chromatographic separation process

专利摘要:
CHROMATOGRAPHIC SEPARATION PROCESS, PUFA PRODUCT, AND, STORAGE MEDIA The present invention provides a chromatographic separation process to recover a polyunsaturated fatty acid (PUFA) product from a feed mixture that is a fish oil or which is derived from fish oil, a process that comprises the steps of: (i) purifying the feed mixture in a chromatographic separation step, to obtain a first intermediate product; and (ii) purifying the first intermediate product obtained in (i) in a simulated or real moving bed chromatographic separation step, to obtain a second intermediate product; and (iii) purifying the second intermediate product obtained in (ii) in a simulated or real moving bed chromatographic separation step, to obtain the PUFA product; wherein an aqueous organic solvent is used as an eluent in each separation step; saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step; the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii); and the PUFA product obtained in the third separation step contains EPA or one (...).
公开号:BR112014000162B1
申请号:R112014000162-6
申请日:2012-07-06
公开日:2021-01-05
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:

[0001] The present invention relates to an improved chromatographic separation process to purify EPA polyunsaturated fatty acid or a derivative thereof.
[0002] EPA and its 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, EPA and its derivatives can be therapeutically useful in the treatment of a wide range of pathological conditions that include CNS conditions; neuropathies, which include diabetic neuropathy; cardiovascular diseases; general immune and inflammatory conditions, which include inflammatory skin diseases.
[0003] EPA is found in natural raw materials, and in particular in fish oils. EPA in fish oils, however, is present in such oils in admixture with saturated fatty acids and numerous other impurities.
[0004] The purification of EPA from fish oils is particularly challenging. Thus, fish oils are extremely complex mixtures that contain a large number of different components with very similar retention times in chromatography devices. E1es represent a much more challenging feed stock than to purify EPA than, for example, a seaweed oil feed stock. However, a very high degree of purity of EPA is required, particularly for pharmaceutical and nutraceutical applications. Historically, therefore, distillation has been used to purify EPA for therapeutic applications.
[0005] Unfortunately, EPA is extremely fragile. Thus, when heated in the presence of oxygen, it is prone to isomerization, peroxidation and oligomerization. The fractionation and purification of EPA to prepare pure fatty acids is therefore difficult. Distillation, even under vacuum, can lead to unacceptable degradation of the product.
[0006] Simulated and real moving bed chromatography are known techniques, familiar to those of skill in the art. The operating principle involves countercurrent movement of a liquid eluting phase and a solid absorbing 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.
[0007] As is well known, in a conventional stationary bed chromatographic system, a mixture whose components must 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 (generally called the stationary phase) that exhibits a high permeability to fluids. The rate of percolation of each component of the mixture depends on the physical properties of this component so that the components come out of the column successively and selectively. Thus, some of the components tend to stick strongly to the stationary phase and thus will seep slowly, while others tend to stick 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.
[0008] In contrast, a simulated moving bed chromatography apparatus consists of several individual columns that contain absorbers that are connected together in series. The eluent is passed through the columns in a first direction. The injection points of the feed stock and the eluent, and the separate component collection points in the system, are periodically changed through a series of valves. The overall effect is to simulate the operation of a single column containing a moving bed of the solid absorber, the solid absorber moving in a direction countercurrent to the eluent flow. Thus, a simulated moving bed system consists of columns that, as in a conventional stationary bed system, contain solid absorber stationary beds through which the eluent is passed, but in a simulated moving bed system the operation is just like simulating a moving bed in continuous countercurrent.
[0009] The processes and equipment for simulated moving bed chromatography are described in several patents, which include 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, all of which are incorporated herein by reference. The topic is also covered in detail in “Preparative and Production Scale Chromatography”, edited by Ganetsos and Barker, Marcel Dekker Inc, New York, 1993, all of which is incorporated by reference.
[00010] A real moving bed system is similar in operation to a simulated moving bed system. However, instead of changing 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 absorption units (ie, columns) are physically moved in relation to the feeding and removal points. Again, the operation is like simulating a moving bed in continuous countercurrent.
[00011] The processes and equipment for real moving bed chromatography are described in several patents, which include US 6,979,402, US 5,069,883 and US 4,764,276, all of which are incorporated herein by reference.
[00012] A typical simulated moving bed chromatography apparatus is illustrated with reference to Figure 1. The concept of a simulated or actual moving bed chromatographic separation process is explained by considering a vertical chromatographic column containing the divided S stationary phase. in sections, more precisely in four overlapping sub-areas I, II, III and IV going from the bottom to the top of the column. The eluent is introduced to the bottom in IE by means of a P pump. The mixture of components A and B that must 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 subarea IV.
[00013] In the case of a simulated moving bed system, a simulated downward movement of the stationary phase S is caused by the movement of the points of introduction and collection in relation to the solid phase. In the case of a real moving bed system, the simulated downward movement of the stationary phase S is caused by the movement of the various chromatographic columns in relation to the points of introduction and collection. In Figure 1, the upward eluent flows and the A + B mixture are injected between sub-area II and sub-area III. The components will move according to their chromatographic interactions with the stationary phase, for example adsorption in a porous medium. Component B that exhibits the strongest affinity for the stationary phase (the component that moves the slowest) will be more slowly trapped by the eluent and will follow it with delay. Component A that exhibits the weakest affinity for the stationary phase (the component that moves the fastest) will be easily trapped by the eluent. If the right set of parameters, especially the flow rate in each sub-area, is correctly estimated and controlled, component A that exhibits the weakest affinity with the stationary phase will be collected between sub-area III and sub-area IV as a raffinate and component B that exhibits the strongest affinity for the stationary phase will be collected between sub-area I and sub-area II as an extract.
[00014] To obtain high-purity EPA or EPA ethyl ester in purities of more than 90%, for example greater than 95 or 97%, it is possible to use a simulated moving bed separation process that performs two stages of simultaneous separation. Such a process is described in international patent application no. PCT / GB10 / 002339, all of which are incorporated by reference.
[00015] In general, all chromatographic separation techniques for separating PUFAs, which include SMB processes, use large volumes of organic solvents as eluents. After the chromatographic separation process is completed, the PUFAs must be recovered from the solution in the eluent. Typically a large expenditure of time and energy is involved in recovering PUFAs from the solution in the eluent. In addition, organic solvents used as eluents in the chromatographic separation processes are often harmful to the environment or to the workers who handle them. Therefore, a chromatographic separation process that reduces the amount of organic solvent that needs to be used is required.
[00016] It has now been advantageously discovered that EPA or an EPA derivative can be produced in a similarly high purity as described in PCT / GB10 / 002339 by a three step separation process that uses a much lower volume of solvent than the process two-step. The improved process of the present invention uses almost 50% less solvent than the two-step process described in PCT / GB10 / 002339. This is clearly advantageous in terms of cost, ease of product recovery, and environmental impact. Summary of the invention
[00017] It has surprisingly been found that EPA or an EPA derivative can be effectively purified from commercially available feed stocks such as fish oils by the simulated or real moving bed apparatus using a relatively low volume of an aqueous organic solvent eluent . The present invention therefore provides a chromatographic separation process to recover a polyunsaturated fatty acid product (PUFA) from a feed mixture that is a fish oil or that is derived from fish oil, a process that comprises the steps of:
[00018] (i) purify the feed mixture in a chromatographic separation step, to obtain a first intermediate product; and
[00019] (ii) purify the first intermediate product obtained in (i) in a simulated or real moving bed chromatographic separation step, to obtain a second intermediate product; and
[00020] (iii) purify the second intermediate product obtained in (ii) in a simulated or real moving bed chromatographic separation step, to obtain the PUFA product; wherein an aqueous organic solvent is used as an eluent in each separation step;
[00021] the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step;
[00022] the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii); and
[00023] the PUFA product obtained in the third separation step contains EPA or an EPA derivative in an amount greater than 90% by weight.
[00024] A PUFA Product obtainable by the process of the present invention is also provided. Description of the Figures
[00025] Figure 1 illustrates the basic principles of a simulated or real moving bed process to separate a binary mixture.
[00026] Figure 2 illustrates three ways in which the chromatographic separation process of the invention can be carried out.
[00027] Figure 3 illustrates a preferred embodiment of the invention that is suitable for producing high purity EPA.
[00028] Figure 4 illustrates in more detail the embodiment of Figure 2.
[00029] Figure 5 illustrates a more preferred embodiment of the embodiment shown in Figure 2.
[00030] Figure 6 illustrates a two-stage separation process to produce EPA (not in accordance with the present invention).
[00031] Figure 7 shows a GC trace of a food supply suitable for use according to the process of the present invention.
[00032] Figure 8 shows a GC trace of a first intermediate product produced according to the process of the present invention.
[00033] Figure 9 shows a GC trace of a second intermediate product produced according to the process of the present invention.
[00034] Figure 10 shows a GC trace of a PUFA product produced according to the process of the present invention. Detailed description of the invention
[00035] As used herein, the term "PUFA product" refers to a product comprising one or more polyunsaturated fatty acids (PUFAs), and / or derivatives thereof, typically of nutritional or pharmaceutical significance. The PUFA product obtained in the process of the present invention contains EPA or an EPA derivative in an amount greater than 90% by weight, that is, EPA or an EPA derivative is present in 90% by weight of purity in relation to all the components in the final PUFA product not including the aqueous organic solvent eluent. Thus EPA or an EPA derivative is present in the PUFA product in an amount of at least 90% by weight based on all components of the PUFA product that originated in the feed mixture.
[00036] An EPA derivative is EPA 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 most preferred.
[00037] Typically, the PUFA product contains EPA or an EPA derivative in an amount greater than 95% by weight, preferably greater than 97% by weight.
[00038] In one embodiment, the PUFA product contains EPA in an amount greater than 90% by weight, preferably greater than 95% by weight, more preferably greater than 97% by weight. As explained above, EPAs are present in the specified weight% relative to the total amount of all components of the PUFA product that originated in the feed mixture.
[00039] In another embodiment, the PUFA product contains EPA ethyl ester in an amount greater than 90% by weight, preferably greater than 95% by weight, more preferably greater than 97% by weight. As explained above, EPAs are present in the specified weight% with respect to the total amount of all components of the PUFA product that originated in the feed mixture.
[00040] Feed mixtures suitable for fractionation by the process of the present invention are fish oils, or feed stocks derived from fish oils. Fish oils suitable for use in the process of the present invention are well known to the skilled person. Typical fish oils contain EPA, DHA, SDA, and typically a range of other PUFAs, both more and less polar than EPA, saturated fatty acids and monounsaturated fatty acids.
[00041] 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, silver nitrate extraction or other metallic salt solutions, iodolactonization or supercritical fluid fractionation . Alternatively, a feed mixture can be used directly without any initial treatment steps.
[00042] 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 absorber used in the process of the present invention than the PUFA product. During operation, such less polar components typically move with the solid absorber phase in preference to the liquid eluting phase. The more polar components have weaker adhesion to the absorber 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 absorbing phase. In general, the more polar components will be separated in a raffinate stream, and the less polar components will be separated in an extract stream.
[00043] Examples of more and less polar components include (1) other compounds that occur in natural oils (for example, marine oils), (2) by-products formed during storage, refining and previous concentration steps, and (3) contaminants of solvents or reagents that are used during the previous concentration or purification steps.
[00044] Examples of (1) include other unwanted PUFAs; saturated fatty acids; sterols, for example, cholesterol; vitamins; and environmental pollutants, such as polychlorinated biphenyl (PCB), polyaromatic hydrocarbon pesticides (PAH), chlorinated pesticides, dioxins and heavy metals. PCBs, PAHs, dioxins and chlorinated pesticides are all highly non-polar components.
[00045] Examples of (2) include isomers and products of the oxidation or decomposition of the PUFA product, for example, polymeric auto-oxidation products of fatty acids or their derivatives.
[00046] Examples of (3) include urea that can be added to remove saturated or monounsaturated fatty acids from the feed mixture.
[00047] Preferably, the feed mixture is a marine oil containing PUFA (for example a fish oil), more preferably a marine oil (for example a fish oil) comprising EPA and / or DHA.
[00048] A typical feed mixture for preparing concentrated EPA (EE) by the process of the present invention comprises 50 to 75% EPA (EE), 0 to 10% DHA (EE), and other components including other fatty acids w -3 and co-6 essentials.
[00049] A preferred feed mixture for preparing concentrated EPA (EE) by the process of the present invention comprises 55% EPA (EE), 5% DHA (EE), and other components including other w-3 and co fatty acids -6 essentials. DHA (EE) is less polar than EPA (EE).
[00050] The process of the present invention involves multiple chromatography separation steps.
[00051] The first separation step is effective for removing saturated and / or monounsaturated fatty acids present in the feed mixture and can be performed using a stationary bed chromatography device or a simulated or real moving bed.
[00052] When the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, there are several ways in which the three separation steps can be performed. Four preferred ways of carrying out the process are given as the first, second, third and fourth embodiments below.
[00053] In a first embodiment, the first, second and third separation steps are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, a solvent aqueous organic, the first, second and third separation steps being carried out in the first, second and third zones respectively, where each zone has one or more injection points for a feed mixture stream, one or more injection points for water and / or organic solvent, a raffinate withdrawal stream from which liquid can be collected from said zone, and an extract withdrawal stream from which liquid can be collected from said zone.
[00054] Typically, each zone has only one injection point for a feed mix. In one embodiment, each zone has only one injection point for the aqueous organic solvent eluent. In another embodiment, each zone has two or more injection points for water and / or organic solvent.
[00055] Typically each zone used has a unique array of chromatography columns connected in series that contain, as an eluent, an aqueous organic solvent. Typically, each of the chromatography columns in a zone is linked to the two columns in the apparatus adjacent to this column. Thus, the output of a given column in a zone is connected to the input of the adjacent column, for example, in the zone, which is downstream with respect to the flow of eluent in the system. Typically, none of the chromatography columns in a zone are linked to non-adjacent columns in the same zone.
[00056] The term "raffinate" is well known to the person 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 absorbing phase. Thus, a raffinate stream is typically enriched with more polar components, and depleted of less polar components compared to a feed stream.
[00057] The term "extract" is well known to the person 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 absorber phase compared to the liquid eluent phase. Thus, an extract stream is typically enriched with less polar components, and depleted of the more polar components compared to a feed stream.
[00058] As used herein the term "non-adjacent" refers to columns, for example, in the same apparatus, separated by one or more columns, preferably 3 or more columns, more preferably 5 or more columns, most preferably around 5 columns.
[00059] In a second embodiment, the first and second separation steps are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent , the first and second separation steps being carried out in the first and second zones respectively, in which each zone is as defined herein, and in which the third separation step is carried out in a separate simulated or real moving bed chromatography apparatus.
[00060] In the second embodiment, the third separation step is typically performed on a simulated or real moving bed chromatography apparatus comprising a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, and having one or more injection points for a feed mixture stream, one or more injection points for water and / or organic solvent, a raffinate withdrawal stream from which liquid can be collected from said plurality of linked chromatography columns , and an extract withdrawal stream from which liquid can be collected from said plurality of linked chromatography columns. This chromatography apparatus typically has only one injection point for a feed mixture. In one embodiment, this chromatography apparatus has only one injection point for the aqueous organic solvent eluent. In another embodiment, this chromatography apparatus has two or more injection points for water and / or organic solvent.
[00061] The chromatography apparatus used in the third separation step in the second embodiment typically has a single array of chromatography columns connected in series that contain, as an eluent, an aqueous organic solvent. Typically, each of the chromatography columns is linked to the two columns in the apparatus adjacent to this column. Thus, the output of a given column is connected to the input of the adjacent column, which is downstream with respect to the flow of eluent in the system. Typically, none of the chromatography columns are linked to non-adjacent columns on the chromatography apparatus.
[00062] The chromatography apparatus used in the third separation step in the second embodiment is an apparatus separate from the apparatus used in the first and second separation steps. Thus, two separate devices are used. The eluent circulates separately in separate chromatographic devices. Thus, the eluent is not shared between the separate chromatographic apparatus other than that eluent that may be present as a solvent in the second intermediate product that is produced in the second step, and which is then introduced into the chromatographic apparatus used in the third separation step. Chromatographic columns are not shared between separate chromatographic devices.
[00063] After the second intermediate product is obtained in the second separation step, the aqueous organic solvent eluent can be partially or totally removed before the second intermediate product is further purified in the third separation step. Alternatively, the intermediate product can be purified in the third step without removing any solvent present.
[00064] The chromatography apparatus used in the third separation step in the second embodiment is similar to the chromatography apparatus illustrated in Figure 1.
[00065] In a third embodiment, the second and third separation steps are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent , the second and third separation steps being carried out in the first and second zones respectively, in which each zone is as defined herein, and in which the first separation step is carried out in a separate simulated or real moving bed chromatography apparatus.
[00066] In the third embodiment, the first separation step is typically performed on a simulated or real moving bed chromatography apparatus comprising a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, and having one or more injection points for a feed mixture stream, one or more injection points for water and / or organic solvent, a raffinate withdrawal stream from which liquid can be collected from said plurality of linked chromatography columns , and an extract withdrawal stream from which liquid can be collected from said plurality of linked chromatography columns. This chromatography apparatus typically has only one injection point for a feed mixture. In one embodiment, this chromatography apparatus has only one injection point for the aqueous organic solvent eluent. In another embodiment, this chromatography apparatus has two or more injection points for water and / or organic solvent.
[00067] The chromatography apparatus used in the first separation step in the third embodiment typically has a single array of chromatography columns connected in series that contain, as an eluent, an aqueous organic solvent. Typically, each of the chromatography columns is linked to the two columns in the apparatus adjacent to this column. Thus, the output of a given column is connected to the input of the adjacent column, which is downstream with respect to the flow of eluent in the system. Typically, none of the chromatography columns are linked to non-adjacent columns on the chromatography apparatus.
[00068] The chromatography apparatus used in the first separation step in the third embodiment is an apparatus separate from the apparatus used in the second and third separation steps. Thus, two separate devices are used. The eluent is not shared between the separate chromatographic apparatus in addition to that eluent which may be present as a solvent in the first intermediate product that is produced in the first step, and which is introduced into the chromatographic apparatus used in the second separation step. Chromatographic columns are not shared between separate chromatographic devices.
[00069] After the first intermediate product is obtained in the first separation step, the aqueous organic solvent eluent can be partially or totally removed before the intermediate product is further purified in the next separation step. Alternatively, the first intermediate product can be purified even in the second separation step without removing any solvent present.
[00070] The chromatography apparatus used in the first separation step in the third embodiment is similar to the chromatography apparatus illustrated in Figure 1.
[00071] It will be assessed that in the first, second and third embodiments above two or more separation steps can occur simultaneously in a single simulated or real moving bed chromatography apparatus having two or three zones, where one zone is as defined above. A typical chromatography apparatus having two or more zones, for example two or three zones, is as described, for example, in PCT / GB10 / 002339, which is incorporated herein by reference.
[00072] In a fourth embodiment, (a) the first, second and third separation steps are carried out sequentially on the same chromatography apparatus, the first and second intermediate products being recovered between the first and second, and second and third stages of separation respectively, and the process conditions in the chromatography apparatus being adjusted between the first and second, and second and third separation steps such that saturated fatty acids and / or monounsaturated present in the feed mixture are removed in the first separation step , and the PUFA product is separated from the components other than the feed mixture in steps (ii) and (iii); or (b) the second separation step is carried out using a chromatographic apparatus different from that used in the first separation step, and / or the third separation step is carried out using a chromatographic apparatus different from that used in the second separation step.
[00073] In the fourth embodiment, each of the chromatography apparatus used to carry out the first, second and third separation steps is typically as defined above for the third separation step in the embodiment (2).
[00074] In option (b) of the fourth embodiment, all three steps are performed on separate chromatographic devices. Two or three of the first, second and third separation steps are performed in two or three separate chromatographic devices. These can be operated sequentially or simultaneously.
[00075] In particular, in option (b) of the fourth embodiment, two separate chromatography apparatus can be operated sequentially to carry out the first and second separation steps. In this case, the first intermediate product is recovered between the first and second separation steps and the process conditions in the first and second chromatography devices are adjusted such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii).
[00076] In particular, in option (b) of the fourth embodiment, two separate chromatography devices can be operated sequentially to carry out the second and third separation steps. In this case, the second intermediate product is recovered between the second and third separation steps and the process conditions in the second and third chromatography apparatus are adjusted such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii).
[00077] In particular, in option (b) of the fourth embodiment, three separate chromatography devices can be operated sequentially to carry out the first, second and third separation steps. In this case, the first intermediate product is recovered between the first and second separation steps, the second intermediate product is recovered between the second and third separation steps and the process conditions in the first, second and third chromatography devices are adjusted such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii).
[00078] In particular, in option (b) of the fourth embodiment, two separate chromatography devices can be operated simultaneously to carry out the first and second separation steps. The first and second stages of separation are carried out in separate chromatography apparatus, the first intermediate product obtained in the first stage being introduced into the chromatography apparatus used in the second separation stage, and the process conditions in the chromatography apparatus being adjusted such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii).
[00079] In particular, in option (b) of the fourth embodiment, two separate chromatography devices can be operated simultaneously to carry out the second and third separation steps. The second and third stages of separation are carried out in separate chromatography apparatus, the second intermediate product obtained in the second stage being introduced into the chromatography apparatus used in the third separation stage, and the process conditions in the chromatography apparatus being adjusted such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii).
[00080] In particular, in option (b) of the fourth embodiment, three separate chromatography devices can be operated simultaneously to perform the first, second and third separation steps. The first, second and third stages of separation are carried out in separate chromatography apparatus, the first intermediate product obtained in the first stage being introduced into the chromatography apparatus used in the second stage of separation, the second intermediate product obtained in the second stage being introduced into from the chromatography apparatus used in the third separation step, and the process conditions in the chromatography apparatus being adjusted such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step and the PUFA product is removed. separate from the components other than the feed mixture in steps (ii) and (iii).
[00081] In particular, in option (b) of the fourth embodiment, two or three separate chromatographic devices are operated. The eluent circulates separately in separate chromatographic devices. Thus, the eluent is not shared between the separate chromatographic apparatus other than that eluent which may be present as a solvent in the intermediate product which is purified in the first and / or second steps, and which is introduced into the chromatographic apparatus used in the next separation step. The chromatographic columns are not shared between the separate chromatographic apparatus used in the first and second and / or second and third separation steps.
[00082] After the intermediate product is obtained in the first and / or second separation steps, the aqueous organic solvent eluent can be partially or totally removed before the intermediate product is further purified in the next separation step. Alternatively, the intermediate product can be purified without removing any solvent present. These considerations also apply to the second intermediate product obtained in the second separation step in the above embodiment (2), and to the first intermediate product obtained in the first separation step in the above embodiment (3).
[00083] In general, any known stationary bed or mobile simulated or actual bed chromatography apparatus can be used for the purposes of the method of the present invention, provided that the apparatus is used in accordance with the process of the present invention. Those devices described in PCT / GB10 / 002339, US 2,985,589, US 3,696,107, US 3,706,812, US 3,761,533, FR-A-2103302, FR-A2651148, 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.
[00084] The above second, third and fourth embodiments are preferred. The third and fourth embodiments are more preferred. For certain applications, the third embodiment will be the most suitable. In other applications, the fourth embodiment will be the most suitable.
[00085] The first to the fourth embodiments are illustrated in more detail with reference to Figure 2. In all four embodiments in Figure 2, the eluent flow is from right to left, and the absorber flow is effective is from left to right. It can be seen in all cases that the first intermediate product obtained from the first separation step is used as the feed mixture for the second separation step, and the second intermediate product is used as the feed mixture for the third separation step .
[00086] Referring now to Figure 2A, this illustrates the first embodiment above, that is, where the first, second and third separation steps are performed in a single simulated or real moving bed chromatography apparatus in the first, second and third zones respectively. The first stage of separation takes place in the first zone. Then the first intermediate product of the first separation step carried out in the first zone is passed into the second zone as the feed mixture. The second separation step is then carried out in the second zone. The second intermediate product is then passed from the second separation step carried out in the second zone into the third zone as the feed mixture. The third stage of separation is then carried out in the third zone.
[00087] Referring now to Figure 2B, this illustrates the second embodiment above, that is, where the first and second separation steps are performed simultaneously on a single simulated or real moving bed chromatography apparatus on the first and second zones respectively, and the third step of separation is carried out in a separate simulated or real mobile bed chromatography apparatus. The first stage of separation takes place in the first zone. Then the first intermediate product of the first separation step carried out in the first zone is passed into the second zone as the feed mixture. The second stage of separation is carried out in the second zone. The second intermediate product is collected from the second zone. This is then introduced into a chromatography apparatus as the feed mixture for the third separation step.
[00088] Referring now to Figure 2C, this illustrates the third embodiment above, that is, where the second and third separation steps are performed simultaneously on a single simulated or real moving bed chromatography apparatus on the first and second zones respectively, and the first separation step is performed on a separate simulated or real moving bed chromatography apparatus. The first step of separation takes place in a chromatography apparatus. The first intermediate product is collected from the first device. This is then introduced into a separate chromatography apparatus as the feed mixture for the second separation step. The second separation step is carried out in the first zone of the chromatographic apparatus in which the second and third separation steps take place. The second intermediate product of the second separation step carried out in the first zone is passed into the second zone as the feed mixture for the third separation step. The third stage of separation takes place in the second zone.
[00089] Referring now to Figure 2D, this illustrates the fourth embodiment above, that is, where (a) the first, second and third separation steps are performed sequentially on the same chromatography apparatus, the first and second products intermediates being recovered between the first and second, and second and third stages of separation respectively, and the process conditions in the chromatography apparatus being adjusted between the first and second, and second and third stages of separation such that saturated fatty acids and / or monounsaturated substances present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii); or (b) two or three of the first, second and third separation steps are performed on two or three different separate devices; wherein the second separation step is carried out using a chromatographic apparatus different from that used in the first separation step, and / or the third separation step is carried out using a chromatographic apparatus different from that used in the second separation step.
[00090] When the first separation step comprises purifying the feed mixture in a stationary bed chromatography apparatus, there are several ways in which the three separation steps can be carried out. Thus typically, (a) the second and third separation steps are carried out simultaneously on a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, the second and third separation steps being carried out in the first and second zones respectively, where each zone is as defined herein; or (b) the second and third separation steps are carried out sequentially on the same chromatography apparatus, the second intermediate product being recovered between the second and third separation steps and the process conditions on the chromatography apparatus being adjusted between the second and third separation steps such that the PUFA product is separated from the components other than the feed mixture in steps (ii) and (iii); or (c) the second and third separation steps are performed in separate chromatography apparatus respectively, the intermediate product obtained from the second separation step being introduced into the chromatography apparatus used in the third separation step.
[00091] The above embodiment (a) is carried out in a manner similar to the second and third separation steps in the above embodiment (3).
[00092] The chromatography apparatus used in the above embodiments (b) and (c) are typically as defined above for the third separation step in the embodiment (2). Embodiments (b) and (c) are typically performed in a manner similar to embodiment (4) above.
[00093] It will be evaluated that in certain embodiments, two or three stages of separation can be performed simultaneously in a single chromatography apparatus having two or three zones respectively. In simulated or real moving bed chromatography devices in which two separation steps are carried out simultaneously in two zones, a stream of raffinate or extract is typically collected from a column in the first zone and introduced into a non-adjacent column in the second zone. In simulated or real moving bed chromatography apparatus in which three separation steps are carried out simultaneously in three zones, a stream of raffinate or extract is typically collected from a column in the first zone and introduced into a non-adjacent column in the second zone, and a stream of raffinate or extract is typically collected from a column in the second zone and introduced into a non-adjacent column in the third zone. This allows the first and / or second intermediate products collected in the first and / or second separation steps to be used as the feed mixture for the next separation step.
[00094] Typically, the second intermediate product is collected as the raffinate stream in the second separation step, and the PUFA product is collected as the extract stream in the third separation step; or the second intermediate product is collected as the extract stream in the second separation step, and the PUFA product is collected as the raffinate stream in the third separation step.
[00095] Preferably, the second intermediate product is collected as the raffinate stream in the second separation step, and the PUFA product is collected as the extract stream in the third separation step.
[00096] Typically, in embodiments where the second and third stages of separation are performed simultaneously on a single simulated or real moving bed chromatography apparatus in the first and second zones respectively, (a) the second intermediate product is collected as a raffinate stream containing the PUFA product together with the more polar components of a column in the first zone and introduced into a non-adjacent column in the second zone, where the PUFA product is then collected as the extract stream in the third separation step carried out in the second zone; or (b) the second intermediate product is collected as an extract stream containing the PUFA product together with the less polar components of a column in the first zone and introduced into a non-adjacent column in the second zone, where the PUFA product is then collected as the raffinate stream in the third separation step performed in the second zone.
[00097] Preferably, in the embodiments where the second and third separation steps are performed simultaneously on a single simulated or real moving bed chromatography apparatus in the first and second zones respectively, the second intermediate product is collected as a raffinate stream containing the PUFA product together with the more polar components of a column in the first zone and introduced into a non-adjacent column in the second zone, where the PUFA product is then collected as the extract stream in the third separation step that is carried out in the second zone.
[00098] When the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, the first intermediate product is typically collected as the raffinate stream in the first separation step.
[00099] When the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, the first intermediate product is typically collected as the raffinate stream in the first separation step and (a) the second intermediate product is collected as the raffinate stream in the second separation step, and the PUFA product is collected as the extract stream in the third separation step; or (b) the second intermediate product is collected as the extract stream in the second separation step, and the PUFA product is collected as the raffinate stream in the third separation step.
[000100] Typically, the first intermediate product obtained in the first separation step is enriched in the PUFA product compared to the feed mixture; and / or the second intermediate product obtained in the second separation step is enriched in the PUFA product compared to the first intermediate product.
[000101] Preferably the first intermediate product obtained in the first separation step is enriched in the PUFA product compared to the feed mixture and the second intermediate product obtained in the second separation step is enriched in the PUFA product compared to the first intermediate product.
[000102] Typically, the first intermediate product obtained in the first separation step is depleted in saturated and / or monounsaturated fatty acids compared to the feed mixture.
[000103] Typically, in the first step the PUFA product is separated from the components of the feed mixture that are less polar than the PUFA product, in the second step the PUFA product is separated 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 separation step, and in the third separation step the PUFA product is separated from the components which are more polar than the PUFA product.
[000104] Alternatively, in the first step the PUFA product is separated from the components of the feed mixture that are less polar than the PUFA product, in the second step the PUFA product is separated from the components of the feed mixture that are more polar than the PUFA product, and in the third separation step the PUFA product is separated from components that are less polar than the PUFA product, but more polar than the components separated in the first separation step.
[000105] The components of the feed mixture separated in the first step that are less polar than the PUFA product are typically unsaturated and / or monounsaturated fatty acids.
[000106] Components of the feed mixture that are less polar than the PUFA product, but more polar than the components separated in the first separation step typically include DHA or a derivative of DHA and / or other PUFAs or PUFA derivatives which are less polar than the PUFA product, but more polar than the components separated in the first separation step.
[000107] Components of the feed mixture that are more polar than the PUFA product include SDA or a derivative of SDA and / or other PUFAs that are more polar than the PUFA product.
[000108] PUFAs other than EPA are well known and include PUFAs o-3 and o-6. Examples of w-3 PUFAs include alpha linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). Examples of w-6 PUFAs include linoleic acid (LA), gamma-linolenic acid (GLA), eicosadienic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (ARA), docosadienic acid, adrenic acid and docosapentaenoic acid ( w-6).
[000109] The number of columns used in each separation step is not particularly limited. A skilled person would easily be able to determine an appropriate number of columns for use. The number of columns is typically 4 or more, preferably 6 or more, more preferably 8 or more, for example 4, 5, 6, 7, 8, 9, or 10 columns. In the preferred embodiment, 5 or 6 columns, more preferably 6 columns are used. In another preferred embodiment, 7 or 8 columns, more preferably 8 columns are used. Typically, there are no more than 25 columns, preferably no more than 20, more preferably no more than 15.
[000110] In embodiments where two separation steps occur simultaneously on a single chromatography apparatus in the first and second zones respectively, the number of columns in each zone is typically 4 or more, preferably 6 or more, more preferably 8 or more , for example 4, 5, 6, 7, 8, 9, or 10 columns.
[000111] In embodiments where three separation steps occur simultaneously on a single chromatography apparatus in the first, second and third zones respectively, the number of columns in each zone is typically 4 or more, preferably 6 or more, more preferably 8 or more, for example 4, 5, 6, 7, 8, 9, or 10 columns.
[000112] The first, second and third separation steps typically involve the same number of columns. For certain applications they may have different numbers of columns.
[000113] The dimensions of the columns used are not particularly limited, and will depend on the volume of the feed mixture to be purified. A skilled person would easily be able to determine columns appropriately sized for use. The diameter of each column is typically between 10 and 1000 mm, preferably between 10 and 500 mm, more preferably between 25 and 250 mm, even more preferably between 50 and 100 mm, and most preferably between 70 and 80 mm. The length of each column is typically between 10 and 300 cm, preferably between 10 and 200 cm, more preferably between 25 and 150 cm, even more preferably between 70 and 110 cm, and most preferably between 80 and 100 cm.
[000114] The first, second and third separation steps typically involve columns having identical dimensions but, for certain applications, can have different dimensions.
[000115] Flow rates for the column are limited by the maximum pressures across the series of columns and will depend on the column dimensions and the particle size of the solid phases. A person skilled in the art will easily be able to establish the required flow rate for each column dimension to ensure efficient desorption. Larger diameter columns will generally need higher flows to maintain linear flow through the columns.
[000116] For the typical column sizes outlined above, typically the flow rate of eluent in the chromatographic apparatus used in the first or second separation steps is 1 to 4.5 L / min., Preferably 1.5 to 2, 5 L / min. Typically, the flow rate of the extract from the chromatographic apparatus used in the first or second separation steps is from 0.1 to 2.5 L / min., Preferably from 0.5 to 2.25 L / min. In embodiments where part of the extract from the first or second separation steps is recycled back into the apparatus used in the first or second separation steps, the recycle flow rate is typically 0.7 to 1.4 L / min., preferably about 1 L / min. Typically, the rafinate flow rate of the chromatographic apparatus used in the first or second separation steps is 0.2 to 2.5 L / min., Preferably 0.3 to 2.0 L / min. In embodiments where part of the raffinate from the first or second separation steps is recycled back into the apparatus used in the first or second separation steps, the recycle flow 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 chromatographic apparatus used in the first or second separation steps is 5 to 150 ml / min., Preferably 10 to 100 ml / min., More preferably 20 to 60 ml / min ..
[000117] For typical column sizes outlined above, typically the flow rate of eluent in the chromatographic apparatus used in the third separation step is 1 to 4 L / min., Preferably 1.5 to 3.5 L / min . Typically, the flow rate of the chromatographic apparatus extract used in the third separation step is 0.5 to 2 L / min., Preferably 0.7 to 1.9 L / min. In embodiments where part of the extract from the third separation step is recycled back into the apparatus used in the third separation step, the recycle flow rate is typically 0.6 to 1.4 L / min., Preferably from 0.7 to 1.1 L / min., more preferably from about 0.9 L / min. Typically, the rafinate flow rate of the chromatographic apparatus used in the third separation step is 0.5 to 2.5 L / min., Preferably 0.7 to 1.8 L / min., More preferably about 1.4 L / min. In embodiments where part of the raffinate from the third separation step is recycled back into the apparatus used in the third separation step, the recycle flow rate is typically 0.3 to 1.0 L / min., Preferably of about 0.5 L / min.
[000118] As the qualified person will assess, references to the rates at which the liquid is collected or removed by means of the various raffinate extracts and streams refer to the volumes of liquid removed in an amount of time, typically L / minute. Similarly, references to the rates at which the liquid is recycled back into an apparatus, typically to an adjacent column in the apparatus, refer to the volumes of liquid recycled in an amount of time, typically L / minute.
[000119] The time of the stage, that is, the time between the change of the injection points of the feed and eluent mixture, and the various points of collection of the collected fractions, is not particularly limited, and will depend on the number and dimensions of the columns used, and the flow rate through the apparatus. A skilled person would easily be able to determine appropriate step times for use in the process of the present invention. The step time is typically from 100 to 1000 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.
[000120] In the process of the present invention, real moving bed chromatography is preferred.
[000121] Conventional absorbers 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 or a different absorber. Typically, each column contains the same absorber. Examples of such commonly used materials are polymer beads, preferably DVB cross-linked polystyrene (divinylbenzene); and silica gel, preferably silica gel bound in reverse phase with C8 or C18 alkanes, especially C18. the C18 reverse phase bonded silica gel is preferred. The absorber used in the process of the present invention is preferably non-polar.
[000122] The shape of the absorber material of the stationary phase can be, for example, spherical or non-spherical beads, preferably substantially spherical beads. Such beads typically have a diameter of 5 to 500 microns, preferably 10 to 500 microns, more preferably 15 to 500 microns, more preferably 40 to 500 microns, more preferably 100 to 500 microns, most preferably 250 to 500 microns , even more preferably from 250 to 400 microns, most preferably from 250 to 350 microns. In some embodiments, beads with a diameter of 5 to 35 microns can be used, typically 10 to 30 microns, preferably 15 to 25 microns. Some preferred particle sizes are slightly larger than the bead particle sizes used in the past in simulated and real moving bed processes. The use of larger particles allows a lower eluent pressure to be used in the system. This, in turn, has advantages in terms of cost savings, efficiency and lifetime of the device. It has surprisingly been discovered that large particle size absorber beads can be used in the process of the present invention (with its associated advantages) without any loss in resolution.
[000123] The absorber typically has a pore size of 10 to 50 nm, preferably 15 to 45 nm, more preferably 20 to 40 nm, most preferably 25 to 35 nm.
[000124] Typically, the process of the present invention is conducted at 15 to 55 ° C, preferably from 20 to 40 ° C, more preferably around 30 ° C. Thus, the process is typically carried out at room temperature, but can be conducted at elevated temperatures.
[000125] As mentioned above, the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii) . This is typically done by adjusting the process conditions on the chromatography apparatus, or zone on a chromatography apparatus in which the first, second and third separation steps are performed.
[000126] Thus, the process conditions in the first, second and third stages of separation typically vary. The varying process conditions may include, for example, the size of the columns used, the number of columns used, the packaging used in the columns, the stage time of the SMB apparatus, the temperature of the apparatus, the eluent used in the separation, or the flow rates used in the apparatus, in particular the recycling rate of the liquid collected through the extract or raffinate streams.
[000127] Preferably the process conditions that vary are the water ratio: organic solvent of the eluent used in the separation steps, and / or the recycling rate of the liquid collected through the extract or raffinate streams in the separation steps. Both of these options are discussed in more detail below.
[000128] Typically, part of the extract stream from the apparatus used in the second separation step is recycled back into the apparatus used in the second separation step; and / or part of the raffinate stream from the apparatus used in the second separation step is recycled back into the apparatus used in the second separation step; and / or part of the extract stream from the apparatus used in the third separation step is recycled back into the apparatus used in the third separation step; and / or part of the raffinate stream from the apparatus used in the third separation step is recycled back into the apparatus used in the third separation step.
[000129] Preferably, part of the extract stream from the apparatus used in the second separation step is recycled back into the apparatus used in the second separation step; and part of the raffinate stream from the apparatus used in the second separation step is recycled back into the apparatus used in the second separation step; and part of the extract stream from the apparatus used in the third separation step is recycled back into the apparatus used in the third separation step; and part of the raffinate stream from the apparatus used in the third separation step is recycled back into the apparatus used in the third separation step.
[000130] When the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, typically part of the extract stream from the apparatus used in the first separation step is recycled back into the used apparatus in the first stage of separation; and / or part of the raffinate stream from the apparatus used in the first separation step is recycled back into the apparatus used in the first separation step.
[000131] When the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, preferably part of the extract stream from the apparatus used in the first separation step is recycled back into the used apparatus in the first stage of separation; and part of the raffinate stream from the apparatus used in the first separation step is recycled back into the apparatus used in the first separation step; and part of the extract stream from the apparatus used in the second separation step is recycled back into the apparatus used in the second separation step; and part of the raffinate stream from the apparatus used in the second separation step is recycled back into the apparatus used in the second separation step; and part of the extract stream from the apparatus used in the third separation step is recycled back into the apparatus used in the third separation step; and part of the raffinate stream from the apparatus used in the third separation step is recycled back into the apparatus used in the third separation step.
[000132] This recycling involves feeding part of the extract or raffinate stream outside the chromatography apparatus used in the first, second or third separation steps back into the apparatus used in this step, typically within an adjacent column. This adjacent column is the adjacent column that is downstream with respect to the flow of eluent in the system.
[000133] When two or three separation steps are carried out simultaneously in a single chromatography in two or three zones respectively, this recycling involves recycling the particular raffinate extract or stream removed from a back zone into the same zone.
[000134] The rate at which the liquid collected via the raffinate extract or stream in a particular separation step is recycled back into a chromatography apparatus or zone used in this separation step is the rate at which the collected liquid through this stream it is fed back into the apparatus used in this step, typically within an adjacent column, that is, the column downstream with respect to the flow of eluent in the system.
[000135] This can be seen with reference to a preferred embodiment in Figure 5. The extract recycling rate in the first separation step is the rate at which the extract collected from the bottom of column 2 of the chromatographic apparatus used in the first step Separation is fed to the top of column 3 of the chromatographic apparatus used in the first separation step, that is, the flow rate of the liquid at the top of column 3 of the chromatographic apparatus used in the first separation step.
[000136] The extract recycling rate in the second separation step is the rate at which the extract collected at the bottom of column 10 of the chromatographic apparatus used in the second separation step is fed at the top of column 11 of the chromatographic apparatus used in the second stage of separation, that is, the flow rate of the liquid at the top of column 11 of the chromatographic apparatus used in the second separation step.
[000137] The extract recycling rate in the third separation step is the rate at which the extract collected at the bottom of column 19 of the chromatographic apparatus used in the second separation step is fed at the top of column 19 of the chromatographic apparatus used in the second stage of separation, that is, the flow rate of the liquid at the top of column 19 of the chromatographic apparatus used in the second separation step.
[000138] The recycling of extract and / or raffinate streams in the first, second and / or third separation steps is typically carried out by feeding the liquid collected through this stream in this separation step into a container, and then pumping an amount of this liquid from the container back into the apparatus or zone used in this separation step, typically within an adjacent column. In this case, the rate of liquid recycling collected through a particular extract or raffinate stream in the first and / or second separation steps, typically back into an adjacent column, is the rate at which the liquid is pumped to outside the container back into the chromatography apparatus or zone, typically within an adjacent column.
[000139] As the qualified person will assess, the amount of liquid that is introduced into a chromatography apparatus through the eluent and feed stock currents is balanced with the amount of liquid removed from the apparatus, and recycled back into the device.
[000140] Thus, with reference to Figure 5, for the extract stream, the flow rate of eluent (desorbent) in the chromatographic apparatus (s) used in the second and third separation steps ( D) is equal to the rate at which the liquid collected through the extract stream in this separation step accumulates in a container (E2 and E3) added at the rate at which the extract is recycled back into the chromatographic apparatus used in this particular separation step (D-E2 and D-E3).
[000141] For the raffinate current of a separation step, the rate at which the extract is recycled back into the chromatographic apparatus used in this particular separation step (DE le D-E2) added to the rate at which the feed stock is introduced into the chromatographic apparatus used in this particular separation step (F and R1) is equal to the rate at which the liquid collected through the raffinate stream in this particular separation step accumulates in a container (R1 and R2) added at the rate at which raffinate is recycled back into the chromatographic apparatus used in this particular separation step (D + F-E1-R1 and D + R1-E2-R2).
[000142] The rate at which the liquid collected from a particular extract or raffinate stream from a chromatography apparatus or zone accumulates in a container can also be considered as the net removal rate of this extract or raffinate stream from this apparatus. chromatography.
[000143] Typically, the rate at which the liquid collected through one or both of the extract and raffinate streams in the second separation step is recycled back into the apparatus used in this separation step is adjusted such that the acids saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii); and / or at which the rate at which the liquid collected through one or both of the extract and raffinate streams in the third separation step is recycled back into the apparatus used in this separation step is adjusted such that the acids saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii).
[000144] Preferably, the rate at which the liquid collected through one or both of the extract and raffinate streams in the second separation step is recycled back into the apparatus used in this separation step is adjusted such that the acids saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii); and in which the rate at which the liquid collected through one or both of the extract and raffinate streams in the third separation step is recycled back into the apparatus used in this separation step is adjusted such that saturated fatty acids and / or monounsaturated substances present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii).
[000145] When the first separation step comprises purifying the feed mixture in a simulated or real mobile bed chromatography apparatus, the rate at which the liquid collected through one or both of the extract and raffinate streams in the first separation step is recycled back into the apparatus used in this separation step is typically adjusted such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii).
[000146] Typically, the rate at which the liquid collected via the extract stream in the second separation step is recycled back into the chromatography apparatus used in the second separation step differs from the rate at which the liquid collected via the extract stream in the third separation step is recycled back into the chromatography apparatus used in the third separation step; and / or the rate at which the liquid collected via the raffinate stream in the second separation step is recycled back into the chromatography apparatus used in the second separation step differs from the rate at which the liquid collected via the stream raffinate in the third separation step is recycled back into the chromatography apparatus used in the third separation step.
[000147] When the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, the rate at which the liquid collected through the extract stream in the first separation step is recycled back to inside the chromatography apparatus used in the first separation step typically differs from the rate at which the liquid collected through the extract stream in the second separation stage is recycled back into the chromatography apparatus used in the second separation stage; and / or the rate at which the liquid collected through the raffinate stream in the first separation step is recycled back into the chromatography apparatus used in the first separation step typically differs from the rate at which the liquid collected through the stream of raffinate in the second separation step is recycled back into the chromatography apparatus used in the second separation step
[000148] Varying the rate at which the liquid collected through the extract and / or raffinate streams in the first, second and / or third separation steps is recycled back into the apparatus used in this particular separation step has the effect of vary the amount of the more polar and less polar components present in the extract and raffinate streams. Thus, for example, a lower extract recycling rate results in less of the less polar components in this separation step being carried through the raffinate stream. A higher extract recycling rate results in more of the less polar components in this separation step being carried through the raffinate stream.
[000149] This can be seen, for example, in the specific embodiment of the invention shown in Figure 5. The rate at which the liquid collected through the extract stream in the second separation step is recycled back into the chromatographic apparatus used in this separation step (D-E2) will affect the extent to which any of the components A are loaded through the raffinate stream in the second separation step (R2).
[000150] Typically, the rate at which the liquid collected via the extract stream in the second separation step is recycled back into the chromatographic apparatus used in the second separation step is faster than the rate at which the liquid collected through of the extract corente in the third separation stage is recycled back into the chromatographic apparatus used in the third separation stage. Preferably, a raffinate stream containing the PUFA product together with the more polar components is collected from the second separation step and purified in the third separation step, and the rate at which the liquid collected through the extract stream in the second step The separation liquid is recycled back into the chromatographic apparatus used in the second separation step is faster than the rate at which the liquid collected through the extract stream in the third separation step is recycled back into the chromatographic apparatus used in the third separation step. separation.
[000151] Alternatively, the rate at which the liquid collected via the raffinate stream in the second separation step is recycled back into the chromatographic apparatus used in the second separation step is faster than the rate at which the liquid collected through of the raffinate stream in the third separation step is recycled back into the chromatographic apparatus used in the third separation step. Preferably, an extract stream containing the PUFA product together with the less polar components is collected from the second separation step and purified in the third separation step, and the rate at which the liquid is collected via the raffinate stream in the second step The separation fluid is recycled back into the chromatographic apparatus used in the second separation step is faster than the rate at which the liquid collected through the raffinate stream in the third separation step is recycled back into the chromatographic apparatus used in the third separation step. separation.
[000152] Where recycling rates are adjusted such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the different components of the feed mixture in the steps ( ii) and (iii) the water: organic solvent ratio of the eluent used in the separation steps where the recycling rates differ may be the same or different.
[000153] The eluent used in the process of the present invention is an aqueous organic solvent.
[000154] The aqueous organic solvent typically comprises water and one or more alcohols, ethers, esters, ketones or nitriles, or mixtures thereof.
[000155] Alcohol solvents are well known to the person skilled in the art. Alcohols are typically short-chain alcohols. Alcohols are typically of the formula ROH, where R is a straight and branched C1-C6 alkyl group. The C1-C6 alkyl group is preferably unsubstituted. Examples of alcohols include methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol and t-butanol. Methanol and ethanol are preferred. Methanol is most preferred.
[000156] Ether solvents are well known to the person skilled in the art. Ethers are typically short-chain ethers. The ethers are typically of the formula R-O-R ', where R and R' are the same or different and represent a straight and branched C1-C6 alkyl group. The C1-C6 alkyl group is preferably unsubstituted. Preferred ethers include diethyl ether, diisopropyl ether, and methyl t-butyl ether (MTBE).
[000157] Ester solvents are well known to the person skilled in the art. Esters are typically short-chain esters. The esters are typically of the formula R- (C = O) O-R ', where R and R' are the same or different and represent a straight and branched C1-C6 alkyl group.
[000158] Preferred esters include methyl acetate and ethyl acetate.
[000159] Ketone solvents are well known to the person skilled in the art. Ketones are typically short-chain ketones. Ketones are typically of the formula R- (C = O) -R ', where R and R' are the same or different and represent a straight and branched C1-C6 alkyl group. The C1-C6 alkyl group is preferably unsubstituted. Preferred ketones include acetone, methyl ethyl ketone and methyl isobutyl ketone (MIBK).
[000160] Nitrile solvents are well known to the person skilled in the art. Nitriles are typically short-chain nitriles. Nitriles are typically of the formula R-CN, where R represents a straight and branched C1-C6 alkyl group. The C1-C6 alkyl group is preferably unsubstituted. Preferred nitriles include acetonitrile.
[000161] Typically, the aqueous organic solvent is aqueous alcohol or aqueous acetonitrile.
[000162] The aqueous organic solvent is preferably aqueous methanol or aqueous acetonitrile. Aqueous methanol is most preferred.
[000163] Typically, the eluent is not in a supercritical state. Typically, the eluent is a liquid.
[000164] Typically the average water: organic solvent ratio, for example the water: methanol ratio, of the eluent in the entire apparatus is 0.1: 99.9 to 9:91% by weight, preferably 0.25 : 99.75 to 7:93% by weight, more preferably from 0.5: 99.5 to 6:94% by weight.
[000165] When the aqueous organic solvent is aqueous acetonitrile, the eluent typically contains up to 30% by weight of water, the rest acetonitrile. Preferably, the eluent contains from 5 to 25% by weight of water, the rest acetonitrile. More preferably, the eluent contains 10 to 20% by weight of water, the remainder acetonitrile. Even more preferably, the eluent contains from 15 to 25% by weight of water, the rest acetonitrile.
[000166] Typically, the water: organic solvent ratio used in each separation step is adjusted such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step; and the PUFA product is separated from the components other than the feed mixture in steps (ii) and (iii).
[000167] Typically, the aqueous organic solvent eluent used in two or more of the separation steps has a different water: organic solvent ratio. In one embodiment, the water: organic solvent ratio used in each separation step has a different water: organic solvent ratio.
[000168] The eluting strength of the eluent used in two or more of the separation steps is typically different. Depending on the choice of the organic solvent, they can be more powerful desorbers than water. Alternatively, they can be less powerful absorbers than water. Acetonitrile and alcohols, for example, are more powerful absorbers than water.
[000169] In a preferred embodiment, the aqueous organic solvent eluent used in the second and third separation steps has the same water ratio: organic solvent, and the aqueous organic solvent eluent used in the first separation step has a reason water: organic solvent different from the organic solvent eluent used in the second and third separation steps.
[000170] In this preferred embodiment, the eluting strength of the eluent used in the second and third separation steps is the same; and / or the eluting strength of the eluent used in the first separation step is greater than that of the eluent used in the second separation step. Preferably in this embodiment, the eluting strength of the eluent used in the second and third stages of separation is the same; and the eluting strength of the eluent used in the first separation step is greater than that of the eluent used in the second and third separation steps. In this embodiment, when the aqueous organic solvent is aqueous alcohol or acetonitrile, the amount of alcohol or acetonitrile in the eluent used in the second and third stages of separation is typically the same, and the amount of alcohol or acetonitrile in the eluent used in the first step The separation phase is typically greater than the amount of alcohol or acetonitrile in the eluent used in the second and third stages of separation. Thus, in this embodiment, the water: organic solvent ratio of the eluent in the second and third separation steps is typically the same, and the water: organic solvent ratio of the eluent in the first separation step is typically lower than the water ratio: organic solvent of the eluent in the second and third stages of separation.
[000171] In this preferred embodiment, the water: organic solvent ratio of the eluent in the first separation step is typically 0: 100 to 5:95% by weight, preferably 0.1: 99.9 to 2.5 : 97.5% by weight, more preferably from 0.1: 99.9 to 2:98% by weight, even more preferably from 0.1: 99.9 to 1:99% by weight, even more preferably from 0 , 25: 99.75 to 0.75: 99.25% by weight, and most preferably around 0.5: 99.5. In this preferred embodiment, the water: organic solvent ratio of the eluent in the second and third separation steps is typically 5:95 to 11:89% by weight, preferably from 6:94 to 10:90% by weight, plus preferably from 7:93 to 9:91% by weight, even more preferably from 7.5: 92.5 to 8.5: 91.5% by weight, and most preferably around 8:92% by weight.
[000172] In this preferred embodiment, the water: organic solvent ratio of the eluent used in the first separation step is preferably 0.1: 99.9 to 1:99% by weight, and the water: organic solvent ratio of the eluent used in the second and third separation steps is preferably from 7:93 to 9:91% by weight.
[000173] In an alternative embodiment, the aqueous organic solvent eluent used in each separation step has a different water: organic solvent ratio.
[000174] In this alternative embodiment, the eluting strength of the eluent used in the first separation step is greater than that of the eluent used in the second separation step; and / or the eluting strength of the eluent used in the second separation step is greater than that of the eluent used in the third separation step. Preferably, a raffinate stream containing the PUFA product together with the more polar components is collected from the second separation step and purified in the third separation step and the eluting strength of the eluent used in the second separation step is greater than that of the eluent used in the third separation step. Alternatively, an extract stream containing the PUFA product together with the less polar components is collected from the second separation step and purified in the third separation step and the elution strength of the eluent used in the second separation step is lower than that of the eluent used in the third separation step.
[000175] In practice this is achieved by varying the relative amounts of water and organic solvent used in each separation step. In this embodiment, when the aqueous organic solvent is aqueous alcohol or acetonitrile, the amount of alcohol or acetonitrile in the eluent used in the first separation step is typically greater than the amount of alcohol or acetonitrile in the eluent used in the second separation step; and / or the amount of alcohol or acetonitrile in the eluent used in the second separation step is typically greater than the amount of alcohol or acetonitrile in the eluent used in the third separation step. Thus, in this embodiment, the water: organic solvent ratio of the eluent in the first separation step is typically lower than the water: organic solvent ratio of the eluent in the second separation stage; and / or the water: organic solvent ratio of the eluent in the second separation step is typically lower than the water: organic solvent ratio of the eluent in the third separation step.
[000176] It will be evaluated that the water and organic solvent ratios in each separation step mentioned above are average ratios within the entire chromatographic apparatus.
[000177] Typically, the water: organic solvent ratio of the eluent in each separation step is controlled by introducing water and / or organic solvent into one or more columns in the chromatographic apparatus used in the separation steps. Thus, for example, to obtain a lower water: organic solvent ratio in the first separation step than in the second and third separation steps, water is typically introduced more slowly into the chromatographic apparatus used in the first separation step than in the second and third separation steps.
[000178] In some embodiments, essentially pure organic solvent and essentially pure water can be introduced at different points in the chromatographic apparatus used in each separation step. The relative flow rates of these two streams will determine the overall solvent profile in the chromatographic apparatus. In other embodiments, different organic solvent / water mixtures can be introduced at different points in each chromatographic apparatus used in each separation step. This will involve introducing two or more different organic solvent / water mixtures into the chromatographic apparatus used in a particular separation step, each mixture of organic solvent / water having a different organic solvent: water ratio. The relative flow rates and relative concentrations of the organic solvent / water mixtures in this embodiment will determine the overall solvent profile in the chromatographic apparatus used in this separation step.
[000179] Preferably, the aqueous organic solvent eluent used in the second and third separation steps has the same water ratio: organic solvent, and the aqueous organic solvent eluent used in the first separation step has a water: organic solvent ratio different from the organic solvent eluent used in the second and third separation steps; and the rate at which the liquid collected via the extract stream in the second separation step is recycled back into the chromatography apparatus used in the second separation step differs from the rate at which the liquid collected via the extract stream in the the third separation step is recycled back into the chromatography apparatus used in the third separation step.
[000180] More preferably, the water: organic solvent ratio of the eluent in the second and third separation steps is the same, and the water: organic solvent ratio of the eluent in the first separation step is lower than the water ratio : organic solvent of the eluent in the second and third stages of separation; and the rate at which the liquid collected via the extract stream in the second separation step is recycled back into the chromatographic apparatus used in the second separation step is faster than the rate at which the liquid collected via the extract stream in the third separation stage it is recycled back into the chromatographic apparatus used in the third separation stage.
[000181] Even more preferably,
[000182] the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus;
[000183] the second and third stages of separation are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, the second and third stages of separation being carried out in the first and second zones respectively, where each zone is as defined herein, and where the first separation step is carried out in a separate simulated or real moving bed chromatography apparatus;
[000184] the first intermediate product is collected as the raffinate stream in the first separation step, the second intermediate product is collected as the raffinate stream in the second separation step, and the PUFA product is collected as the extract stream in the third stage of separation;
[000185] the second intermediate product raffinate stream containing the PUFA product together with the more polar components is collected from a column in the first zone and introduced into a non-adjacent column in the second zone;
[000186] the aqueous organic solvent eluent used in the second and third separation steps has the same water ratio: organic solvent, and the water: organic solvent ratio of the eluent used in the first separation step is lower than the ratio water: organic solvent of the eluent used in the second and third stages of separation; and
[000187] the rate at which the liquid collected via the extract stream in the second separation step is recycled back into the chromatographic apparatus used in the second separation step is faster than the rate at which the liquid collected via the stream of extract in the third stage of separation is recycled back into the chromatographic apparatus used in the third stage of separation.
[000188] It is preferred that the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus; and the second and third separation steps are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, the second and third separation steps being carried out in the first and second zones respectively, in which each zone is as defined herein, and in which the first separation step is carried out in a separate simulated or real moving bed chromatography apparatus. A preferred embodiment of this is illustrated in Figure 3.
[000189] A feed mixture F comprising the PUFA product (B) and the most polar (C) and the least polar (A ') and the components (a) are purified in the first separation step. In the first separation step, the less polar components (for example saturated and / or monounsaturated) (A ') are removed as an E1 extract stream. The PUFA product (B), the more polar components (C) and the less polar components (but more polar than (A ')) (a) are collected as a raffinate stream R1. The rafinate stream R1 is the intermediate product which is then purified in the second separation step.
[000190] In the second separation step, the less polar components (a) are removed as an E2 extract stream. The PUFA product (B) and the more polar components (C) are collected as a rafinate stream R2. The rafinate stream R2 is the intermediate product which is then purified in the third separation step.
[000191] In the third separation step, the more polar components (C) are removed as a R3 rafinate chain. The PUFA product (B) is collected as an E3 extract stream. The second and third stages of separation take place in two zones on a single SMB chromatographic apparatus.
[000192] This embodiment is illustrated in more detail in Figure 4. Figure 4 is identical to Figure 2, except that the points of introduction of the aqueous organic solvent desorbent (D) into each chromatographic apparatus are shown.
[000193] Typical solvents for use in this most preferred embodiment are aqueous alcohols or aqueous acetonitrile, preferably aqueous methanol.
[000194] Typically in this preferred embodiment, the aqueous organic solvent eluent used in the second and third separation steps has the same water ratio: organic solvent, and the aqueous organic solvent eluent used in the first separation step has a reason water: organic solvent different from the organic solvent eluent used in the second and third separation steps; and the rate at which the liquid collected via the extract stream in the second separation step is recycled back into the chromatography apparatus used in the second separation step differs from the rate at which the liquid collected via the extract stream in the the third separation step is recycled back into the chromatography apparatus used in the third separation step.
[000195] Preferably, in this preferred embodiment, the water: organic solvent ratio of the eluent in the second and third separation steps is the same, and the water: organic solvent ratio of the eluant in the first separation step is lower than that the water: organic solvent ratio of the eluent in the second and third stages of separation; and the rate at which the liquid collected via the extract stream in the second separation step is recycled back into the chromatographic apparatus used in the second separation step is faster than the rate at which the liquid collected via the extract stream in the third separation stage it is recycled back into the chromatographic apparatus used in the third separation stage.
[000196] In this preferred embodiment the first rafinate stream in the first separation step is typically removed downstream from the point of introduction of the feed mixture into the chromatographic apparatus used in the first separation step, with respect to the eluent flow.
[000197] In this particularly preferred embodiment, the first extract stream in the first separation step is typically removed upstream of the point of introduction of the feed mixture into the chromatographic apparatus used in the first separation step, with respect to the eluent flow.
[000198] In this particularly preferred embodiment, the second raffinate stream in the second separation step is typically removed downstream of the point of introduction of the first intermediate product into the chromatographic apparatus used in the second separation step, with respect to the eluent flow.
[000199] In this particularly preferred embodiment, the second extract stream in the second separation step is typically collected upstream of the point of introduction of the first intermediate product into the chromatographic apparatus used in the second separation step, with respect to the eluent flow.
[000200] In this particularly preferred embodiment, the third rafinate stream in the third separation step is typically removed downstream from the point of introduction of the second intermediate product into the chromatographic apparatus used in the third separation step, with respect to the eluent flow.
[000201] In this particularly preferred embodiment, the third extract stream in the third separation step is typically collected upstream of the point of introduction of the second intermediate product in the chromatographic apparatus used in the third separation step, with respect to the eluent flow.
[000202] Typically in this preferred embodiment, the aqueous organic solvent is introduced into the chromatographic apparatus used in the first separation step upstream of the removal point of the first extract stream, with respect to the eluent flow.
[000203] Typically in this preferred embodiment, the aqueous organic solvent is introduced into the chromatographic apparatus used in the second separation step upstream of the removal point of the second extract stream, with respect to the eluent flow.
[000204] Typically in this preferred embodiment, the aqueous organic solvent is introduced into the chromatographic apparatus used in the third separation step upstream of the removal point of the third extract stream, with respect to the eluent flow.
[000205] A more preferred embodiment of the invention illustrated in Figures 3 and 4 is shown in Figure 5. This illustrates the number of columns used in each separation step, and shows typical points of introduction of mixtures of feed and eluents, and typical extract removal points and raffinate chains.
[000206] Thus, in this most preferred embodiment, the SMB chromatography apparatus used in the first separation stage consists of eight chromatographic columns, from 1 to 8. The SMB chromatography apparatus used in the second separation stage consists of eight chromatographic columns. , from 9 to 16. The SMB chromatography apparatus used in the third separation step consists of seven chromatographic columns, from 17 to 23.
[000207] In each apparatus the columns are typically arranged in series so that (in the case of the first separation step) the bottom of column 1 is linked to the top of column 2, the bottom of column 2 is linked to the top of column 3 ... etc ... and the bottom of the column 8 is connected to the top of the column 1. These connections can be optionally via a containment container, with a recycling current inside the next column. The flow of eluent through the system is from column 1 to column 2 to column 3 etc. The effective absorber flow through the system is from column 8 to column 7 to column 6 etc.
[000208] In this most preferred embodiment, a feed mixture F comprising the product of PUFA (B) and the most polar (C) and the least polar (A ') and the components (a) are introduced at the top of the column 5 in the chromatographic apparatus used in the first separation step. The desorbent aqueous organic solvent is introduced at the top of column 1 of the chromatographic apparatus used in the first separation step. In the first separation step, the less polar components (for example, saturated and / or monounsaturated) (A ') are removed as an extract stream E1 from the bottom of column 2. The PUFA product (B), the most polar components ( C) and the less polar components (but more polar than (A ')) (a) are collected as a raffinate stream R1 from the bottom of column 6.
[000209] The rafinate stream R1 is the first intermediate product that is then purified in the second separation step, being introduced into the chromatographic apparatus used in the second separation step at the top of column 13. The aqueous organic solvent is introduced into the top of the column D in the chromatographic apparatus used in the second separation step.
[000210] In the second separation step, the less polar components (a) are removed as an extract stream E2 at the bottom of column 10. The PUFA product (B) and the more polar components (C) are collected as raffinate stream R2 at the bottom of column 14. The rafinate stream R2 is the intermediate product which is then purified in the third separation step, being introduced into the chromatographic apparatus used in the second separation step at the top of column 21.
[000211] In the third separation step, the more polar components (C) are removed as a rafinate stream R3 at the bottom of column 22. The PUFA product (B) is collected as an extract stream E3 at the bottom of column 18. A second and third separation steps take place in two zones on a single SMB chromatographic apparatus.
[000212] In this most preferred embodiment, the aqueous organic solvent is typically introduced at the top of column 1 of the chromatographic apparatus used in the first separation step.
[000213] In this most preferred embodiment, the aqueous organic solvent is typically introduced at the top of column 9 of the chromatographic apparatus used in the second separation step.
[000214] In this most preferred embodiment, the aqueous organic solvent is typically introduced at the top of column 17 of the chromatographic apparatus used in the third separation step.
[000215] In this most preferred embodiment, the supply current is typically introduced at the top of column 5 of the chromatographic apparatus used in the first separation step.
[000216] In this most preferred embodiment, a first stream of raffinate is typically collected as the first intermediate product from the bottom of column 6 of the chromatographic apparatus used in the first separation step. this first intermediate product is then purified in the second separation step and is typically introduced at the top of column 13 of the chromatographic apparatus used in the second separation step. the first rafinate stream can optionally be collected in a container before being purified in the second separation step.
[000217] In this most preferred embodiment, a first extract stream is typically removed from the bottom of column 2 of the chromatographic apparatus used in the first separation step. the first extract stream can optionally be collected in a container and reintroduced at the top of column 3 of the chromatographic apparatus used in the first separation step.
[000218] In this most preferred embodiment, a second stream of raffinate is typically collected as the second intermediate product from the bottom of column 14 of the chromatographic apparatus used in the second separation step. This second intermediate product is then purified in the third separation step and is typically introduced at the top of column 21 of the chromatographic apparatus used in the third separation step. the second rafinate stream can optionally be collected in a container before being purified in the second separation step.
[000219] In this most preferred embodiment, a second stream of extract is typically removed from the bottom of column 10 of the chromatographic apparatus used in the second separation step.
[000220] In this most preferred embodiment, a third stream of extract is typically collected from the bottom of column 18 of the chromatographic apparatus used in the third separation step. this third extract stream typically contains the purified PUFA product. The third extract stream can optionally be collected in a container and reintroduced at the top of column 19 of the chromatographic apparatus used in the third separation step.
[000221] In this most preferred embodiment, a third stream of raffinate is typically removed from the bottom of column 22 of the chromatographic apparatus used in the third separation step.
[000222] Typically in this most preferred embodiment, the aqueous organic solvent eluent used in the second and third separation steps has the same water ratio: organic solvent, and the aqueous organic solvent eluent used in the first separation step has a water ratio: organic solvent different from the organic solvent eluent used in the second and third separation steps; and the rate at which the liquid collected via the extract stream in the second separation step is recycled back into the chromatography apparatus used in the second separation step differs from the rate at which the liquid collected via the extract stream in the the third separation step is recycled back into the chromatography apparatus used in the third separation step.
[000223] Preferably in this most preferred embodiment, the water: organic solvent ratio of the eluent in the second and third separation steps is the same, and the water: organic solvent ratio of the eluent in the first separation step is lower than that the water: organic solvent ratio of the eluent in the second and third stages of separation; and the rate at which the liquid collected via the extract stream in the second separation step is recycled back into the chromatographic apparatus used in the second separation step is faster than the rate at which the liquid collected via the extract stream in the third stage of separation it is recycled back into the chromatographic apparatus used in the third stage of separation.
[000224] In this most preferred embodiment, the water: organic solvent ratio of the eluent used in the second and third separation steps is the same and is from 7:93 to 9:91% by weight, and the water ratio: organic solvent of the eluent in the first separation step is 0.1: 99.9 to 1:99% by weight.
[000225] Although these preferred and most preferred embodiments are shown as for Figure 2C discussed above, they can also be performed with devices configured such that:
[000226] the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, and the first, second and third separation steps are carried out simultaneously in a single simulated moving bed chromatography apparatus or real having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, the first, second and third separation steps being carried out in the first, second and third zones respectively, where each zone is as defined herein; or
[000227] the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, and the second and third separation steps are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, the second and third separation steps being carried out in the first and second zones respectively, where each zone is as defined herein, and in which the first step of separation is performed in a separate simulated or real mobile bed chromatography apparatus; or
[000228] the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, and (a) the first, second and third separation steps are carried out sequentially in the same chromatography apparatus, the first and second intermediate products being recovered between the first and second, and second and third separation steps respectively, and the process conditions in the chromatography apparatus being adjusted between the first and second, and second and third separation steps such that the fatty acids saturated and / or monounsaturated substances present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii); or
[000229] (b) the second separation step is carried out using a chromatographic apparatus different from that used in the first separation step, and / or the third separation step is carried out using a chromatographic apparatus different from that used in the second separation step; or
[000230] the first separation step comprises purifying the feed mixture in a stationary bed chromatography apparatus, and the second and third separation steps are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, the second and third separation steps being carried out in the first and second zones respectively, where each zone is as defined herein; or
[000231] the first separation step comprises purifying the feed mixture in a stationary bed chromatography apparatus, and the second and third separation steps are carried out sequentially in the same chromatography apparatus, the second intermediate product being recovered between the second and third separation steps and the process conditions in the chromatography apparatus being adjusted between the second and third separation steps such that the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii); or
[000232] the first separation step comprises purifying the feed mixture in a stationary bed chromatography apparatus, and the second and third separation stages are carried out in separate chromatography apparatus respectively, the intermediate product obtained from the second separation stage being introduced into the chromatography apparatus used in the third separation step.
[000233] The process of the invention allows much higher purities of PUFA product to be obtained than has been 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.
[000234] In practice, the process of the present invention will in general be controlled by a computer. The present invention therefore also provides a computer program for controlling a chromatographic apparatus as defined herein, the computer program containing encoding means which, when executed, instructs the apparatus to carry out the process of the invention.
[000235] The Examples that follow illustrate the invention. EXAMPLES Example 1
[000236] 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 C18 silica gel bound as a stationary phase and aqueous methanol as eluent according to the system schematically illustrated in Figure 5. A GC trace of the feed mixture is shown as Figure 7.
[000237] In a first separation step, the feed mixture was passed through an SMB device having 8 columns from 1 to 8 (diameter: 152 mm, length: 813 mm) connected in series as shown in Figure 5. The conditions process parameters were adjusted to remove saturated and monounsaturated components from the feed mixture such as the extract stream. An eluent at 0.5: 99.5% by weight of water: methanol was used. The raffinate stream was retained as the first intermediate product. A GC trace of the first intermediate product is shown as Figure 8.
[000238] The first intermediate product was passed through an SMB device having two zones with eight columns, columns 9 to 16, in the first zone and seven columns, columns 17 to 23, in the second zone. An eluent at 8:92% by weight of water: methanol was used in both the first and second zones, that is, both in the second and third separation steps. The process conditions in the first zone were adjusted to purify EPA from the slowest moving components such as DHA, which were removed as the extract stream. The raffinate stream was retained as the second intermediate product. A GC trace of the second intermediate product is shown as Figure 9.
[000239] The second intermediate product was then introduced into the second zone and separated from the faster moving components, which were removed as a raffinate stream. High purity EPA was collected as the extract stream from the second zone. A GC trace of the PUFA EPA product is shown as Figure 10.
[000240] EPA was produced with a final purity of more than 97%.
[000241] It can be seen that for the three stages of separation as far as together, the overall rate of extract accumulation (E1 + E2 + E3) 3876 ml / min ..
[000242] The process conditions for each separation step are as follows: First separation step
[000243] Feed rate of the feed stock: 94 ml / min.
[000244] Feed rate of the desorbent: 6250 ml / min.
[000245] Extract accumulation rate: 1250 ml / min.
[000246] Extract recycling rate: 5000 ml / min.
[000247] Raffinate accumulation rate: 1688 ml / min.
[000248] Cycle time: 600 s Second separation step
[000249] Feed rate of the first intermediate product: 40 ml / min.
[000250] Desorbent feed rate: 6313 ml / min.
[000251] Extract accumulation rate: 1188 ml / min.
[000252] Extract recycling rate: 5125 ml / min.
[000253] Raffinate accumulation rate: 1625 ml / min.
[000254] Cycle time: 1200 s Third separation step
[000255] Feed rate of the second intermediate product: 40 ml / min.
[000256] Feed rate of the desorbent: 6189 ml / min.
[000257] Extract accumulation rate: 1438 ml / min.
[000258] Extract recycling rate: 4750 ml / min.
[000259] Raffinate accumulation rate: 1438 ml / min.
[000260] Cycle time: 1080 s Comparative Example 1
[000261] An experiment was carried out to produce a PUFA product that contains more than 97% EPA from the same feed mixture as used in Example 1. However, instead of using a three-step separation process, according to the present invention, only two separation steps were used. Thus, the process was carried out in accordance with the process disclosed in PCT / GB10 / 002339, and as illustrated in Figure 6.
[000262] A single chromatographic apparatus having two zones was used as shown in Figure 6. The first zone contains 8 columns (diameter: 24 ”(61 cm), length: 32” (81 cm)) and the second zone 7 columns ( diameter: 24 ”(61 cm), length: 32” (81 cm)). The process conditions were adjusted to separate the EFA PUFA product from the less polar components of the feed mixture in the first zone, and the more polar components of the feed mixture in the second zone. An eluent of 8:92% by weight of water: methanol was used in both zones.
[000263] EPA was produced with a final purity of more than 97%.
[000264] It can be seen that for the two separation steps when together, the overall rate of extract accumulation (E1 + E2) was 10571 ml / min. Thus, it can be seen that a much higher volume of organic solvent aqueous is required to recover the PUFA product compared to the three-step process of the invention.

权利要求:
Claims (29)
[0001]
1. Chromatographic separation process to recover a polyunsaturated fatty acid product (PUFA) from a feed mixture that is a fish oil or that is derived from fish oil, characterized by the fact that the process comprises the steps of: (i) purifying the feed mixture in a chromatographic separation step of stationary bed or simulated or real moving bed, to obtain a first intermediate product; and (ii) purifying the first intermediate product obtained in (i) in a simulated or real moving bed chromatographic separation step, to obtain a second intermediate product; and (iii) purifying the second intermediate product obtained in (ii) in a simulated or real moving bed chromatographic separation step, to obtain the PUFA product; wherein an aqueous organic solvent is used as an eluent in each separation step; the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step; the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii); in the first stage the PUFA product is separated from the components of the feed mixture which are less polar than the PUFA product, in the second step the PUFA product is separated from the components of the feed mixture which are less polar than the product of PUFA. PUFA but more polar than the components separated in the first separation step, and in the third separation step the PUFA product is separated from the more polar components of the feed mixture; and the PUFA product obtained in the third separation step contains EPA or an EPA derivative in an amount greater than 90% by weight.
[0002]
2. Process according to claim 1, characterized by the fact that the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus; and wherein the first, second and third separation steps are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, the first, second and third separation steps being carried out in the first, second and third zones respectively, where each zone has one or more injection points for a feed mixture stream, one or more injection points for water and / or organic solvent, one raffinate withdrawal stream from which liquid can be collected from said zone, and an extract withdrawal stream from which liquid can be collected from said zone.
[0003]
Process according to claim 1, characterized by the fact that the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus; and wherein the first and second separation steps are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, the first and second steps of separation being carried out in the first and second zones respectively, where each zone has one or more injection points for a feed mixture stream, one or more injection points for water and / or organic solvent, a raffinate removal stream a from which liquid can be collected from said zone, and an extract withdrawal stream from which liquid can be collected from said zone, and in which the third step of separation is carried out in a simulated moving bed chromatography apparatus or separate real.
[0004]
Process according to claim 1, characterized in that the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus; and wherein the second and third separation steps are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, the second and third steps of separation being carried out in the first and second zones respectively, where each zone has one or more injection points for a feed mixture stream, one or more injection points for water and / or organic solvent, a raffinate removal stream a from which liquid can be collected from said zone, and an extract withdrawal stream from which liquid can be collected from said zone, and in which the first separation step is performed in a simulated moving bed chromatography apparatus or separate real.
[0005]
Process according to claim 1, characterized by the fact that the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus; and where (a) the first, second and third separation steps are carried out sequentially in the same chromatography apparatus, the first and second intermediate products being recovered between the first and second, and second and third separation steps respectively, and the conditions process in the chromatography apparatus being adjusted between the first and second, and second and third separation steps such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the components other than the feed mixture in steps (ii) and (iii); or (b) the second separation step is carried out using a chromatographic apparatus different from that used in the first separation step, and / or the third separation step is carried out using a chromatographic apparatus different from that used in the second separation step.
[0006]
Process according to claim 1, characterized in that the first separation step comprises purifying the feed mixture in a stationary bed chromatography apparatus; and wherein (a) the second and third separation steps are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, the second and third separation steps being carried out in the first and second zones respectively, where each zone has one or more injection points for a feed mix stream, one or more injection points for water and / or organic solvent, a withdrawal stream raffinate from which liquid can be collected from said zone, and an extract withdrawal stream from which liquid can be collected from said zone; or (b) the second and third separation steps are carried out sequentially on the same chromatography apparatus, the second intermediate product being recovered between the second and third separation steps and the process conditions on the chromatography apparatus being adjusted between the second and third separation steps such that the PUFA product is separated from the components other than the feed mixture in steps (ii) and (iii); or (c) the second and third separation steps are performed in separate chromatography apparatus respectively, the intermediate product obtained from the second separation step being introduced into the chromatography apparatus used in the third separation step.
[0007]
7. Process according to any one of claims 2 to 6, characterized by the fact that in simulated or real mobile bed chromatography apparatus in which two separation steps are carried out simultaneously in two zones, a stream of raffinate or extract is collected from a column in the first zone and introduced into a non-adjacent column in the second zone; and / or where in the simulated or real moving bed chromatography apparatus in which three separation steps are carried out simultaneously in three zones, a stream of raffinate or extract is collected from a column in the first zone and introduced in a non-adjacent column in the second zone, and a stream of raffinate or extract is collected from a column in the second zone and introduced into a non-adjacent column in the third zone.
[0008]
Process according to any one of the preceding claims, characterized by the fact that the first intermediate product obtained in the first separation step is enriched in the PUFA product compared to the feed mixture; and the second intermediate product obtained in the second separation step is enriched in the PUFA product compared to the first intermediate product.
[0009]
Process according to any one of the preceding claims, characterized in that the separate components of the PUFA product in the second separation step include DHA or a derivative of DHA and / or other PUFAs or PUFA derivatives that are less polar than that the PUFA product; and / or the separate components of the PUFA product in the third separation step include SDA or a derivative of SDA and / or other PUFAs that are more polar than the PUFA product.
[0010]
10. Process according to any of the preceding claims, characterized by the fact that the second intermediate product is collected as the raffinate stream in the second separation step, and the PUFA product is collected as the extract stream in the third separation step. separation.
[0011]
Process according to claim 10, characterized by the fact that the first separation step comprises purifying the feed mixture in a simulated or real mobile bed chromatography apparatus, and in which the first intermediate product is collected as the current rafinate in the first separation step.
[0012]
Process according to any one of the preceding claims, characterized in that the eluent is a mixture of water and an alcohol, an ether, an ester, a ketone or a nitrile.
[0013]
Process according to claim 12, characterized in that the eluent is a mixture of water and methanol.
[0014]
Process according to any one of the preceding claims, characterized in that the PUFA product contains EPA or an EPA derivative in an amount greater than 95% by weight.
[0015]
Process according to claim 14, characterized in that the PUFA product contains EPA or an EPA derivative in an amount greater than 97% by weight.
[0016]
16. Process according to any of the preceding claims, characterized by the fact that the EPA derivative is EPA ethyl ester (EE).
[0017]
17. Process according to any of the preceding claims, characterized in that - part of the extract stream from the apparatus used in the second separation step is recycled back into the apparatus used in the second separation step; and / or - part of the raffinate stream from the apparatus used in the second separation step is recycled back into the apparatus used in the second separation step; and / or - part of the extract stream from the apparatus used in the third separation step is recycled back into the apparatus used in the third separation step; and / or - part of the raffinate stream from the apparatus used in the third separation step is recycled back into the apparatus used in the third separation step.
[0018]
18. Process according to claim 17, characterized in that the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, and in which - part of the extract stream of the apparatus used in the first separation step it is recycled back into the apparatus used in the first separation step; and / or - part of the raffinate stream from the apparatus used in the first separation step is recycled back into the apparatus used in the first separation step.
[0019]
19. Process according to any of the preceding claims, characterized by the fact that the water: organic solvent ratio used in each separation step is adjusted such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the feed mixture. first separation step; and the PUFA product is separated from the components other than the feed mixture in steps (ii) and (iii).
[0020]
20. Process according to any one of the preceding claims, characterized in that the aqueous organic solvent eluent used in each separation step has a different water: organic solvent ratio.
[0021]
21. Process according to any one of claims 1 to 19, characterized in that the aqueous organic solvent eluent used in the second and third separation steps has the same water ratio: organic solvent, and the aqueous organic solvent eluent used in the first separation step has a water: organic solvent ratio different from the organic solvent eluent used in the second and third separation steps.
[0022]
22. Process according to claim 21, characterized in that the water: organic solvent ratio of the aqueous organic solvent eluent used in the first separation step is lower than the water: organic solvent ratio of the solvent eluent aqueous organic used in the second and third separation steps.
[0023]
23. Process according to claim 22, characterized in that the water: organic solvent ratio of the eluent used in the first separation step is 0.1: 99.9 to 1:99% by weight, and the ratio water: organic solvent of the eluent used in the second and third separation steps is from 7:93 to 9:91% by weight.
[0024]
24. Process according to any one of claims 17 to 23, characterized in that the rate at which the liquid collected through one or both of the extract and raffinate streams in the second separation step is recycled back into the apparatus used in this separation step it is adjusted so that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the different components of the feed mixture in the steps (ii) and (iii); and / or at which the rate at which the liquid collected through one or both of the extract and raffinate streams in the third separation step is recycled back into the apparatus used in this separation step is adjusted such that the acids saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii).
[0025]
25. Process according to claim 21, characterized in that the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, and at which the rate at which the liquid collected through one or both of the extract and raffinate streams in the first separation step is recycled back into the apparatus used in this separation step is adjusted such that the saturated and / or monounsaturated fatty acids present in the feed mixture are removed in the first separation step, and the PUFA product is separated from the different components of the feed mixture in steps (ii) and (iii).
[0026]
26. Process according to any of claims 17 to 25, characterized in that the rate at which the liquid collected through the extract stream in the second separation step is recycled back into the chromatography apparatus used in the second separation stage differs from the rate at which the liquid collected through the extract stream in the third separation stage is recycled back into the chromatography apparatus used in the third separation stage.
[0027]
27. Process according to any one of claims 17 to 26, characterized in that the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus, and at which the rate at which the liquid collected via the extract stream in the first separation step is recycled back into the chromatography apparatus used in the first separation step differs from the rate at which the liquid collected through the extract stream in the second separation step is recycled back into the chromatography apparatus used in the second separation step.
[0028]
28. Process according to any one of claims 17 to 27, characterized in that the rate at which the liquid collected through the extract stream in the second separation step is recycled back into the chromatographic apparatus used in the second separation is faster than the rate at which the liquid collected via the extract stream in the third separation step is recycled back into the chromatographic apparatus used in the third separation step.
[0029]
29. Process according to claim 13, characterized by the fact that: - the first separation step comprises purifying the feed mixture in a simulated or real moving bed chromatography apparatus; - the second and third separation steps are carried out simultaneously in a single simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, the second and third separation steps being carried out in the first and second zones respectively, where each zone has one or more injection points for a feed mixture stream, one or more injection points for water and / or organic solvent, a raffinate withdrawal stream from the which liquid can be collected from said zone, and an extract withdrawal stream from which liquid can be collected from said zone; and wherein the first separation step is carried out in a separate simulated or real moving bed chromatography apparatus; - the first intermediate product is collected as the raffinate stream in the first separation step, the second intermediate product is collected as the raffinate stream in the second separation step, and the PUFA product is collected as the extract stream in the third step separation; - the second stream of intermediate product raffinate containing the PUFA product together with the more polar components is collected from a column in the first zone and introduced into a non-adjacent column in the second zone; - the aqueous organic solvent eluent used in the second and third separation steps has the same water ratio: organic solvent, and the water: organic solvent ratio of the eluent used in the first separation step is lower than the water ratio : organic solvent of the eluent used in the second and third separation steps; and - the rate at which the liquid collected via the extract stream in the second separation step is recycled back into the chromatographic apparatus used in the second separation step is faster than the rate at which the liquid collected via the extract stream extract in the third stage of separation is recycled back into the chromatographic apparatus used in the third stage of separation.

类似技术:

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同族专利:

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公开号 | 公开日

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JP6248143B2|2017-12-13|

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HUE037030T2|2018-08-28|

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CN103826714B|2016-03-09|

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US20150166929A1|2015-06-18|

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KR20140034922A|2014-03-20|

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JP2016212110A|2016-12-15|

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EP2886176B1|2017-12-27|

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ES2536913T3|2015-05-29|

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CN103826714A|2014-05-28|

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KR101757132B1|2017-07-12|

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HUE025265T2|2016-02-29|

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CA2815300C|2015-08-25|

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ES2661261T3|2018-03-28|

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US20160186092A1|2016-06-30|

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NO2886176T3|2018-05-26|

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GB201111595D0|2011-08-24|

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US9315762B2|2016-04-19|

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JP5951764B2|2016-07-13|

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CL2013003798A1|2014-07-04|

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KR20160033793A|2016-03-28|

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KR101604930B1|2016-03-18|

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TR201802797T4|2018-03-21|

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PL2613860T3|2015-08-31|

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PE20140807A1|2014-07-17|

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DK2886176T3|2018-03-26|

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EP2613860A1|2013-07-17|

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EP2886176A1|2015-06-24|

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CA2815300A1|2013-01-10|

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DK2613860T3|2015-07-06|

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JP2014518313A|2014-07-28|

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WO2013005048A1|2013-01-10|

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AU2012280067A1|2013-05-09|

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BR112014000162A2|2017-02-07|

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PL2886176T3|2018-06-29|

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US9695382B2|2017-07-04|

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AU2012280067B2|2015-07-09|

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EP2613860B1|2015-03-25|

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法律状态:
2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-08-06| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-06-30| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|

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2020-10-27| B09A| Decision: intention to grant|

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2021-01-05| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/07/2012, 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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GB1111595.3|2011-07-06|

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GBGB1111595.3A|GB201111595D0|2011-07-06|2011-07-06|Improved process|

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PCT/GB2012/051593|WO2013005048A1|2011-07-06|2012-07-06|Smb process for producing highly pure epa from fish oil|

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