chromatographic separation process

专利摘要:
CHROMATOGRAPHIC SEPARATION PROCESS, AND, STORAGE MEDIA. The present invention provides a chromatographic separation process to recover a polyunsaturated fatty acid product (PUFA) from a feed mixture, which comprises the steps of: (i) purifying the feed mixture in a first step of separating in a simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, to obtain an intermediate product; and (ii) purifying the intermediate product obtained in (i) in a second separation step using a simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, for obtain the PUFA product; where (a) the first and second separation steps are carried out sequentially on the same chromatography apparatus, the intermediate product being recovered between the first and second separation steps and the process conditions on the chromatography apparatus being adjusted between the first and second separation steps such that the PUFA product is separated from the components other than the feed mixture in (...).
公开号:BR112014000152B1
申请号:R112014000152-9
申请日:2012-07-06
公开日:2020-10-27
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 polyunsaturated fatty acids (PUFAs) and derivatives thereof. In particular, the present invention relates to an improved simulated or actual moving bed chromatographic separation process for purifying PUFAs and derivatives thereof.
[0002] Fatty acids, in particular PUFAs, and their derivatives are precursors to biologically important molecules, which play an important role in regulating biological functions such as platelet aggregation, inflammation and immune responses. Thus, PUFAs and their derivatives can be therapeutically useful in the treatment of a wide range of pathological conditions that include CNS conditions; neuropathies, which include diabetic neuropathy; cardiovascular diseases; general immune and inflammatory conditions, which include inflammatory skin diseases.
[0003] PUFAs are found in natural raw materials, such as vegetable oils and marine oils. Such PUFAs are, however, often present in such oils in admixture with saturated fatty acids and numerous other impurities. PUFAs, therefore, should desirably be purified before nutritional or pharmaceutical uses.
[0004] Unfortunately, PUFAs are extremely fragile. Thus, when heated in the presence of oxygen, they are prone to isomerization, peroxidation and oligomerization. The fractionation and purification of PUFA products to prepare pure fatty acids is therefore difficult. Distillation, even under vacuum, can lead to unacceptable degradation of the product.
[0005] Simulated and real moving bed chromatography are known techniques, familiar to those skilled in the art. The operating principle involves countercurrent movement of a liquid eluent phase and a solid absorbent phase. This operation allows the minimum 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.
[0006] 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 percolation speed of each component of the mixture depends on the physical properties of this component so that the components leave the column successively and selectively. Thus, some of the components tend to attach strongly to the stationary phase and thus seep slowly, while others tend to attach 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.
[0007] In contrast, a simulated moving bed chromatography apparatus consists of several individual columns containing absorbent which 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 absorbent, the solid absorbent 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 absorbent 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.
[0008] 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.
[0009] 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.
[00010] 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.
[00011] The purification of PUFA products is particularly challenging. Thus, many feed stocks suitable for preparing PUFA products are extremely complex mixtures containing a large number of different components with very similar retention times in chromatography devices. It is therefore very difficult to separate certain PUFAs from such feed stocks. However, a high degree of purity of PUFA products is required, particularly for pharmaceutical and nutraceutical applications. Historically, therefore, distillation has been used when high purity of PUFA products is required. There are, however, significant disadvantages to using distillation as a separation technique for delicate PUF As discussed above.
[00012] Until now, no chromatographic technique has been made available to obtain PUFA products with high purity, for example, greater than 95 or 97% purity, in particular from commercially available feed stocks such as fish.
[00013] 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 LE by means of a P pump. The mixture of the A and B components 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 sub-area 1 and sub-area II, and a raft containing mainly A is collected in SA between sub-area III and sub-area IV.
[00014] 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 with 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.
[00015] Therefore, it will be evaluated that the conventional simulated moving bed system schematically illustrated in Fig. 1 is limited to binary fractionation.
[00016] Consequently, there is a need for a simulated or real moving bed chromatographic separation process that can separate PUF As or its derivatives from components that move both faster and slower (ie, the more polar impurities and less polar), to produce high purity PUF A products from commercially available feed stocks such as fish oils. It is still desirable that the process involves cheap eluents that operate under conditions of standard temperature and pressure. Summary of the invention
[00017] It has now surprisingly been discovered that a PUF A product can be effectively purified from commercially available feed stocks such as fish oils by the simulated or real moving bed apparatus using 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, a process that comprises the steps of:
[00018] (i) purifying the feed mixture in a first separation step in a simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, to obtain an intermediate product; and
[00019] (ii) purify the intermediate product obtained in (i) in a second separation step using a simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent , to obtain the PUFA product; on what
[00020] (a) the first and second separation steps are performed sequentially on the same chromatography apparatus, the intermediate product being recovered between the first and second separation steps and the process conditions on the chromatography apparatus being adjusted between the first and second separation steps such that the PUFA product is separated from the different components of the feed mixture in each separation step; or
[00021] (b) the first and second separation steps are carried out on the separate primary and secondary chromatography apparatus respectively, the intermediate product obtained from the first separation step being introduced into the second chromatography apparatus, and the PUFA product being separated different components of the feed mixture at each separation step.
[00022] A PUFA product obtainable by the process of the present invention is also provided.
[00023] PUFA products produced by the process of the present invention are produced in high yield, and have high purity. In addition, the content of the distinctive impurities that typically arise from the distillation of PUFAs is very low. As used herein, the term ‘“ isomeric impurities ”is used to indicate those impurities typically produced during the distillation of natural oils that contain PUFA. These include isomers, peroxidation products and PUF A oligomerization. Description of the Figures
[00024] Figure 1 illustrates the basic principles of a simulated or real moving bed process to separate a binary mixture.
[00025] Figure 2 illustrates a first preferred embodiment of the invention that is suitable for separating EPA from components that move faster and slower (i.e., more polar and less polar impurities).
[00026] Figure 3 illustrates a second preferred embodiment of the invention that is suitable for separating DHA from components that move faster and slower (i.e., more polar and less polar impurities).
[00027] Figure 4 illustrates in more detail the first preferred embodiment of the invention that is suitable for separating EPA from the components that move faster and slower (i.e., more polar and less polar impurities).
[00028] Figure 5 illustrates in more detail the second preferred embodiment of the invention that is suitable for separating DHA from components that move faster and slower (i.e., more polar and less polar impurities).
[00029] Figure 6 illustrates in more detail an alternative method for the first preferred embodiment of the invention that is suitable for separating EPA from components that move faster and slower (i.e., more polar and less polar impurities).
[00030] Figure 7 illustrates in more detail an alternative method for the second preferred embodiment of the invention that is suitable for separating DHA from components that move faster and slower (i.e., more polar and less polar impurities).
[00031] Figure 8 illustrates a particularly preferred embodiment of the invention for purifying EPA from components that move faster and slower (i.e., more polar and less polar impurities).
[00032] Figure 9 illustrates an alternative method for a particularly preferred embodiment of the invention for purifying EPA from components that move faster and slower (i.e., more polar and less polar impurities).
[00033] Figure 10 illustrates three ways in which the chromatographic separation process of the invention can be carried out.
[00034] Figure 11 shows an HPLC analysis of an EPA-rich feed stock that can be properly used as the feed mixture in the process of the present invention.
[00035] Figure 12 shows HPLC analyzes of the raffinate (R) and extract (E) streams of the first separation step of a process according to the present invention.
[00036] Figure 13 shows HPLC analyzes of the raffinate (R) and extract (E) streams of the second separation step of a process according to the present invention.
[00037] Figure 14 shows a more detailed HPLC analysis of the extract stream from the second stage of separation of a process according to the present invention.
[00038] Figure 15 shows a trace of GC FAMES from a DHA product produced by SMB.
[00039] Figure 16 shows a trace of GC FAMES from a DHA product produced by distillation. Detailed description of the invention
[00040] The chromatographic separation process of the invention is typically other than a chromatographic separation process for recovering a polyunsaturated fatty acid product (PUFA) from a feed mixture, a process which comprises introducing the mixture of feeding into a simulated or real moving bed chromatography apparatus having a plurality of connected chromatography columns containing, as an eluent, an aqueous alcohol, in which the apparatus has a plurality of zones comprising at least a first zone and a second zone, each zone having an extract stream and a raffinate stream from which liquid can be collected from said plurality of linked chromatography columns, and in which (a) a raffinate stream containing the PUFA product along with more polar components is collected from a column in the first zone and introduced into a non-adjacent column in the second zone, and / or (b) an extract stream containing the PU product FA together with the less polar components is collected from a column in the second zone and introduced into a non-adjacent column in the first zone, said PUFA product being separated from the different components of the feed mixture in each zone.
[00041] As used in this embodiment, the term "zone" refers to a plurality of linked chromatography columns that contain, as an eluent, an aqueous alcohol, and having one or more injection points for a mixing stream feed, one or more injection points for water and / or alcohol, 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. 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 alcohol eluent.
[00042] In another embodiment, each zone has two or more injection points for water and / or alcohol.
[00043] Further details of this embodiment are to be found in international patent application no. PCT / GB 10/002339, all of which are incorporated by reference. The chromatographic separation process of the invention is typically other than the processes disclosed in PCT / GB 10/002339.
[00044] 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. Typically, the PUFA product is a single PUFA or derivative thereof. Alternatively, the PUFA product is a mixture of two or more PUFAs or derivatives thereof, for example, two.
[00045] The term "polyunsaturated fatty acid" (PUFA) refers to fatty acids that contain more than one double bond. Such PUFAs are well known to the person skilled in the art. As used herein, a PUFA Derivative is a PUFA in the form of a mono-, di- or triglyceride, ester, phospholipid, amide, lactone, or salt. Triglycerides and esters are preferred. Esters are more preferred. Esters are typically alkyl esters, preferably C) -C6 alkyl esters, more preferably C] -C4 alkyl esters. Examples of esters include methyl and ethyl esters. Ethyl esters are most preferred.
[00046] Typically, the PUFA product comprises at least one PUFA (0-3 or ω-6, preferably at least one PUFA ω-3. Examples of ω-3 PUFAs include alpha-linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) .SDA. EPA, DPA and DITA are preferred. EPA and DHA are more preferred. Examples of ω-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 (ω-6) LA, ARA, GLA and DGLA are preferred.
[00047] In one embodiment, the PUFA product is EPA and / or EPA ethyl ester (EE)
[00048] In another embodiment, the PUFA product is DHA and / or DHA ethyl ester (EE).
[00049] In another embodiment, the PUFA product is a mixture of EPA and DHA and / or EPA EE and DHA EE.
[00050] In a more preferred embodiment, the PUFA product is EPA or EPA ethyl ester which is produced in greater than 90% purity, preferably more than 95% purity, and more preferably more than 97 % purity.
[00051] Typically, in addition to said PUFA product, an additional secondary PUFA product is collected in the chromatographic separation process of the invention. Preferably, the PUFA product is EPA and the additional secondary PUFA product is DHA.
[00052] In another embodiment of the invention, the apparatus is configured to collect a PUFA product which is a concentrated mixture of EPA and DHA. Thus, a feed mixture is used that contains EPA, DHA, components that are more polar than EPA and DHA, and components that are less polar than EPA and DHA. In the first separation step, material less polar than EPA and DHA is typically removed. In the second separation step, material that is more polar than EPA and DHA is typically removed, and a concentrated mixture of EPA and DHA is collected as the PUFA product.
[00053] Feed mixtures suitable for fractionation by the process of the present invention can be obtained from natural sources including vegetable and animal oils and fats, and from synthetic sources including oils obtained from plants, animals and genetically modified microorganisms that include yeast. Examples include fish oils, seaweed and microalgae oils and vegetable oils, for example borage oil, Echium oil and onagraceous oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is an algae oil. Seaweed oils are particularly suitable when the desired PUFA product is EPA and / or DHA. Genetically modified saffron oil is particularly suitable when the desired PUFA product is GLA. Genetically modified yeast is particularly suitable when the desired PUFA product is EPA.
[00054] In a particularly preferred embodiment the feed mixture is a fish oil or feed stock derived from fish oil. It has been advantageously discovered that when a fish oil or fish oil derived feed stock is used, an EPA product or PUFA EPA ethyl ester can be produced by the process of the present invention in more than 90% purity, preferably greater than 95% purity, and more preferably greater than 97% purity.
[00055] 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, iodolactonisation or supercritical fluid fractionation . Alternatively, a feed mixture can be used directly without any initial treatment steps.
[00056] 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 absorbent used in the process of the present invention than the PUFA product. During operation, such less polar components typically move with the solid absorbent phase in preference to the liquid eluent phase. The more polar components have a weaker adhesion to the absorbent used in the process of the present invention than the PUFA product. During operation, such more polar components typically move with the liquid eluent phase in preference to the solid absorbent phase. In general, the more polar components will be separated into a raffinate stream, and the less polar components will be separated into an extract stream.
[00057] Examples of the more and less polar components include (1) other compounds that occur in natural oils (for example marine oils or vegetable oils), (2) by-products formed during the previous storage, refining and concentration steps and (3 ) solvent contaminants or reagents that are used during the previous concentration or purification steps.
[00058] 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. PCB, PAH, dioxins and chlorinated pesticides are all highly non-polar components.
[00059] Examples of (2) include isomers and oxidation or decomposition products of the PUFA product, for example, polymeric auto-oxidation products of fatty acids or their derivatives.
[00060] Examples of (3) include urea that can be added to remove saturated or monounsaturated fatty acids from the feed mixture.
[00061] 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.
[00062] 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 that include other acids fatty ω-3 and ω-6 essential.
[00063] 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 ω-3 and to fatty acids -6 essentials. DHA (EE) is less polar than EPA (EE).
[00064] A typical feed mixture for preparing concentrated DHA (EE) by the process of the present invention comprises 50 to 75% DHA (EE), 0 to 10% EPA (EE), and other components that include other acids fatty ω-3 and ω-6 essential.
[00065] A preferred feed mixture for preparing concentrated DHA (EE) by the process of the present invention comprises 75% DHA (EE), 7% EPA (EE) and other components including other ω-3 and ω- fatty acids 6 essentials. EPA (EE) is more polar than DEIA (EE).
[00066] A typical feed mixture for preparing a concentrated mixture of EPA (EE) and DHA (EE) by the process of the present invention comprises more than 33% EPA (EE), and more than 22% DHA (EE) ).
[00067] Each step of separation of the process of the present invention is carried out in a simulated or real moving bed chromatography apparatus.
[00068] Any known simulated or real moving bed chromatography apparatus may 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 apparatus described in US 2,985,589, US 3,696,107, US 3,706,812, US 3,761,533, FR.-A-2103302, FR-A-2651148, FR- A-2651149, US 6,979,402, US 5,069 .883 and US 4,764,276 can all be used if configured according to the process of the present invention.
[00069] As used herein, the term 'simulated or real moving bed chromatography apparatus' * typically refers to a plurality of linked chromatography columns that contain, as an eluent, an aqueous organic solvent, and having one or more points injection for a feed mixture stream, one or more injection points for water and / or organic solvent, a raffinate removal stream from which liquid can be collected from said plurality of linked chromatography columns, and a stream extracting extract from which liquid can be collected from said plurality of linked chromatography columns.
[00070] The chromatography apparatus used in each stage of the process of the present invention has a single array of chromatography columns connected in series which 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 in the array is connected to the input of the adjacent column in the array, which is downstream with respect to the flow of eluent in the array. Thus, the eluent can flow around the array of linked chromatography columns. Typically, none of the chromatography columns are linked to non-adjacent columns in the apparatus.
[00071] 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.
[00072] Typically, each device has only one injection point for a feed mix. In one embodiment, each apparatus has only one injection point for the aqueous organic solvent eluent. In another embodiment, each device has two or more injection points for water and / or organic solvent.
[00073] 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 eluent phase compared to the solid absorbent phase. Thus, a raffinate stream is typically enriched with more polar components, and depleted of less polar components compared to a feed stream.
[00074] 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 absorbent phase compared to the liquid eluent phase. Thus, an extract stream is typically enriched with less polar components, and depleted of more polar components compared to a feed stream.
[00075] The number of speakers used in each device 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.
[00076] The chromatographic apparatus used in the first and second separation steps typically contains the same number of columns. For certain applications they may have different numbers of columns.
[00077] The dimensions of the columns used in the apparatus 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.
[00078] The columns in the chromatographic apparatus used in the first and second stages of separation typically have identical dimensions but may, for certain applications, have different dimensions.
[00079] Flow rates for the column are limited by the maximum pressures across the series of columns and will depend on the column dimensions and 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.
[00080] For the typical column sizes outlined above, typically the flow rate of eluent in the chromatographic apparatus used in the first separation step is 1 to 4.5 L / min., Preferably 1.5 to 2.5 L / min Typically, the flow rate of the chromatographic apparatus extract used in the first separation step 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 separation step is recycled back into the apparatus used in the first separation step, 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 separation step 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 separation step is recycled back into the apparatus used in the first separation step, the recycle flow rate is typically 0.3 to 1.0 L / min., Preferably of about 0.5 L / min. Typically, the flow rate of introducing the feed mixture into the chromatographic apparatus used in the first separation step is from 5 to 150 ml / min., Preferably from 10 to 100 ml / min., More preferably from 20 to 60 ml / min. .
[00081] For typical column sizes outlined above, typically the flow rate of eluent in the chromatographic apparatus used in the second 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 second 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 second separation step is recycled back into the apparatus used in the second 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 second 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 second separation step is recycled back into the apparatus used in the second separation step, the recycle flow rate is typically 0.3 to 1.0 L / min., Preferably of about 0.5 L / min.
[00082] As the qualified person will evaluate, references to the rates at which liquid is collected or removed through the various streams of extract and raffinate refer to the volumes of liquid removed in an amount of time, typically L / minute. Similarly, references to rates at which liquid is recycled back into an apparatus, typically to an adjacent column in the apparatus, refer to volumes of liquid recycled in an amount of time, typically L / minute.
[00083] The time of the stage, that is, the time between changing 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.
[00084] In the process of the present invention, real moving bed chromatography is preferred.
[00085] Conventional absorbents 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 absorbent. Typically, each column contains the same absorbent. Examples of such commonly used materials are polymeric beads, preferably DVB cross-linked polystyrene (divinylbenzene); and silica gel, preferably reversed-bonded silica gel with C8 or Cig alkanes, especially C18. Reverse-bonded silica gel Cl8 is preferred. The absorbent used in the process of the present invention is preferably non-polar.
[00086] The shape of the absorbent 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, more 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 particle sizes of the beads 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 absorbent beads can be used in the process of the present invention (with its associated advantages) without any loss in resolution.
[00087] The absorbent 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.
[00088] Typically, the process of the present invention is conducted at 15 to 55 ° C, preferably 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.
[00089] The process of the present invention comprises a first and a second separation step.
[00090] These two steps can be easily performed in a single chromatographic apparatus. Thus, in an embodiment, (a) the first and second separation steps are carried out sequentially on the same chromatography apparatus, the intermediate product being recovered between the first and second separation steps and the process conditions on the chromatography apparatus being adjusted between the first and second separation steps such that the PUFA product is separated from the different components of the feed mixture in each separation step. A preferred embodiment of this separation process is shown as Figure 10a. Thus, the first separation step (left side) is performed on an SMB device having 8 columns. Between the first and second separation steps the intermediate product is recovered, for example, in a container, the process conditions in the chromatography apparatus are adjusted such that the PUFA product is separated from the different components of the feed mixture at each step of separation. The second separation step (right side) is then performed on the same SMB device having 8 columns.
[00091] In embodiment (a), adjusting the process conditions typically refers to adjusting the process conditions on the apparatus as a whole, that is, physically modifying the apparatus so that the conditions are different. This does not refer to simply reintroducing the intermediate product back into a different part of the same apparatus where the process conditions could happen to be different.
[00092] Alternatively, the first and second separate chromatographic apparatus can be used in the first and second separation steps. Thus, in another embodiment, (b) the first and second separation steps are carried out on the primary and secondary chromatography apparatus separated respectively, the intermediate product obtained from the first separation step being introduced into the second chromatography apparatus, and the PUFA product being separated from the different components of the feed mixture at each separation step.
[00093] In embodiment (b), the two separation steps can each be carried out sequentially or simultaneously.
[00094] Thus, in embodiment (b) in the case where the two separation steps are carried out sequentially, the first and second separation steps are carried out sequentially on the primary and secondary chromatography apparatus separated respectively, the intermediate product being recovered between the first and second separation steps and the process conditions in the first and second chromatography apparatus being adjusted such that the PUFA product is separated from the different components of the feed mixture in each separation step. A preferred embodiment of this separation process is shown as Figure 10b. Thus, the first step of separation (left side) is performed on an SMB device having 8 columns, from one to eight. Between the first and second separation steps the intermediate product is recovered, for example, in a container, and then introduced into a second separate SMB device. The second separation step (right side) is performed on the second separate SMB device that has 8 columns, nine to sixteen. The process conditions in the two chromatography devices are adjusted such that the PUFA product is separated from the different components of the feed mixture in each separation step.
[00095] In embodiment (b) in the case where the two separation steps are carried out simultaneously, the first and second separation steps are carried out in the primary and secondary chromatography apparatus separated respectively, the intermediate product being introduced into the apparatus chromatography used in the second separation step, and the process conditions in the first and second chromatography apparatus being adjusted such that the PUFA product is separated from the different components of the feed mixture in each separation step. A preferred embodiment of this separation process is shown as Figure 10c. Thus, the first step of separation (left side) is performed on an SMB device having 8 columns, from one to eight. The intermediate product obtained in the first separation step is then introduced into the second separate chromatography apparatus used in the second separation step. The intermediate product can be passed from the first separation step to the second separation step directly or indirectly, for example, via a container. The second separation step (right side) is performed on the second separate SMB device that has 8 columns, from nine to sixteen. The process conditions in the two chromatography devices are adjusted such that the PUFA product is separated from the different components of the feed mixture in each separation step.
[00096] In embodiment (b) in the case where the two separation steps are carried out simultaneously, the eluent circulates separately in the two separate chromatographic apparatus. Thus, the eluent is not shared between the two separate chromatographic apparatus in addition to that eluent which may be present as a solvent in the intermediate product which is purified in the second separation step, and which is introduced into the chromatographic apparatus used in the second separation step. Chromatographic columns are not shared between the two separate chromatographic devices used in the first and second separation steps.
[00097] After the 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 purified in the second separation step. Alternatively, the intermediate product can be purified in the second separation step without removing any solvent present.
[00098] As mentioned above, the PUFA product is separated from the different components of the feed mixture in each separation step. In embodiment (a), the process conditions of the single SMB apparatus used in both separation steps are adjusted between the first and second separation steps such that the PUFA product is separated from the different components of the feed mixture in each separation step. In embodiment (b), the process conditions in the two separate chromatography apparatus used in the first and second separation steps are adjusted such that the PUFA product is separated from the different components of the feed mixture in each separation step.
[00099] Thus, the process conditions in the first and second stages of separation 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, or the eluent used in the steps separation.
[000100] The intermediate product obtained in the first separation step is typically enriched in the PUFA product compared to the feed mixture.
[000101] The intermediate product obtained in the first separation step is then introduced into the chromatographic apparatus used in the second separation step.
[000102] The intermediate product is typically collected as the raffinate stream or extract from the chromatographic apparatus used in the first separation process.
[000103] Typically, the intermediate product is collected as the raffinate stream in the first separation step, and the PUFA product is collected as the extract stream in the second separation step. Thus, the raffinate current collected in the first separation step is used as the feed mixture in the second separation step. The raffinate stream collected in the first separation step typically contains the PUFA product together with the most polar components.
[000104] Alternatively, the intermediate product is collected as the extract stream in the first separation step, and the PUFA product is collected as the raffinate stream in the second separation step. Thus, the extract stream collected in the first separation step is used as the feed mixture in the second separation step. The extract stream collected in the first separation step typically contains the PUFA product along with the less polar components.
[000105] The PUFA product is separated from the different components of the feed mixture in each separation step. Typically, the components separated at each stage of separation of the process of the present invention have different polarities.
[000106] Preferably, the PUFA product is separated from the less polar components of the feed mixture in the first separation step, and the PUFA product is separated from the more polar components of the feed mixture in the second separation step.
[000107] Typically, (a) part of the extract stream from the apparatus used in the first separation step is recycled back into the apparatus used in the first separation step; and / or
[000108] (b) 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 / or
[000109] (c) 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
[000110] (d) 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.
[000111] Preferably, (a) part of the extract stream from the apparatus used in the first separation step is recycled back into the apparatus used in the first separation step; and
[000112] (b) 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
[000113] (c) 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
[000114] (d) 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.
[000115] This recycling involves feeding part of the extract or raffinate stream outside the chromatography apparatus used in the first or second 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.
[000116] The rate at which the liquid collected via the extract or raffinate stream in the first or second separation steps is recycled back into the chromatography apparatus used in this step is the rate at which the liquid collected via 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.
[000117] This can be seen with reference to a preferred embodiment in Figure 9. The rate of extract recycling 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.
[000118] The extract recycling rate in the second separation step is the rate at which the extract collected at the bottom of column 2 of the chromatographic apparatus used in the second separation step is fed at the top of column 3 of the chromatographic apparatus used in the second stage of separation, that is, the flow rate of the liquid at the top of column 3 of the chromatographic apparatus used in the second separation step.
[000119] The recycling of extract and / or raffinate streams in the first and / or second separation steps is typically carried out by feeding the liquid collected through this stream into this separation step into a container, and then pumping an amount of this liquid of the container back into the apparatus 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 out of the container back into the chromatography apparatus, typically within an adjacent column,
[000120] As the qualified person will assess, the amount of liquid that is introduced into a chromatography apparatus through the eluent streams and feed stock is balanced with the amount of liquid removed from the apparatus, and recycled back into the device.
[000121] Thus, with reference to Figure 9, for the extract stream, the flow rate of eluent (desorbent) in the chromatographic apparatus (s) used in the first and second 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 (E1 and E2) added at the rate at which the extract is recycled back into the chromatographic apparatus used in this particular separation step (D-El and D-E2).
[000122] 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 (D-El and D-E2) added at the rate at which the stock of feed is introduced into the chromatographic apparatus used in this particular separation step (F and Rl) is equal to the rate at which the liquid collected through the raffinate stream in this particular separation step accumulates in a container (Rl and R2) added to the rate at which rafinate is recycled back into the chromatographic apparatus used in this particular separation step (D + FE1-R1 and D + R1-E2-R2).
[000123] The rate at which the liquid collected from a particular extract or raffinate stream from a chromatography apparatus accumulates in a container can also be considered as the net rate of removal of this extract or raffinate stream from this chromatography apparatus.
[000124] The eluent used in the process of the present invention is an aqueous organic solvent.
[000125] The aqueous organic solvent typically comprises water and one or more alcohols, ethers, esters, ketones or nitriles, or mixtures thereof.
[000126] 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 C 1 -C 6 alkyl group. The C1 -C2 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.
[000127] 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 C [-C ()) alkyl group. The C1 -C6 alkyl group is preferably unsubstituted. Preferred ethers include diethyl ether, diisopropyl ether, and methyl t-butyl ether (MTBE).
[000128] 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 C 1 -C 6 alkyl group.
[000129] Preferred esters include methyl acetate and ethyl acetate.
[000130] 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 C 1 -C 6 alkyl group. The C 1 -C 6 alkyl group is preferably unsubstituted. Preferred ketones include acetone, methyl ethyl ketone and methyl isobutyl ketone (MTBK).
[000131] 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 C) -Cò alkyl group. The C 1 -C 6 alkyl group is preferably unsubstituted. Preferred nitriles include acetonitrile.
[000132] Typically, the aqueous organic solvent is aqueous alcohol or aqueous acetonitrile.
[000133] The aqueous organic solvent is preferably aqueous methanol or aqueous acetonitrile. Aqueous methanol is most preferred.
[000134] Typically, the eluent is not in a supercritical state. Typically, the eluent is a liquid.
[000135] 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 parts by volume, preferably 0.25 : 99.75 to 7:93 parts by volume, more preferably 0.5: 99.5 to 6:94 parts by volume.
[000136] 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.
[000137] Typically, the aqueous organic solvent eluent used in each separation step has a different water: organic solvent ratio. The water: organic solvent ratio used in each separation step is preferably adjusted such that the PUFA product can be separated from the different components of the feed mixture in each separation step.
[000138] The eluting strength of the eluent used in each of the separation steps is typically different. Preferably, 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. In practice, this is achieved by varying the relative amounts of water and organic solvent used in each separation step.
[000139] Depending on the choice of organic solvent, they can be more powerful absorbers than water. Alternatively, they can be less powerful absorbers than water. Acetonitrile and alcohols, for example, are more powerful desorbers than water.
[000140] Thus, 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.
[000141] Typically, the water: organic solvent ratio of the eluent in the first separation step is lower than the water: organic solvent ratio of the eluent in the second separation stage. Thus, the eluent in the first separation step typically contains more organic solvent, preferably alcohol, more preferably methanol, than the eluent in the second separation step.
[000142] In embodiments where the aqueous organic solvent used in each separation step has a water: different organic solvent ratio, the water ratio of the organic solvent to the eluent in the first separation step is typically 0: 100 to 5:95 parts by volume, preferably from 0.1: 99.9 to 2.5: 97.5 parts by volume, more preferably from 0.25: 99.75 to 2:98 parts by volume, and most preferably from 0.5: 99.5 to 1.5: 98.5 parts by volume. In these embodiments, the water: organic solvent ratio of the eluent in the second separation step is typically from 2:98 to 8:92 parts by volume, preferably from 3:97 to 7:93 parts by volume, more preferably from 4 : 96 to 6:94 parts by volume, and even more preferably from 4.5: 95.5 to 5.5: 94.5 parts by volume.
[000143] In a particularly preferred embodiment where the aqueous organic solvent used in each separation step has a different water and organic solvent content, the water: organic solvent ratio of the eluent in the first separation step is 0.5 : 99.5 to 1.5: 98.5 parts by volume, and the water: organic solvent ratio of the eluent in the second separation step is 4.5: 95: 5 to 5.5: 94.5 parts in volume.
[000144] 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.
[000145] 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 separation step, water is typically introduced more slowly into the chromatographic apparatus used in the first separation step than in the second separation step.
[000146] 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.
[000147] In a particularly preferred embodiment, (1) the intermediate product containing the PUFA product together with the more polar components is collected as the raffinate stream in the first separation step, and the PUFA product is collected as the extract stream in the second separation step; or
[000148] (2) the intermediate product containing the PUFA product together with the less polar components is collected as the extract stream in the first separation step, and the PUFA product is collected as the raffinate stream in the second separation.
[000149] The particularly preferred embodiment (1) is suitable for purifying EPA from a feed mixture.
[000150] This particularly preferred embodiment (1) is illustrated in Figure 2. A feed mixture F comprising the PUFA product (B) and the more polar (C) and the less polar (a) components is purified on first separation step. In the first separation step, the less polar components (a) are removed as an extract stream El. The PUFA product (B) and the more polar components (C) are collected as a raffinate stream RI. The raffinate stream RI is the intermediate product which is then purified in the second separation step. In the second separation step, the more polar components (C) are removed as a rafinate stream R2. The PUFA product (B) is collected as an E2 extract stream.
[000151] 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 desorbent organic solvent (D) and water (W) in each chromatographic apparatus are shown. The organic solvent desorbent (D) and water (W) together make up the eluent. Phase (D) is typically essentially pure organic solvent, but may, in certain embodiments, be an organic solvent / water mixture that comprises mainly organic solvent. The (W) phase is typically essentially pure water, but may, in certain embodiments, be an organic solvent / water mixture that mainly comprises water, for example, a 98% water / 2% methanol mixture.
[000152] Another illustration of this particularly preferred embodiment is shown in Figure 6. Here there is no separate water injection point, and instead a desorbent aqueous organic solvent is injected in (D).
[000153] The current separation of raffinate and extract can be aided by varying the desorption force of the eluent within each chromatographic apparatus. This can be achieved by introducing the organic solvent component (or rich in organic solvent) of the eluent and the water component (or rich in water) at different points in each chromatographic apparatus. Thus, typically, the organic solvent is introduced upstream of the extract collection point and water is introduced between the extract collection point and the feed introduction point in the chromatographic apparatus, in relation to the eluent flow in the system. This is shown in Figure 4.
[000154] Typical solvents for use in this most preferred embodiment are aqueous alcohols or aqueous acetonitrile, preferably aqueous methanol.
[000155] Typically, in this particularly preferred embodiment, the aqueous organic solvent eluent used in the first separation step contains more organic solvent than the eluent used in the second separation step, i.e. the water: organic solvent ratio in the first stage it is lower than the water: organic solvent ratio in the second stage.
[000156] In this particularly 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.
[000157] 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.
[000158] In this particularly preferred embodiment, the second rafinate stream in the second separation step is typically removed downstream from the point of introduction of the intermediate product in the chromatographic apparatus used in the second separation step, with respect to the eluent flow.
[000159] 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 intermediate product in the chromatographic apparatus used in the second separation step, with respect to the eluent flow.
[000160] Typically in this particularly preferred embodiment, the organic solvent or 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.
[000161] Typically in this particularly preferred embodiment, when water is introduced into the chromatographic apparatus used in the first separation step, water is introduced into the chromatographic apparatus used in the first separation step upstream of the feed mixture introduction point but at downstream of the removal point of the first extract stream, with respect to the eluent flow.
[000162] Typically in this particularly preferred embodiment, the organic solvent or 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.
[000163] Typically in this particularly preferred embodiment, when water is introduced into the chromatographic apparatus used in the second separation step, water is introduced into the chromatographic apparatus used in the second separation step upstream of the intermediate product introduction point, but the downstream of the removal point of the second extract stream, with respect to the eluent flow.
[000164] The particularly preferred embodiment (2) is suitable for purifying DHA from a feed mixture.
[000165] The particularly preferred embodiment (2) is illustrated in Figure 3. A feed mixture F comprising PUFA product (B) and the more polar (C) and less polar components (a) is purified in the first separation step. In the first separation step, the more polar components (C) are removed as a raffinate stream RI. The PUFA product (B) and the less polar components (a) are collected as an extract stream El. The extract stream EI is the intermediate product that is then purified in the second separation step. In the second separation step, the less polar components (a) are removed as an E2 extract stream. The PUFA product (B) is collected as a rafinate stream R2.
[000166] This embodiment is illustrated in more detail in Figure 5. Figure 5 is identical to Figure 3, except that the points of introduction of the desorbent organic solvent (D) and water (W) within each chromatographic apparatus are shown . As above, phase (D) is typically essentially pure organic solvent, but may, in certain embodiments, be an organic solvent / water mixture that comprises mainly organic solvent. The (W) phase is typically essentially pure water, but may, in certain embodiments, be an organic solvent / water mixture that mainly comprises water, for example, a 98% water / 2% methanol mixture.
[000167] Another illustration of this particularly preferred embodiment is shown in Figure 7. There is no separate water injection point here, and instead a desorbent aqueous organic solvent is injected into (D).
[000168] Typical solvents for use in this most preferred embodiment are aqueous alcohols or aqueous acetonitrile, preferably aqueous methanol.
[000169] Typically in this embodiment, the aqueous organic solvent eluent used in the first separation step contains less organic solvent than the eluent used in the second separation step, that is, the water: organic solvent ratio in the first separation step separation is higher than in the second separation step.
[000170] In this 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.
[000171] In this 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.
[000172] In this embodiment, the second rafinate stream in the second separation step is typically removed downstream from the point of introduction of the intermediate product into the chromatographic apparatus used in the second separation step, with respect to the eluent flow.
[000173] In this embodiment, the second extract stream in the second separation stage is typically collected upstream of the point of introduction of the intermediate product in the chromatographic apparatus used in the second separation stage, with respect to the eluent flow.
[000174] Typically in this embodiment, the organic solvent or 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.
[000175] Typically in this embodiment, when water is introduced into the chromatographic apparatus used in the first separation step, water is introduced into the chromatographic apparatus used in the first separation step upstream of the point of introduction of the feed mixture but downstream of the removal point of the first extract stream, with respect to the eluent flow.
[000176] Typically in this embodiment, the organic solvent or 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.
[000177] Typically in this embodiment, when water is introduced into the chromatographic apparatus used in the second separation step, water is introduced into the chromatographic apparatus used in the second separation step upstream from the point of introduction of the intermediate product but downstream from the point of removing the second extract stream, with respect to the eluent flow.
[000178] In a preferred embodiment of the invention, each of the simulated or real moving bed chromatography apparatus used in the first and second separation steps consists of eight chromatographic columns. These are referred to as columns 1 through 8. In each apparatus the eight columns are arranged in series so that 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 column 8 is connected to the top of column 1. These connections can optionally be via a containment container, with a recycling stream inside the next column. The flow of eluent through the system is from column 1 to column 2 to column 3 etc. The effective absorbent flow through the system is from column 8 to column 7 to column 6 etc.
[000179] A more preferred embodiment is illustrated in Figure 8. A feed mixture F comprising the PUFA product (B) and the more polar (C) and the less polar (a) components are introduced at the top of the column 5 in the chromatographic apparatus used in the first separation step. The desorbent organic solvent is introduced at the top of column 1 of the chromatographic apparatus used in the first separation step. Water is introduced at the top of column 4 of the chromatographic apparatus used in the first separation step. In the first separation step, the less polar components (a) are removed as a stream of extract of phtindo from column 2. The PUFA product (B) and the more polar components (C) are removed as a raffinate stream RI from the bottom. from column 7. The raffinate stream RI is the intermediate product which 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 5. The desorbent organic solvent is introduced at the top of the column l in the chromatographic apparatus used in the second separation step. The water is introduced at the top of column 4 in the chromatographic apparatus used in the second separation step. In the second separation step, the more polar components (C) are removed as a raffinate stream R2 at the bottom of column 7. The PUFA product (B) is collected as an extract stream E2 at the bottom of column 2.
[000180] In this most preferred embodiment, the organic solvent is typically introduced at the top of column 1 of the chromatographic apparatus used in the first separation step.
[000181] In this most preferred embodiment, water is typically introduced at the top of column 4 of the chromatographic apparatus used in the first separation step.
[000182] In this most preferred embodiment, the organic solvent is typically introduced at the top of column 1 of the chromatographic apparatus used in the second separation step.
[000183] In this most preferred embodiment, the organic solvent is typically introduced at the top of column 4 of the chromatographic apparatus used in the second separation step.
[000184] 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.
[000185] In this most preferred embodiment, a first stream of raffinate is typically collected as the intermediate product from the bottom of column 7 of the chromatographic apparatus used in the first separation step. This intermediate product is then purified in the second separation step and is typically introduced at the top of column 5 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.
[000186] In this most preferred embodiment, a first stream of extract 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.
[000187] In this most preferred embodiment, a second stream of raffinate is typically removed from the bottom of column 7 of the chromatographic apparatus used in the second separation step.
[000188] In this most preferred embodiment, a second stream of extract is typically collected from the end of column 2 of the chromatographic apparatus used in the second separation step. This second extract stream typically contains the purified PUFA product. The second extract stream can optionally be collected in a container and reintroduced at the top of column 3 of the chromatographic apparatus used in the second separation step.
[000189] In this most preferred embodiment, the eluent used is typically aqueous alcohol, preferably aqueous methanol. The water-alcohol ratio is typically 0.5: 99.5 to 6:94 parts by volume.
[000190] Typically, in this most preferred embodiment, the water: organic solvent ratio in the chromatographic apparatus used in the first separation step is lower than the water: organic solvent ratio in the chromatographic apparatus used in the second separation step . Thus, the eluent in the first separation step typically contains more organic solvent than the eluent used in the second separation step.
[000191] The ratio of organic solvent water in the first separation step is typically 0.5: 99.5 to 1.5: 98.5 parts by volume. The water: organic solvent ratio in the second separation step is typically 2:98 to 6:94 parts by volume.
[000192] Although the embodiment of Figure 8 is configured as shown in Figure 10a, the configurations shown in Figures 10b and 10c can also be used in this embodiment.
[000193] Another more preferred embodiment is illustrated in Figure 9. A feed mixture F comprising the PUFA product (B) and the more polar (C) and the less polar (a) components 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 in the chromatographic apparatus used in the first separation step. In the first separation step, the less polar components (a) are removed as an extract stream El from the bottom of column 2. The PUFA product (B) and the more polar components (C) are removed as a raffinate stream RI from the bottom. from column 7. The rafinate stream R1 is the intermediate product that is purified in the second separation step being introduced at the top of column 4 of the chromatographic apparatus used in the second separation step. The desorbent aqueous organic solvent is introduced at the top of column 1 in the chromatographic apparatus used in the second separation step. In the second separation step, the more polar components (C) are removed as a raffinate stream R2 at the bottom of column 7. The PUFA product (B) is collected as an extract stream E2 at the bottom of column 2.
[000194] In this most preferred embodiment, the aqueous organic solvent is typically introduced at the top of column 1 in the chromatographic apparatus used in the first separation step.
[000195] In this most preferred embodiment, the aqueous organic solvent is typically introduced at the top of column 9 into the chromatographic apparatus used in the second separation step.
[000196] In this most preferred embodiment, the supply current is typically introduced at the top of column 5 in the chromatographic apparatus used in the first separation step.
[000197] In this most preferred embodiment, a first stream of raffinate is typically collected as the intermediate product from the bottom of column 7 of the chromatographic apparatus used in the first separation step. This intermediate product is then purified in the second separation step and is typically introduced at the top of column 5 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.
[000198] 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 a portion reintroduced at the top of column 3 of the chromatographic apparatus used in the first separation step. The rate of liquid recycling collected through the extract stream in the first separation step back to the chromatographic apparatus used in the first separation step is the rate at which the liquid is pumped from this container to the top of column 3.
[000199] In this most preferred embodiment, a second stream of raffinate is typically removed from the bottom of column 7 of the chromatographic apparatus used in the first separation step.
[000200] In this most preferred embodiment, a second stream of extract is typically collected from the bottom of column 2 of the chromatographic apparatus used in the first separation step. This second extract stream typically contains the purified PUFA product. The second extract stream can optionally be collected in a container and a portion reintroduced at the top of column 3 of the chromatographic apparatus used in the first separation step. The rate of recycling of liquid collected through the extract stream from the second separation step back to the chromatographic apparatus used in the second separation step is the rate at which the liquid is pumped from this container to the top of column 3.
[000201] In this most preferred embodiment, the eluent used is typically aqueous alcohol, preferably aqueous methanol. The water-alcohol ratio is typically 0.5: 99.5 to 6:94 parts by volume.
[000202] Typically, in this most preferred embodiment, the water: organic solvent ratio in the chromatographic apparatus used in the first separation step is lower than the water: organic solvent ratio in the chromatographic apparatus used in the second separation step. Thus, the eluent used in the first separation step typically contains more organic solvent than the eluent used in the second separation step.
[000203] The water: organic solvent ratio in the first separation step is typically 0.5: 99.5 to 1.5: 98.5 parts by volume. The water: organic solvent ratio in the second separation step is typically 2:98 to 6:94 parts by volume.
[000204] Although the embodiment of Figure 9 is configured as shown in Figure 10a, the configurations shown in Figures 10b and 10c can also be used in this embodiment.
[000205] 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 concerns compositions comprising a PUFA product, for example one obtainable by the process of the present invention.
[000206] 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.
[000207] The Examples that follow illustrate the invention. EXAMPLES Example 1
[000208] 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 silica gel C) s bound (size particle size 5 pm) as a stationary phase and aqueous methanol as eluent according to the system schematically illustrated in Figure 9. 8 columns (diameter: 4.6 mm, length 250 mm) are connected in series as shown in Figure 9.
[000209] The feed mixture was passed through the SMB device in a first separation step. In the first separation step, the process conditions were adjusted to purify EPA from DHA and other slowly moving impurities. EPA along with other impurities that move fast was collected in the raffinate stream as the intermediate product. The extract stream containing DHA and other slowly moving impurities was rejected.
[000210] The process conditions of the SMB device were then adjusted for the second separation step. In the second separation step, the process conditions were adjusted to purify EPA from the impurities that move faster. The aqueous methanol eluent used in the second separation step had a higher water: organic solvent ratio than the aqueous methanol eluent used in the first separation step. The intermediate product was introduced into the SMB device as the feed mixture in the second separation step. High purity EPA was collected as the extract stream. The stream of raffinate containing impurities that move faster was rejected.
[000211] EPA was produced with a final purity of ~ 95% and recovery of -95%.
[000212] An HPLC analysis of the feed stock is shown as Figure 11.
[000213] HPLC analyzes of the raffinate (R) and extract (E) streams from the first separation step are shown as Figure 12.
[000214] HPLC analyzes of the raffinate (R) and extract (E) streams of the second separation step are shown as Figure 13. A more detailed HPLC analysis of the extract stream of the second separation step is shown as Figure 14.
1000215] Figures 12 and 13 also show the process conditions for the first and second separation steps. Reference Example 1
[000216] An experiment was carried out to compare the amount of environmental pollutants present in two of the PUFA products produced by SMB with similar oils prepared by distillation. The pollutant profiles of the oils are shown in Table 1 below.
Dioxin limits include the sum of polychlorinated dibenzene-to-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) and expressed in toxic equivalents from the World Health Organization (WHO) using WHO toxic equivalent factors (TEFs). This means that the analytical results concerning the 7 individual dioxin counterparts of toxicological interest are expressed in a single quantifiable unit: TCDD or TEQ 21 toxic equivalent concentration The maximum for dioxin and Furans remains at 2 pg / g Reference Example 2
[000217] An experiment was carried out to determine the amount of isomeric impurities present in an oil prepared by SMB compared to an equivalent oil prepared by distillation,
[000218] A GC trace of the DHA-rich oil prepared by SMB is shown as Figure 15. There is no evidence of isomeric impurities in the GC trace.
[000219] A GC trace of the oil prepared by distillation is shown as Figure 16. The four peaks with elution times longer than the DHA peak correspond to the DHA isomers. From the GC trace it can be seen that the oil prepared by distillation contains about 1.5% by weight of isomeric impurities. Reference Example 3
[000220] Two EPA-rich products produced by SMB have been compared with EPA-rich oils produced by distillation. The analysis by weight% of its PUFA components is shown below.
Reference Example 4
[000221] A product rich in EPA / DHA produced by SMB has been compared with an oil rich in EPA / DHA produced by distillation. The analysis by weight% of its PUFA components is shown below.

权利要求:
Claims (14)
[0001]
1. Chromatographic separation process to recover a polyunsaturated fatty acid product (PUFA) from a feed mixture, a process that comprises the steps of: (i) purifying the feed mixture in a first separation step in a simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns which contain, as an eluent, an aqueous organic solvent, to obtain an intermediate product; and (ii) purifying the intermediate product obtained in (i) in a second separation step using a simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as an eluent, an aqueous organic solvent, for obtain the PUFA product; where (a) the first and second separation steps are carried out sequentially on the same chromatography apparatus, the intermediate product being recovered between the first and second separation steps and the process conditions on the chromatography apparatus being adjusted between the first and second separation steps such that the PUFA product is separated from the different components of the feed mixture at each separation step; or (b) the first and second separation steps are performed on the separate primary and secondary chromatography apparatus respectively, the intermediate product being recovered from the first separation step and introduced into the second chromatography apparatus, and the PUFA product being separated from the different components of the feed mixture in each separation step, and in which the aqueous organic solvent eluent used in each separation step has a ratio by volume of water: different organic solvent, characterized by the fact that (1) the intermediate product it is collected as the raffinate stream in the first separation step and the PUFA product is collected as the extract stream in the second separation step; or (2) the intermediate product is collected as the extract stream in the first separation step, and the PUFA product is collected as the refine stream in the second separation step.
[0002]
2. Process according to claim 1, characterized by the fact that each apparatus has an extract stream and a raffinate stream from which liquid can be collected from said plurality of linked chromatography columns.
[0003]
Process according to claim 1 or 2, characterized by the fact that the intermediate product recovered from the first separation step is enriched in the PUFA product compared to the feed mixture.
[0004]
Process according to any one of claims 1 to 3, characterized in that the PUFA product is separated from the less polar components of the feed mixture in the first separation step, and the PUFA product is separated from the more polar components of the feed mixture in the second separation step.
[0005]
Process according to any one of claims 1 to 4, characterized in that the PUFA product comprises at least one PUFA ω-3.
[0006]
Process according to claim 5, characterized in that the PUFA product comprises eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA).
[0007]
Process according to any one of claims 1 to 6, characterized in that the eluent is a mixture of water and an alcohol, an ether, an ester, a ketone or a nitrile.
[0008]
8. Process according to claim 7, characterized by the fact that the eluent is a mixture of water and methanol.
[0009]
Process according to any one of claims 1 to 8, characterized in that the feed mixture is a fish oil or feed stock derived from fish oil, the PUFA product is EPA or EPA ethyl ester, and the PUFA product is produced in a purity greater than 90% purity, preferably greater than 95% purity, and more preferably greater than 97% purity.
[0010]
Process according to any one of claims 1 to 9, characterized in that (a) part of the extract stream from the apparatus used in the first separation step is recycled back into the apparatus used in the first separation step; and / or (b) 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 / or (c) 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 (d) 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.
[0011]
Process according to any one of claims 1 to 10, characterized in that the ratio by volume of water: organic solvent used in each separation step is adjusted such that the PUFA product can be separated from the different components of the mixture at each separation step.
[0012]
Process according to any one of claims 1 to all, characterized in that the ratio by volume of water: organic solvent of the eluent in the first separation step is lower than the ratio by volume of water: organic solvent of the eluent in the second stage of separation.
[0013]
Process according to any one of claims 1 to 12, characterized in that the ratio by volume of water: organic solvent of the eluent in the first separation step is 0.5: 99.5 to 1.5: 98 , 5 parts by volume, and the water: organic solvent ratio of the eluent in the second separation step is 4.5: 95: 5 to 5.5: 94.5 parts by volume.
[0014]
Process according to any one of claims 1 to 13, characterized in that the ratio by volume of water: organic solvent of the eluent in the first and second separation steps is controlled by the introduction of water and / or organic solvent into one or more columns in the apparatus used in the first and second separation steps.

类似技术:

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公开号 | 公开日 | 专利标题

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BR112014000152B1|2020-10-27|chromatographic separation process

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US9695382B2|2017-07-04|SMB process for producing highly pure EPA from fish oil

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BR112014000147B1|2020-10-27|chromatographic separation process, pufa product

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JP6535052B2|2019-06-26|Compositions Comprising PUFA Products

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JP2017020031A|2017-01-26|Smb process

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BR112014000133B1|2020-12-01|CHROMATOGRAPHIC SEPARATION PROCESS TO RECOVER A POLYINSATURATED FATTY ACID | PRODUCT FROM A FOOD MIXTURE

同族专利:

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公开号 | 公开日

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HUE043139T2|2019-07-29|

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CA2815301A1|2013-01-10|

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GB201111589D0|2011-08-24|

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JP2014523944A|2014-09-18|

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CA2815301C|2015-09-29|

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TR201819866T4|2019-01-21|

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US20140128627A1|2014-05-08|

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KR101603448B1|2016-03-14|

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CN103796724B|2015-07-15|

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KR20140034923A|2014-03-20|

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AU2012279993A1|2013-05-02|

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US9260677B2|2016-02-16|

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WO2013005051A1|2013-01-10|

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JP5990580B2|2016-09-14|

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AU2012279993B2|2015-07-23|

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BR112014000152A2|2017-02-14|

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CL2013003799A1|2014-07-04|

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EP2613862B1|2018-12-19|

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ES2710652T3|2019-04-26|

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PE20142014A1|2014-12-24|

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PL2613862T3|2019-05-31|

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EP2613862A1|2013-07-17|

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DK2613862T3|2019-03-11|

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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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2019-07-30| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-06-30| B09A| Decision: intention to grant|

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2020-10-27| 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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GB1111589.6|2011-07-06|

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GBGB1111589.6A|GB201111589D0|2011-07-06|2011-07-06|New modified process|

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PCT/GB2012/051596|WO2013005051A1|2011-07-06|2012-07-06|New smb process|

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