can be removed in a single process.
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The PUFA product of the present invention typically has a purity greater than 80% by weight, preferably greater than 85%, by weight more preferably greater than 90% by weight, even more preferably greater than 95% by weight, even more preferably greater than 97%. by weight, and above all preferably greater than 99% by weight. When the PUFA product is a single PUFA or derivative thereof, the above concentrations refer to the concentration of this PUFA or derivative. When the PUFA product is a mixture of two or more PUFAs or their derivatives, for example, two, the above concentrations refer to the combined concentration of the PUFAs, or their derivatives.
The method of the present invention also avoids the problems of isomerization, peroxidation and oligomerization associated with distilled oils. The PUFA product of the present invention typically has an isomeric impurity content of less than 5% by weight, preferably less than 3% by weight, and more preferably less than 1% by weight. As mentioned earlier, isomeric impurities include products of PUFA isomers peroxidation and oligomerization. PUFA isomers include positional and / or geometric isomers. Examples of positional and / or geometric isomers of EPA include 17E-EPA, 5E-EPA, 5E, 8E-EPA, 8E, 11E-EPA, 5E, 14E-EPA, and 5E, 8E, 11E, 17E-EPA, Such isomers they are discussed in more detail in Wijesundera, RC, et al, Journal of the American Oil. Chemists Society, 1989, vol. 66, no: 12, 1822-1830, whose full text is incorporated herein by reference.
In practice, the process of the present invention will generally be controlled by a computer. The present invention therefore also relates to a computer program for controlling a chromatographic apparatus as defined herein, the computer program containing a code device which when executed instructs the apparatus to carry out the process of the invention.
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The following examples illustrate the invention.
EXAMPLES
Example 1
A feed stock derived from fish oil (55% by weight of EPA EE, 5% by weight of DHA EE) is fractionated using a real moving bed chromatography system using bound C18 silica gel (particle size 300 pm) as stationary phase and aqueous methanol as eluent according to the system schematically illustrated in the figure
8. 15 columns (diameter: 76.29 mm, length: 914.40 mm) are connected in series as shown in figure 8.
The operating parameters and flow rates are as follows for eight different cases. For the following conditions, EPA EE is produced at a high level of purity (85 to 98% by GC FAMES). FAMES GC traces of the extract and raffinate from zone 1 are shown as figures 11 and 12 respectively.
Example 1a
Step time: 750 s
Cycle time: 200 min
Feed rate of feed stock (F): 70 mL / min
Desorbent feed rate (D): 850 mL / min
Extract rate: 425 mL / min
Raffinate rate: 495 mL / min
Example 1b
Step time: 250 s
Cycle time: 66.67 min
Feed rate of feed stock (F): 210 mL / min
Desorbent feed rate (D): 2,550 mL / min
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Extract rate: 1,275 mL / min
Raffinate rate: 1,485 mL / min
Example 1c
Step time; 500 s
Cycle time: 13,133 min
Feed rate of feed stock (F): 25 mL / min
Desorbent feed rate (D1) in the first zone:
2,050 mL / min
Accumulation rate of the extract container (E1) in the first zone: 1,125 mL / min
Extract recycling rate (D1-E1) in the first zone: 925 mL / min
Rate of raffinate (R1) in the first zone: 950 mL / min
Desorbent feed rate (D2) in the second zone:
1,700 mL / min
Accumulation rate of the extract container (E2) in the second zone: 900 mL / min
Extract recycling rate (D2-E2) in the second zone: 800 mL / min
Rate of raffinate (R2) in the second zone: 800 mL / min
Example 1d
Step time: 250 s
Cycle time: 66.67 min
Feed rate of feed stock (F): 50 mL / min
Desorbent feed rate (D1) in the first zone:
4,125 ml / min.
Accumulation rate of the extract container (E1) in the first
Petition 870190102397, of 11/10/2019, p. 67/176 zone: 2,250 mL / min
Extract recycling rate (D1-E1) in the first zone:
1,875 mL / min
Rate of raffinate (R1) in the first zone: 1,925 mL / min
Desorbent feed rate (D2) in the second zone
3,375 mL / min
Accumulation rate of the extract container (E2) in the second zone: 1,800 mL / min
Extract recycling rate (D2-E2) in the second zone 1.575 mL / min
Rate of raffinate (R2) in the second zone: 1,575 mL / min
Example 1e
Step time: 500 s
Cycle time: 133.33 min
Feed rate of feed stock (F): 50 mL / min
Desorbent feed rate (D1) in the first zone: 4,000 mL / min
Accumulation rate of the extract container (E1) in the first zone: 2,250 mL / min
Extract recycling rate (D1-E1) in the first zone:
1,750 mL / min
Rate of raffinate (IR) in the first zone: 18.00 mL / min
Desorbent feed rate (D2) in the second zone: 3,200 mL / min.
Accumulation rate of liquid extract (E2) in the second zone:
1,750 mL / min
Extract recycling rate (D2-E2) in the second zone:
1,450 mL / min
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Rate of raffinate (R2) in the second zone: 1,450 mL / min
Example 1f
Step time: 250 s
Cycle time: 66.67 min
Feed rate of feed stock (F): 100 mL / min
Desorbent feed rate (D1) in the first zone:
4,050 mL / min
Accumulation rate of the extract container (E1) in the first zone: 2,100 mL / min
Extract recycling rate (D1-E1) in the first zone;
1,950 mL / min
Rate of raffinate (R1) in the first zone: 2,050 mL / min
Desorbent feed rate (D2) in the second zone:
3,300 mL / min
Accumulation rate of liquid extract (E2) in the second zone:
1,700 mL / min
Extract recycling rate (D2-E2) in the second zone:
1,600 mL / min
Rate of raffinate (R2) in the second zone: 1,600 mL / min
Example 1g
Step time: 500 s
Cycle time: 133.33 min
Feed rate of feed stock (F): 25 mL / min
Desorbent feed rate (D1) in the first zone:
1275 mL / min
Accumulation rate of the extract container (E1), in the first zone: 750 mL / min
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Extract recycling rate (D1-E1) in the first zone: 550 mL / min
Rate of raffinate (IR) in the first zone: 575 mL / min
Desorbent feed rate (D2) in the second zone:
1,275 mL / min
Accumulation rate of liquid extract (E2) in the second zone: 950 mL / min
Extract recycling rate (D2-E2) in the second zone: 325 mL / min
Rate of raffinate (R2) in the second zone: 325 mL / min
Example 1h
Step time: 25.0 s
Cycle time: 66.67 min
Feed rate of feed stock (F): 50 mL / min
Desorbent feed rate (D1) in the first zone:
2,550 mL / min
Accumulation rate of the extract container (E1) in the first zone: 1,500 mL / min
Extract recycling rate (D1-E1) in the first zone: 950 mL / min
Rate of raffinate (R1) in the first zone: 1,000 mL / min
Desorbent feed rate (D2) in the second zone:
2,000
Accumulation rate of liquid extract (E2) in the second zone: 900 mL / min
Extract recycling rate (D2-E2) in the second zone: 600 mL / min
Rate of raffinate (R2) in the second zone: 600 mL / min
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Example 2
A fish oil-derived feed stock comprising eicosatetraenoic acid ethyl ester (ETA EE), EPA EE, its isomers and DHA EE were fractionated using a real moving bed chromatography system using bound C18 silica gel (particle size 40- 604 pm) as a stationary phase and aqueous methanol as eluent according to the system schematically illustrated in figure 10. 19 columns (diameter: 10 mm, length: 250 mm) are connected in series as shown in figure 10.
The operating parameters and flow rates are as follows.
Cycle time: 600 s.
Feed rate of feed stock (F): 0.5 mL / min.
Desorbent (Dl, 100% methanol) feed rate in the first zone: 6 mL / min
Desorbent (D2, 99% methanol / 1% water) feed rate in the first zone: 6 mL / min.
Extract rate (E1) of the first zone: 3 mL / min
Extract rate (E1) of the first zone: 1.9 mL / min
Rate of raffinate (R1) of the first zone: 4.6 mL / min
Desorbent (D2, 97% methanol / 3% water) feed rate in the second zone 6 mL / min
Extract rate (E2) of the second zone: 2.4 mL / min
Rate of raffinate (R2) of the second zone: 4.6 mL / min
Again, EPA EE was produced at a high level of purity (more than 90% by weight, more than 95% by weight, more than 98% by weight).
Example 3
A feed stock derived from fish oil (55% by weight of EPA EE, 5% by weight of DHA EE) was fractionated using a
Petition 870190102397, of 11/10/2019, p. 71/176 real moving bed chromatography system using bonded C18 silica gel (particle size 300 pm, particle porosity 150 angstroms) as a stationary phase and aqueous methanol as eluent according to the system schematically illustrated in figure 8. 15 columns ( diameter: 10 mm, length: 250 mm) are connected in series as shown in figure 8.
The operating parameters and flow rates are as follows.
Cycle time: 380 s
Feed rate of feed stock (F): 0.5 mL / min
Desorbent (D, 98.5% methanol / 1.5% water) feed rate in the first zone: 9 mL / min
Water-rich phase (W, 85% methanol / 15% water) feed rate in the first zone: 3.1 mL / min
Extract rate (E1) of the first zone: 4 mL / min
Rate of raffinate (R1) of the first zone: 8.6 mL / min
Desorbent (D, 97% methanol / 3% water) feed rate in the second zone: 10.8 mL / min
Water-rich phase (W, 85% methanol / 15% water): feed rate in the second zone: 3.1 mL / min
Extract rate (E2) of the second zone: 4.1 mL / min
Rate of raffinate (R2) of the second zone: 10.3 mL / min
EPA EE was produced at a high level of purity (> 95% purity). A GC trace of the product is shown as figure 13
Example 4
A feed stock derived from fish oil (70 wt% DHA EE, 7 wt% EPA EE) is fractionated using an actual moving bed chromatography system using bound C18 silica gel (particle size 300 μm) as stationary phase and aqueous methanol
Petition 870190102397, of 11/10/2019, p. 72/176 as eluent according to the system schematically illustrated in the figure
8. 15 columns (diameter: 76.29 mm, length: 914.40 mm) are connected in series as shown in figure 8.
The operating parameters and flow rates are as follows.
Step time: 600 s
Cycle time: 160 min
Feed rate of feed stock (F): 25 mL / min
Desorbent feed rate (D1) in the first zone 2,062.5 mL / min
Extract rate (E1) in the first zone: 900 mL / min
Rate of raffinate (R1) in the first zone: 1,187.5 mL / min
Desorbent feed rate (D2) in the second zone:
1,500 mL / min
Extract rate (E2) in the second zone: 450 mL / min
Rate of raffinate (R2) in the second zone: 1,050 mL / min
DHA EE is produced at a high level of purity (> 97% by GC FAMES). A GC FAMES trace of zone 2 extract is shown as a figure
14.
Example 5
A feed stock (3.3 wt% EPA EE 22, wt% DHA EE) is fractionated using a real moving bed chromatography system using bound C18 silica gel (particle size 300 qm) as a stationary phase and aqueous methanol as eluent according to the system schematically illustrated in figure 8. 15 columns (diameter: 76.29 mm, length 914.40 mm) are connected in series as shown in figure 8.
The operating parameters and flow rates are as follows.
Step time: 380 s
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Cycle time: 101.33 min
Feed rate of feed stock (F): 40 mL / min
Desorbent feed rate (D1) in the first zone:
1,950 mL / min
Extract rate (E1) in the first zone: 825 mL / min
Rate of raffinate (R1) in the first zone: 1,165 mL / min
Desorbent feed rate (D2) in the second zone:
1,425 mL / min
Extract rate (E2) in the second zone: 787.5 mL / min
Rate of raffinate (R2) in the second zone: 637.5 mL / min.
A mixture of EPA EE and DHA EE is produced at a high level of purity> 80% EPA EE and total DHA EE).
Example 6
An experiment was carried out to compare the amount of environmental pollutants present in two PUFA products according to the present invention with similar oils prepared by distillation. The polluting profiles of the oils are shown in table 1 below.
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Table 1
Parameter Release specification gives Distilled oil [1] Distilled oil [2] PUFA product according to the invention [1] PUFA product according to the invention [2] Polyaromatic hydrocarbons (PAH) (pg / kg)Benzo (a) pyrene NMT 2.00.90 0.90 <0.05 <0.05 Dioxins and furans impurities PCDDs and PCDFs ¹ NMT 2.00.46 0.37 0.2 0.184 (page WHO-PCDD / F-TEQ / g) NMT 0.090.0037 0.0103 0.0007 0.0012 PCBs (mg / kg) NMT 10.01.03 0.466 0.30 0.298 Sum of dioxins, furans and dioxin-like PCBS ² (pg WHO- PCDD / F-PCB-TEQ / g)
1) Dioxin limits include the sum of polychlorinated dibenzo-paradioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) and expressed in the World Health Organization (WHO) toxic equivalence using WHO-equivalence factors (TEFs). This means that analytical results related to the 17 individual dioxin congeners of toxicological concern are expressed in a single quantifiable unit: TCDD or TEQ toxic equivalence concentration.
2) maximum for dioxin and furans remain at 2pg / g.
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Example 7
An experiment was carried out to determine the amount of isomeric impurities present in an oil prepared according to the present invention compared to an equivalent oil prepared by distillation.
A GC trace of the DHA-rich oil prepared according to the invention is shown as figure 14. There is no evidence of isomeric impurities in the GC trace.
A GC trace of the oil prepared by distillation is shown as figure 15. The four peaks with longer elution times than the DHA peak correspond to the DHA isomers. From the GC trace it can be seen that the oil prepared by distillation contains about 1.5% by weight of isomeric impurities.
Example 8
Two EPA-rich products from the process of the present invention were compared with EPA-rich oils produced by distillation. The analysis by weight of its component PUFAs is shown below.
Fatty acid PUFA product according to the invention [1] PUFA product according to the invention [2] Distilled oil [1] Distilled oil [2] EPA (C20: 5n-3) 98.33 97.04 98.09 98.14 DHA (C22: 6n-3) 0.15 <LOD 0.34 <LOD C18: 3 n-3 <LOD 0.28 0.24 <LOD C18: 4 n-3 0.33 0.20 0.14 0.26 C20: 4 n-3 0.14 0.45 0.18 0.46 C21: 5 n-3 <LOD <LOD <LOD <LOD C22: 5 n-3 0.32 <LOD <LOD <LOD Total omega-3 99.27 97.97 98.94 98.86 C18: 3n-6 <LOD <LOD 0.05 <LOD C20: 3 n-6 <LOD <LOD 0.13 0.11 C20: 4 n-6 <LOD 0.21 0.26 0.37 Total Omega-6 <LOD 0.21 0.44 0.48
Example 9
A product rich in EPA / DHA from the process of the present invention was compared with an oil rich in EPA DHA produced by distillation. The analysis by weight of its component PUFAs is
Petition 870190102397, of 11/10/2019, p. 76/176 shown below.
Fatty acid Maxomega ethyl ester (Omega-3 ethyl esters 90)% area Distilled ethyl ester (Omega-3 ethyl esters 90 ¹ )% area EPA (C20: 5n-3) 53.3 46.6 DHA (C22: 6n-3) 32.9 38.2 TOTAL EPA + DHA 86.2 84.8 C18: 3 n-3 0.3 0.1 C18: 4 n-3 1.2 2.0 C20: 4 n-3 1.8 0.6 C21: 5 n-3 2.7 1.8 C22: 5 n-3 5.0 3.8 Total Omega-3 97.2 93.1 C18: 2 n-6 0.2 0.1 C18: 3n-6 <0.1 0.2 C20: 3 n-6 <0.1 0.1 C20: 4 n-6 2.0 2.6 C22: 4 n-6 <0.1 0.1 C22: 5 n-6 0.6 1.0 Total Omega-6 2.8 4.1
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权利要求:
Claims (21)
[1]
1. Chromatographic separation process to recover a polyunsaturated fatty acid (PUFA) product, from a feed mixture, characterized by the fact that the process comprises introducing the feed mixture into a simulated moving bed chromatography apparatus or real having a plurality of linked chromatography columns containing, as an eluent, an aqueous alcohol, wherein the apparatus has a plurality of zones comprising at least a first zone and a second zone, each zone having an extract stream and a raffinate stream from which the liquid can be collected from said plurality of linked chromatography columns, and in which (a) a stream of raffinate containing the PUFA product together with more polar components is collected from a column in the first zone and introduced into a non-adjacent column in the second zone, and / or (b) an extract stream containing the PUFA product together with less polar components is collected from a column in the second unda zone and introduced into a column not adjacent to the first zone, said PUFA product being separated from different components of the feed mixture in each zone.
[2]
Process according to claim 1, characterized by the fact that part of one or more of the extract chain of the first zone, the raffinate stream of the first zone, the extract stream of the second zone, and the raffinate stream of the second zone are recycled back into the same zone, typically in an adjacent column in the same zone.
[3]
3. Process according to claim 1 or 2, characterized by the fact that (a) the eluent aqueous alcohol present in each zone has a different water: alcohol ratio; and / or (b) the rate at which the liquid collected by the extract streams
Petition 870200012258, of 01/27/2020, p. 11/17 and raffinate in each zone is recycled back in the same zone is adjusted in such a way that the PUFA product can be separated from different components of the feed mixture in each zone.
[4]
4. Process according to claim 3, characterized in that the rate at which the liquid collected by the extract stream outside the first zone is recycled back in the first zone differs from the rate at which the liquid collected by the extract stream outside from the second zone is recycled back into the second zone and / or the rate at which liquid collected by the raffinate stream outside the first zone differs from the rate at which liquid collected by the raffinate stream outside the second zone is recycled back into the second zone.
[5]
Process according to any one of claims 1 to 4, characterized in that the apparatus has a first zone and a second zone, said PUFA product being separated from less polar components of the feed mixture in the first zone, and the said PUFA product being separated from more polar components of the feed mixture in the second zone.
[6]
Process according to any one of claims 1 to 5, characterized in that the PUFA product comprises at least one ω-3 PUFA, and in which PUFA product preferably comprises EPA and / or DHA.
[7]
Process according to any one of claims 1 to 6, characterized in that in addition to said PUFA product, an additional secondary PUFA product is recovered in the chromatographic separation process, and in which the PUFA product is preferably EPA and the product Additional secondary PUFA is preferably DHA.
[8]
Process according to any one of claims 1 to 7, characterized in that the chromatographic columns contain, as an adsorbent, substantially spherical granules, in which the granules are
Petition 870200012258, of 01/27/2020, p. 12/17 preferably formed from C18 bound silica gel; and / or wherein the substantially spherical granules preferably have a diameter of 250 to 500 µm.
[9]
Process according to any one of claims 1 to 8, characterized in that the eluent is a mixture of water and a C1-C6 alcohol, wherein the C1-C6 alcohol is preferably methanol or ethanol.
[10]
Process according to any one of claims 1 to 9, characterized in that the eluent in the first zone contains more alcohol than the eluent in the second zone, and the second zone is downstream of the first zone, with respect to flow of the eluent in the system; and / or where the water: alcohol ratio of the eluent in the first zone is 0.5: 99.5 to 1.5: 98.5 parts by volume, and the water: alcohol ratio of the eluent in the second zone is 0.5 4.5: 95.5 to 5.5: 94.5 parts by volume; and / or where the water: alcohol ratio of the eluent in the first and second zones is controlled by introducing water and / or alcohol into one or more columns in the first and second zones.
[11]
11. Process according to any one of claims 2 to 10, characterized in that the rate at which liquid collected by the extract stream from the first zone is recycled back into the first zone is faster than the rate at which liquid collected by the extract stream from the second zone is recycled back into the second zone.
[12]
12. Process according to any one of claims 5 to 11, characterized by the fact that it comprises:
(a) (i) introducing the feed mixture into the first zone, and removing a first stream of raffinate enriched with the PUFA product and a first exhaust stream of the PUFA product, and (ii) introducing the first stream of raffinate into the second zone, remove a second stream of impoverished raffinate from the PUFA product and collect a second stream of extract to obtain the PUFA product; or
Petition 870200012258, of 01/27/2020, p. 13/17 (b) (i) introducing the feed mixture into the second zone, and removing a first exhausted strand of the PUFA product and a first extract stream enriched in the PUFA product, and (ii) introducing the first extract stream in the first zone, remove a second stream of exhausted extract from the PUFA product and collect a second stream of raffinate to obtain the PUFA product.
[13]
Process according to any one of claims 1 to 12, characterized in that the simulated or real moving bed chromatography apparatus has fifteen chromatographic columns 1 to 15; and / or in which the first zone consists of eight adjacent columns 1 to 8; and / or where the second zone consists of seven adjacent columns 9 to
15.
[14]
Process according to claim 13, characterized in that (i) (a) alcohol is introduced in column 1, and / or (b) alcohol is introduced in column 9, and / or (c) water is introduced in column 4 and / or (d) water is introduced in column 12; or (ii) aqueous alcohol is introduced in column 1 and / or column 9.
[15]
15. Process according to claim 13 or 14, characterized by the fact that the first streak is collected from column 7 and introduced into column 13.
[16]
16. Process according to claim 1, characterized by the fact that the apparatus has a first zone, a second zone and a third zone and in which (a) the eluent in the first zone contains more alcohol than the eluent in the second and third zones and the first zone is upstream of the second and third zones with respect to the flow of the eluent in the system and (b) the eluent in the second zone contains more alcohol than the eluent in the third zone and the second zone is upstream of the third zone with relation to the flow of the eluent in the system, said PUFA product being separated in the first zone of the
Petition 870200012258, of 01/27/2020, p. 14/17 components of the feed mixture that are less polar than the PUFA product, said PUFA product being separated in the second zone from the components of the feed mixture that are less polar than the PUFA product, but more polar than the components separated in the first zone, and said PUFA product being separated in the third zone from the components of the feed mixture which are more polar than the PUFA product.
[17]
17. Composition obtained by the process as defined in any one of claims 1 to 16, characterized by the fact that it comprises a PUFA product, polyunsaturated fatty acids ω-6 and eicosatetraenoic acid ω-3, in which the PUFA product is present EPA in an amount of 96.5 to 99% by weight, the composition comprises an amount of ω-6 polyunsaturated fatty acids of up to 0.40% by weight; and the composition further comprises or:
(a) - an amount of ω-3 eicosatetraenoic acid from 0.1 to 0.75% by weight;
- an amount of DHA of up to 0.6% by weight;
- an amount of α-linoleic up to 0.6% by weight;
- an amount of stearidonic acid from 0.15 to 0.6% by weight;
- an amount of docosapentaenoic acid of up to 0.6% by weight;
an amount of arachidonic acid of up to 0.6% by weight; or (b) - an amount of eicosatetraenoic acid ω-3 of 0.1 to 0.5% by weight;
- an amount of DHA of up to 0.2% by weight;
- an amount of α-linoleic up to 0.3% by weight;
- an amount of stearidonic acid from 0.15 to 0.4% by weight;
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- an amount of docosapentaenoic acid of up to 0.35% by weight; and
- an amount of arachidonic acid of up to 0.25% by weight.
[18]
18. Composition according to claim 17, characterized in that the composition (a) comprises from 98 to 99% by weight of EPA, from 0.1 to 0.3% by weight of DHA, from 0.3 to 0.35% by weight of stearidonic acid, from 0.1 to 0.3% by weight of eicosatetraenoic acid ω-3 and from 0.3 to 0.35% by weight of docosapentaenoic acid; or (b) the composition comprises 96.5 to 99% by weight of EPA, 0.1 to 0.5% by weight of DHA, 0.1 to 0.5% by weight of stearidonic acid, from 0, 1 to 0.5% by weight of eicosatetraenoic acid ω-3, from 0.1 to 0.5% by weight of docosapentaenoic acid, and from 0.1 to 0.3% by weight arachidonic acid; or (c) comprises 98 to 99% EPA, 0.1 to 0.2% by weight DHA, 0.3 to 0.35% stearidonic acid, 0.1 to 0.2% by weight weight of eicosatetraenoic acid ω-3 and 0.3 to 0.35% by weight of docosapentaenoic acid; or (d) comprises 96.5 to 97.5% EPA, 0.25 to 0.35% by weight of α-linoleic acid, 0.18 to 0.24% by weight of stearidonic acid, of 0.4 to 0.46% by weight of ω-3 eicosatetraenoic acid, and from 0.15 to 0.25% by weight arachidonic acid.
[19]
19. Composition according to claim 17 or 18, characterized in that the composition comprises an amount of isomeric impurities of up to 1% by weight.
[20]
20. Composition according to claim 19, characterized in that it comprises an amount of isomeric purities of up to 0.5% by weight, up to 0.25% by weight or up to 0.1% by weight.
[21]
21. Composition according to any one of claims 17 to 20, characterized by the fact that (a) the total amount of
Petition 870200012258, of 01/27/2020, p. 16/17 polyaromatic hydrocarbons in the composition is up to 0.89 pg / kg, (b), the total amount of dioxins, furans, dibenzene-to-dioxins and polychlorinated dibenzofurans is up to 0.35 pg / g (c) the amount total polychlorinated biphenyls is up to 0.0035 mg / kg, and / or (d) the total amount of 5 dioxins, furans, dibenzene-stop-dioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls is up to 1 pg / g.

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法律状态:
2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-12-18| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-07-16| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2019-10-29| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-03-03| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-03-31| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 24/12/2010, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US29118409P| true| 2009-12-30|2009-12-30|

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US61/291184|2009-12-30|

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GB0922707A|GB0922707D0|2009-12-30|2009-12-30|New process|

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GB0922707.5|2009-12-30|

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GBGB1015343.5A|GB201015343D0|2010-09-14|2010-09-14|New process|

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GB1015343.5|2010-09-14|

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PCT/GB2010/002339|WO2011080503A2|2009-12-30|2010-12-24|Simulated moving bed chromatographic separation process|

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