阅读说明：本技术 用于制造富含磷脂组合物的方法 (Process for making phospholipid-enriched compositions ) 是由 皮埃尔·勒米厄 西蒙·德斯平斯 萨里亚·阿齐兹 雷米·拉布雷克 尼科拉斯·比叙埃尔 于 2019-05-15 设计创作，主要内容包括：本申请提供了一种从原料磷虾油产生富含磷脂组合物的方法,其中该方法可以是分批的、连续的,或者包括分批步骤和连续步骤两者。本技术通过该方法提供了高质量的富含磷脂组合物。此外,与分批方法相比,连续方法提供了显著更高的产量,同时提供了相等或更高质量的产品。(The present application provides a process for producing a phospholipid-enriched composition from raw krill oil, wherein the process may be batch, continuous, or include both batch and continuous steps. The present technology provides high quality phospholipid-rich compositions by this method. Furthermore, the continuous process provides significantly higher throughput compared to batch processes, while providing an equal or higher quality product.)

1. A method for producing a phospholipid-enriched composition, the method comprising the steps of:

mixing Raw Krill Oil (RKO) with a mixture comprising at least about 85% by weight of C₃-C₈Organic solvent mixing of ketones to provide an RKO containing mixture containing an aqueous portion; and

fractionating the RKO-containing mixture into a first low density layer and a first higher density phospholipid-containing layer (PCL), and separating the first PCL from the first low density layer, thereby producing the phospholipid-enriched composition.

2. The method of claim 1, further comprising

Separating the first PCL from the first low density layer, thereby producing a first separated PCL;

mixing the first isolated PCL with the organic solvent, thereby producing a PCL-containing mixture;

fractionating the PCL-containing mixture into a second low density layer and a second PCL; and

separating the second PCL from the second low density layer, thereby producing the phospholipid-enriched composition.

3. A method for producing a phospholipid-enriched composition, the method comprising the steps of:

mixing Raw Krill Oil (RKO) with a mixture comprising at least about 85% by weight of C₃-C₈Organic solvent mixing of ketones to provide an RKO containing mixture containing an aqueous portion;

fractionating the RKO-containing mixture into a first low density layer and a first higher density phospholipid-containing layer (PCL);

separating the first PCL from the first low density layer, thereby producing a first separated PCL;

mixing the first isolated PCL with the organic solvent, thereby producing a PCL-containing mixture;

fractionating the PCL-containing mixture into a second low density layer and a second PCL; and

separating the second PCL from the second low density layer, thereby producing the phospholipid-enriched composition.

4. The method of any of claims 1-3, further comprising filtering the RKO-containing mixture prior to fractionating the RKO-containing mixture.

5. A method for producing a phospholipid-enriched composition, the method comprising the steps of:

mixing Raw Krill Oil (RKO) with a mixture comprising at least about 85% by weight of C₃-C₈Organic solvent mixing of ketones to provide an RKO containing mixture containing an aqueous portion; and

mixing the RKO-containing mixture with water and fractionating the RKO-containing mixture and water into a first low-density layer and a first higher-density phospholipid-containing layer (PCL), and separating the first PCL from the first low-density layer, thereby producing the phospholipid-enriched composition.

6. The method of any one of claims 2-4, further comprising combining the second PCL with a stabilizer, a viscosity reducer, or a combination thereof.

7. The method of any one of claims 1-6, wherein the aqueous portion is substantially salt-free.

8. The method of any one of claims 1-7, wherein the aqueous portion is substantially free of carbonates, bicarbonates, or a combination thereof.

9. The method of any of claims 1-8, wherein the method comprises mixing at least 100kg RKO with the organic solvent.

10. The method of any of claims 1-9, wherein the method includes mixing the RKO and the organic solvent in units of volumetric organic solvent kg RKO at a ratio of about 5 to about 15.

11. The method of any of claims 1-10, wherein prior to mixing the RKO with the organic solvent, the method includes heating the RKO to a temperature of about 30 ℃ to about 70 ℃.

12. The method of claim 11, wherein heating the RKO is performed for no more than 72 hours prior to mixing the RKO with the organic solvent.

13. The process according to any one of claims 1-12, wherein the mixing of Raw Krill Oil (RKO) with the organic solvent is performed at a temperature of about 15 ℃ to about 40 ℃ to provide the RKO-containing mixture.

14. The method of any one of claims 1-13, wherein the RKO-containing mixture is directed through a 0.45 μ ι η filter.

15. The method of any of claims 1-14, wherein the RKO-containing mixture is filtered through a first filter and a second filter.

16. The method of claim 15, wherein the first filter is a 10 μ ι η filter and the second filter is a 0.45 μ ι η filter.

17. The method of any one of claims 1-16, wherein the RKO-containing mixture is filtered by centrifugation or decantation.

18. The method of claims 2-4 and 6 further including mixing the RKO-containing mixture with water in units of volumetric aqueous fraction kg RKO in a ratio of the RKO-containing mixture to water to the aqueous fraction of about 0.15 to about 0.40.

19. The method of any one of claims 1-18, wherein the first isolated PCL is mixed with the organic solvent in a volume ratio of about 2 to about 10.

20. The method of any one of claims 2-4, 6, and 18, wherein combining the second PCL with a viscosity reducing agent comprises a volume ratio of viscosity reducing agent to second PCL of about 0.1 to about 0.3.

21. The method of any one of claims 2-4, 6, 18, and 20, wherein the stabilizer comprises vitamin E.

22. The method of claim 21, wherein the stabilizer comprises about 3g to about 5g vitamin E per kg second PCL (based on dry weight of second PCL).

23. The process of any one of claims 1-22, wherein the process is a continuous process.

24. The method of any one of claims 1-23, wherein the phospholipid-enriched composition comprises at least 75% phospholipids on a dry weight basis.

25. The method of any one of claims 1-24, wherein the phospholipid-enriched composition comprises at least 85% phospholipids on a dry weight basis.

Technical Field

The present invention provides a robust and flexible purification process for producing a high quality phospholipid-rich composition from raw krill oil for use in the pharmaceutical industry.

Background

A general process for purifying raw krill oil has been described. U.S.2017/0101600 and 9,650,590 describe a continuous process for refining krill oil extract to remove salts and trimethylamine nitrogen oxides and recover a product containing neutral lipids, polar lipids and astaxanthin by a series of separation processes based on adsorption and chromatographic separation. In US 2016/034561, a method is described for processing lipid rich crustaceans by extraction with a solvent to produce a composition comprising phospholipids, protein nutrients and oils low in fluoride, triethylamine and trimethyl amide oxides. These methods aim at removing impurities and separating specific molecules. Therefore, they do not allow to produce high quality phospholipid-rich compositions for use in the pharmaceutical industry for the treatment of medical conditions such as hypertriglyceridemia.

An extraction process for preparing the concentrated phospholipid krill oil composition is also described. US 2017/0020928 discloses the preparation of a concentrated phospholipid krill oil composition that can be manufactured using a small molecule organic solvent (i.e. acetone or ethanol)/water extraction mixture and/or a subcritical or supercritical fluid extraction at low temperature followed by a drying process. US 2016/0228461 and 2016/0228462 describe an efficient and high-yield process for extracting crude krill lipid compositions using an alcohol fractionation process. These methods allow the extraction of lipid components from marine biomass without any concentration. In order to be economically viable and to meet regulatory requirements for pharmaceutical products, the manufacturing process needs to take into account several variability (i.e. variability of starting materials, etc.) and should allow for the production of commercial quantities of high purity products in a consistent and efficient manner.

Thus, there remains a great need for processes with greater overall throughput that provide commercially economically viable components and compositions derived from raw krill oil that exhibit high purity in a consistent and efficient manner for use in the pharmaceutical industry. These phospholipid-enriched compositions can be used to treat hypertriglyceridemia or other indications in a subject.

Disclosure of Invention

A process for producing a phospholipid-enriched composition is provided, the process comprising the steps of: mixing Raw Krill Oil (RKO) with a mixture comprising at least about 85% by weight of C₃-C₈Organic solvent mixing of ketones to provide an RKO containing mixture containing an aqueous portion; and fractionating the RKO-containing mixture into a first low density layer and a first higher density phospholipid-containing layer (PCL) and separating the first PCL from the first low density layer, thereby producing the phospholipid-enriched composition.

In one embodiment, the methods described herein further comprise detaching the first PCL from the first low density layer, producing a first detached PCL; mixing the first isolated PCL with an organic solvent, thereby producing a PCL-containing mixture; fractionating the PCL-containing mixture into a second low density layer and a second PCL; and separating the second PCL from the second low density layer, thereby producing a phospholipid-enriched composition.

Also provided is a method for producing a phospholipid-rich composition, the method comprising the steps of: mixing Raw Krill Oil (RKO) with a mixture comprising at least about 85% by weight of C₃-C₈Organic solvent mixing of ketones to provide an RKO containing mixture containing an aqueous portion; fractionating the RKO-containing mixture into a first low density layer and a first higher density phospholipid-containing layer (PCL); from the first low densityDelaminating the first PCL, thereby producing a first detached PCL; mixing the first isolated PCL with an organic solvent, thereby producing a PCL-containing mixture; fractionating the PCL-containing mixture into a second low density layer and a second PCL; and separating the second PCL from the second low density layer, thereby producing a phospholipid-enriched composition.

In another embodiment, the methods described herein further comprise filtering the RKO-containing mixture prior to fractionating the RKO-containing mixture.

Further provided is a method for producing a phospholipid-enriched composition, the method comprising the steps of: mixing Raw Krill Oil (RKO) with a mixture comprising at least about 85% by weight of C₃-C₈Organic solvent mixing of ketones to provide an RKO containing mixture containing an aqueous portion; and mixing the RKO-containing mixture with water and fractionating the RKO-containing mixture and water into a first low density layer and a first higher density phospholipid-containing layer (PCL) and separating the first PCL from the first low density layer, thereby producing the phospholipid-enriched composition.

In additional embodiments, the methods described herein further comprise combining the second PCL with a stabilizer, a viscosity reducer, or a combination thereof.

In one embodiment, the aqueous portion is substantially free of salts.

In another embodiment, the aqueous portion is substantially free of carbonates, bicarbonates, or a combination thereof.

In another embodiment, the methods contained herein comprise mixing at least 100kg of RKO with an organic solvent.

In a further embodiment, the methods described herein include mixing RKO with an organic solvent in units of volumetric organic solvent kg RKO at a ratio of about 5 to about 15.

In one embodiment, prior to mixing the RKO and the organic solvent, the method includes heating the RKO to a temperature of from about 30 ℃ to about 70 ℃.

In a further embodiment, the RKO is heated for no more than 72 hours prior to mixing the RKO and the organic solvent.

In a further embodiment, the mixing of the Raw Krill Oil (RKO) with the organic solvent is performed at a temperature of about 15 ℃ to about 40 ℃ to provide the RKO-containing mixture.

In further embodiments, the method further comprises directing the RKO-containing mixture through a 0.45 μm filter.

In one embodiment, the RKO-containing mixture is filtered through a first filter and a second filter.

In another embodiment, the first filter is a 10 μm filter and the second filter is a 0.45 μm filter.

In another embodiment, the RKO containing mixture is filtered by centrifugation or decantation.

In one embodiment, the method further includes mixing the RKO-containing mixture with water in units of volumetric aqueous fraction kg RKO in a ratio of RKO-containing mixture to water to aqueous fraction of about 0.15 to about 0.40.

In one embodiment, the first isolated PCL is mixed with an organic solvent in a volume ratio of about 2 to about 10.

In another embodiment, combining the second PCL with the viscosity reducing agent comprises a volume ratio of the viscosity reducing agent to the second PCL of about 0.1 to about 0.3.

In one embodiment, the stabilizing agent comprises vitamin E.

In one embodiment, the stabilizer comprises about 3g to about 5g vitamin E per kg of the second PCL (based on dry weight of the second PCL).

In other embodiments, the process is a continuous process.

In one embodiment, the phospholipid-enriched composition comprises at least 75% phospholipids on a dry weight basis.

In one embodiment, the phospholipid-enriched composition comprises at least 85% phospholipids on a dry weight basis.

Drawings

Reference will now be made to the drawings.

Fig. 1 shows a flow diagram according to an embodiment of a continuous process as contained herein.

Detailed Description

The present invention provides a process for producing a phospholipid-rich composition from raw krill oil, wherein such process may be batch, continuous, or comprise both batch and continuous steps.

As used herein, "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If the use of a term is not clear to one of ordinary skill in the art, given the context in which it is used, "about" will mean up to plus or minus 5% of the particular term.

As used herein, "raw krill oil" refers to oil isolated from krill, such as by the processes described in U.S. patent nos. 9,475,830, 9,650,590 and 9,068,142, and u.s.2004/0234587, 2009/0074857, 2008/0274203, 2013/0274496, 2017/0020928 and 2017/0101600, the disclosures of which are incorporated herein by reference.

As used herein, "phospholipid" refers to an organic compound having one fatty acid moiety attached at the sn-1 or sn-2 position of glycerol or two fatty acid moieties attached at the sn-1 and sn-2 positions of glycerol and containing a head group linked by a phosphate residue at the sn-3 position of glycerol. Exemplary headgroup moieties include choline, ethanolamine, serine, and inositol. The phospholipid includes phosphatidylcholine, lysophosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, lysophosphatidylinositol, phosphatidic acid and lysophosphatidic acid. The fatty acid moiety is a moiety bonded at the sn-1 or sn-2 position in the fatty acid molecule, for example, by an ester or ether linkage. When the fatty acid moiety is a fatty acyl group, the aliphatic chain of the fatty acyl group is attached via an ester bond, and when the fatty acid moiety is an aliphatic chain of a fatty acid, the aliphatic chain is attached via an ether bond. When referring to a particular fatty acid (e.g., EPA or DHA) in conjunction with a phospholipid as herein, it should be considered to refer to the relevant fatty acyl group or to the aliphatic chain thereof.

As used herein, the term "ether phospholipid" refers to a phospholipid in which the fatty acid moiety in one of the sn-1 or sn-2 positions is an aliphatic chain of fatty acids attached via an ether linkage. Ether phospholipids include, for example, alkanoyl phosphatidylcholine, alkanoyl phosphatidylethanolamine and alkanoyl phosphatidylserine.

As used herein, the term "omega-3 fatty acid" refers to a polyunsaturated fatty acid having a double bond in the hydrocarbon chain between the third and fourth carbon atoms from the methyl end of the molecule. Non-limiting examples of omega-3 fatty acids include 5,8,11,14, 17-eicosapentaenoic acid (EPA), 4,7,10,13,16, 19-docosahexaenoic acid (DHA), and 7,10,13,16, 19-docosapentaenoic acid (DPA).

As used herein, the term "phospholipid-rich composition" refers to a mixture having a concentration of phospholipids of at least 75 wt.%, preferably 85 wt.%, on a dry weight basis.

There is thus provided a method comprising the steps of: fractionating a mixture ("third mixture") comprising raw krill oil ("RKO") into a first low density layer and a first higher density phospholipid-containing layer ("PCL") ("first fractionation step"); separating the first PCL from the first low density layer ("first separation step"); wherein the RKO-containing mixture comprises RKO, comprises at least about 85 wt.% C₃-C₈A first organic solvent of a ketone solvent, and an aqueous portion.

As contained herein, the process optionally further comprises mixing the raw krill oil with a first organic solvent to produce a first mixture ("first mixing step"); optionally directing the first mixture through a filter to provide a second mixture ("first directing step"); and mixing the first mixture or the second mixture (when present) with the aqueous portion to provide the RKO-containing mixture ("second mixing step").

The method can include (in addition to or as an alternative to the optional steps described above) directing the first PCL to a third blending step comprising blending the first PCL with a blend comprising at least about 85 wt.% C₃-C₈A second organic solvent of the ketone solvent to provide a fourth mixture ("second directing step"); fractionating the fourth mixture into a second low density layer and a second PCL ("second fractionation step"); and separating the second PCL from the second low density layer ("second separation step").

In a related aspect, the methods contained hereinComprising fractionating a mixture comprising raw krill oil ("RKO") ("third mixture") into a first low density layer and a first higher density phospholipid-containing layer ("PCL") ("first fractionation step"); separating the first PCL from the first low density layer ("first separation step"); directing the first PCL to a third mixing step comprising admixing the first PCL with a blend comprising at least about 85% by weight of C₃-C₈A second organic solvent of the ketone solvent to provide a fourth mixture ("second directing step"); fractionating the fourth mixture into a second low density layer and a second PCL ("second fractionation step"); and separating the second PCL from the second low density layer ("second separation step"); wherein the RKO-containing mixture comprises RKO, comprises at least about 85 wt.% C₃-C₈A first organic solvent of a ketone solvent, and an aqueous portion.

The process may further comprise mixing the raw krill oil with a first organic solvent to produce a first mixture ("first mixing step"); optionally directing the first mixture through a filter to provide a second mixture ("first directing step"); and mixing the first mixture or the second mixture (when present) with the aqueous portion to provide the RKO-containing mixture ("second mixing step").

Thus, in any embodiment herein, the process may comprise contacting raw krill oil ("RKO") with a composition comprising at least about 85% by weight of C₃-C₈Mixing a first organic solvent of a ketone solvent to produce a first mixture ("first mixing step"); optionally directing the first mixture through a filter to provide a second mixture ("first directing step"); mixing the first mixture or the second mixture (when present) with the aqueous portion to provide a third mixture ("second mixing step"); fractionating the third mixture into a first low density layer and a first higher density phospholipid-containing layer ("PCL") ("first fractionation step"); and separating the first PCL from the first low density layer ("first separation step").

The method can optionally include directing the first PCL to a third mixing step comprising mixing the first PCL with a blend comprising at least about 85 wt% C₃-C₈Ketone solutionsMixing a second organic solvent of the agent to provide a fourth mixture ("second directing step"); fractionating the fourth mixture into a second low density layer and a second PCL ("second fractionation step"); and separating the second PCL from the second low density layer ("second separation step").

In any of the embodiments herein, the process can include contacting raw krill oil ("RKO") with a composition comprising at least about 85% by weight of C₃-C₈Mixing a first organic solvent of a ketone solvent to produce a first mixture ("first mixing step"); optionally directing the first mixture through a filter to provide a second mixture ("first directing step"); mixing the second mixture with the aqueous portion to provide a third mixture ("second mixing step"); fractionating the third mixture into a first low density layer and a first phospholipid-containing layer ("PCL") ("first fractionation step"); separating the first PCL from the first low density layer ("first separation step"); directing the first PCL to a third mixing step comprising admixing the first PCL with a blend comprising at least about 85% by weight of C₃-C₈A second organic solvent of the ketone solvent to provide a fourth mixture ("second directing step"); fractionating the fourth mixture into a second low density layer and a second PCL ("second fractionation step"); and separating the second PCL from the second low density layer ("second separation step").

The first "low density layer" of any embodiment herein is to be understood as having a density less than the first PCL (higher density layer) and the second low density layer having a density less than the second PCL. Such low-density and high-density layers are referred to herein as "light phase" and "heavy phase," respectively, alternately with reference to each other.

In any of the embodiments herein, the process can be a batch process, a continuous process, or a combination thereof. Thus, the methods described herein are robust and flexible, allowing control of variability (i.e., season, material, age, storage, and processing) associated with raw krill oil that may affect its lipid content and lipid profile and have a significant impact on the quality of the phospholipid-rich composition. Furthermore, a continuous process is a dynamic process that provides significantly higher throughput compared to batch processes, while providing equal or higher quality product.

Thus, each step of the process may be performed as part of a batch process, each step of the process may be part of a continuous process, or some steps may be performed batchwise, while other steps may be performed as part of a continuous process. Preferably, the first mixing step, the first directing step (when present), the second mixing step, the first fractionating step, the first separating step, the second directing step, the second fractionating step, and the second separating step are each performed as part of a continuous process. The first fractionating step may include introducing the third mixture into a horizontal settler. The first separating step can include transferring the first low density layer from the horizontal settler via a first port in the horizontal settler and transferring the first PCL from a second port in the horizontal settler. The second fractionating step may comprise introducing the fourth mixture into a vertical settler. The second separating step may comprise pumping the low density layer from the vertical settler via a first port in the vertical settler and simultaneously pumping the second PCL from the vertical settler via a second port in the vertical settler.

As shown in fig. 1, in one embodiment, the process described herein may be a continuous process. RKO 10 and acetone 12 are first mixed 14. The RKO/acetone mixture obtained 16 is filtered using a filter with a pore size of, for example but not limited to, 10 μm and 0.45 μm.

The filtered RKO/acetone feed is directed into a static mixer 18 along with a demineralized water feed 20, where each feed may utilize a calibrated pump to ensure the flow rate of the water and the flow rate of the filtered RKO/acetone feed. The static mixer 18 feeds a horizontal settler 22 in which a light phase 24 and a heavy phase 26 (containing phospholipids) are each continuously collected at the end of the horizontal settler 22.

Heavy phase 26 is then directed into second mixer 28, and in one embodiment, a concurrent feed of acetone 30 is also directed into second mixer 28. Pumps are used to ensure the flow rate of the heavy phase 26 and the flow rate of the acetone 30 into the static mixer 28. The resulting mixture flows from the mixer 28 to a vertical settler 32 which is maintained to provide a light phase 24' and a heavy phase 40 comprising phospholipids. Each phase is discharged continuously from the vertical settler 32.

The addition of ethanol and vitamin E, as well as the general storage conditions involving the heavy (phospholipid-containing) phase, may be carried out as described in a batch process, or may be carried out by a continuous procedure.

The first PCL and the second PCL comprise a phospholipid. The first PCL and the second PCL may each independently comprise at least 75 wt.% (dry weight basis) phospholipids. Thus, the amount of phospholipid (on a dry weight basis) can be about 75 wt.%, about 76 wt.%, about 77 wt.%, about 78 wt.%, about 79 wt.%, about 80 wt.%, about 81 wt.%, about 82 wt.%, about 83 wt.%, about 84 wt.%, about 85 wt.%, about 86 wt.%, about 87 wt.%, about 88 wt.%, about 89 wt.%, about 90 wt.%, about 91 wt.%, about 92 wt.%, about 93 wt.%, about 94 wt.%, about 95 wt.%, or any range including and/or between any two of these values. The first PCL and the second PCL may each independently comprise about 0 wt.% to about 15 wt.% free fatty acid (on a dry weight basis); thus, the amount of free fatty acid can be about 0 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 11 wt.%, about 12 wt.%, about 13 wt.%, about 14 wt.%, about 15 wt.%, or any range including and/or between any two of these values. The first PCL and the second PCL may each independently comprise about 0 wt.% to about 5 wt.% triglycerides (on a dry weight basis); thus, the amount of about 0 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, or any range including and/or between any two of these values. The first PCL and the second PCL may each independently comprise less than about 2 wt.% monoglyceride (on a dry weight basis). The first PCL and the second PCL may each independently comprise about 1.9 wt.%, about 1.8 wt.%, about 1.7 wt.%, about 1.6 wt.%, about 1.5 wt.%, about 1.4 wt.%, about 1.3 wt.%, about 1.2 wt.%, about 1.1 wt.%, about 1.0 wt.%, about 0.9 wt.%, about 0.8 wt.%, about 0.7 wt.%, about 0.6 wt.%, about 0.5 wt.%, about 0.4 wt.%, about 0.3 wt.%, about 0.2 wt.%, about 0.1 wt.%, or monoglycerides (on a dry weight basis) that comprise and/or are in any range between any two of these values, or are below any range of any one of these values. The first PCL and the second PCL may each independently comprise less than about 2 wt.% diglyceride (on a dry weight basis). The first PCL and the second PCL may each independently comprise about 1.9 wt.%, about 1.8 wt.%, about 1.7 wt.%, about 1.6 wt.%, about 1.5 wt.%, about 1.4 wt.%, about 1.3 wt.%, about 1.2 wt.%, about 1.1 wt.%, about 1.0 wt.%, about 0.9 wt.%, about 0.8 wt.%, about 0.7 wt.%, about 0.6 wt.%, about 0.5 wt.%, about 0.4 wt.%, about 0.3 wt.%, about 0.2 wt.%, about 0.1 wt.%, or diglycerides comprising and/or between any two of these values, or any range below any one of these values (on a dry weight basis). The first PCL and the second PCL may each independently comprise about 0 wt.% to about 3 wt.% cholesterol (on a dry weight basis); thus, the amount of cholesterol on a dry weight basis may be about 0 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 1.1 wt.%, about 1.2 wt.%, about 1.3 wt.%, about 1.4 wt.%, about 1.5 wt.%, about 1.6 wt.%, about 1.8 wt.%, about 1.9 wt.%, about 2.0 wt.%, about 2.1 wt.%, about 2.2 wt.%, about 2.3 wt.%, about 2.4 wt.%, about 2.5 wt.%, about 2.6 wt.%, about 2.8 wt.%, about 2.9 wt.%, about 3.0 wt.%, or a range including any two of these values, or a range between any of these.

As previously described, in any embodiment of the method, a first directing step may optionally be included. Such optional step may be employed when the first mixture is found to include visible insoluble components in performing the first mixing step. Such insoluble components are those that cannot pass through a filter of 0.45 μm or less. Filters suitable for use in the method are well known to those of ordinary skill in the art and include, but are not limited to, polyester, nylon, polypropylene, polytetrafluoroethylene, glass microfiber, or a combination of any two or more thereof. Directing the first mixture through a filter to provide the second mixture may include directing the first mixture through a filter of at least 0.45 μm. Directing the first mixture through a filter to provide a second mixture may include directing the first mixture through at least a first filter and a second filter in series. For example, the first filter may be a 10 μm filter and the second filter may be a 0.45 μm filter. Where the filters are in series, the first mixture may be passed through two or more 10 μm filters before being passed through the 0.45 μm filter. Alternatively, any solid/liquid content separation method known in the art, such as centrifugation or decantation, is also included.

C of the first organic solvent, the second organic solvent, or both the first and second organic solvents₃-C₈The ketone can be acetone, butanone, 2-pentanone, 3-pentanone, methyl isobutyl ketone, 2-hexanone, 3-hexanone, acetylacetone, or a combination of any two or more thereof. In the first organic solvent, the second organic solvent, or both the first and second organic solvents₃-C₈The total concentration of ketones (in wt.% of the corresponding organic solvent) may be about 85 wt.%, about 90 wt.%, about 95 wt.%, about 96 wt.%, about 97 wt.%, about 98 wt.%, about 99 wt.%, about 100 wt.%, or any range including and/or between any two of these values. Thus, as a non-limiting example, in any of the embodiments herein, the first organic solvent can be at least about 99 wt.% acetone. As another non-limiting example, the second organic solvent can be at least about 99 wt.% acetone. In addition to one or more C₃-C₈In addition to the ketone, the first organic solvent, the second organic solvent, or both the first and second organic solvents may or may not include a co-solvent. Exemplary co-solvents include alcohols (e.g., methanol (CH)₃OH), ethanol (EtOH), isopropanol (iPrOH), Trifluoroethanol (TFE), butanol (BuOH), ethylene glycol, propylene glycol), carboxylic acids (e.g., formic acid, acetic acid, propionic acid, butyric acidValeric acid, lauric acid, stearic acid, deoxycholic acid, glutamic acid, glucuronic acid), ethers (e.g., Tetrahydrofuran (THF), 2-methyltetrahydrofuran (2Me-THF), Dimethoxyethane (DME), dioxane), esters (e.g., ethyl acetate, isopropyl acetate), nitriles (e.g., acetonitrile (CH)₃CN), propionitrile (CH)₃CH₂CN)), or a mixture of any two or more thereof.

In any of the embodiments herein that include a continuous process, the process can include mixing at least about 4 kg/hour of the raw krill oil with the first organic solvent. The amount of RKO per hour mixed with the first organic solvent may be about 4 kg/hour, about 5 kg/hour, about 6 kg/hour, about 7 kg/hour, about 8 kg/hour, about 9 kg/hour, about 10 kg/hour, about 15 kg/hour, about 20 kg/hour, about 25 kg/hour, about 30 kg/hour, about 35 kg/hour, about 40 kg/hour, about 45 kg/hour, about 50 kg/hour, about 60 kg/hour, about 70 kg/hour, about 80 kg/hour, about 90 kg/hour, about 100 kg/hour, about 110 kg/hour, about 120 kg/hour, about 130 kg/hour, about 140 kg/hour, about 150 kg/hour, about 160 kg/hour, about 170 kg/hour, about, About 180 kg/hour, about 190 kg/hour, about 200 kg/hour, about 220 kg/hour, about 240 kg/hour, about 260 kg/hour, about 280 kg/hour, about 300 kg/hour, about 320 kg/hour, about 340 kg/hour, about 360 kg/hour, about 380 kg/hour, about 400 kg/hour, about 420 kg/hour, about 440 kg/hour, about 460 kg/hour, about 480 kg/hour, about 500 kg/hour, or any range including and/or between any two or more of these values, or any range including and greater than any one of these values.

In any of the embodiments herein, the process may comprise mixing the raw krill oil and the first organic solvent in units of volume organic solvent: kg RKO (volume of organic solvent: kg RKO), in a ratio of about 5 to about 15. Thus, the ratio of the volume of the first organic solvent to kg RKO can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or any range including and/or between any two of these values. The mixing of the raw krill oil with the first organic solvent may be carried out at a temperature as follows: about 15 ℃ to about 40 ℃, such as about 15 ℃, about 16 ℃, about 17 ℃, about 18 ℃, about 19 ℃, about 20 ℃, about 22 ℃, about 24 ℃, about 26 ℃, about 28 ℃, about 30 ℃, about 32 ℃, about 34 ℃, about 36 ℃, about 38 ℃, about 40 ℃, or any range including and/or between any two of these values. In any embodiment including a continuous process, mixing can include combining the first organic solvent at a flow rate of from about 25L/h to about 7,500L/h; thus, the flow rate of the first organic solvent may be about 25L/h, about 30L/h, about 35L/h, about 40L/h, about 45L/h, about 50L/h, about 55L/h, about 60L/h, about 65L/h, about 70L/h, about 75L/h, about 80L/h, about 85L/h, about 90L/h, about 95L/h, about 100L/h, about 110L/h, about 120L/h, about 130L/h, about 140L/h, about 150L/h, about 200L/h, about 250L/h, about 300L/h, about 350L/h, about 400L/h, about 450L/h, about 500L/h, about 550L/h, about 600L/h, about 650L/h, about, About 700L/h, about 750L/h, about 800L/h, about 850L/h, about 900L/h, about 1,000L/h, about 1,200L/h, about 1,400L/h, about 1,600L/h, about 1,800L/h, about 2,000L/h, about 2,200L/h, about 2,400L/h, about 2,600L/h, about 2,800L/h, about 3,000L/h, about 3,200L/h, about 3,400L/h, about 3,600L/h, about 3,800L/h, about 4,000L/h, about 4,200L/h, about 4,400L/h, about 4,600L/h, about 4,800L/h, about 5,000L/h, about 5,500L/h, about 6,000L/h, about 6,500L/h, about 7,000L/h, about 7,500L/h, or any range including and/or between any two of these values.

Prior to mixing the RKO and the first organic solvent, the method may include heating the RKO to a temperature of: from about 30 ℃ to about 70 ℃, such as about 30 ℃, about 35 ℃, about 40 ℃, about 45 ℃, about 50 ℃, about 55 ℃, about 60 ℃, about 65 ℃, about 70 ℃, or a temperature in any range including and/or between any two of these values. In any of the embodiments herein, heating of the RKO can be performed for no more than 72 hours (e.g., from about 24 hours to about 48 hours) (such as no more than about 60 hours, no more than about 48 hours, no more than about 36 hours, no more than about 24 hours, no more than about 12 hours, no more than about 6 hours, no more than about 1 hour, no more than about 30 minutes, or any range including and/or between any two of these values) prior to mixing the RKO and the first organic solvent. In any of the embodiments herein, the RKO may be first heated to one temperature as provided above and further heated to a second higher temperature as provided above en route to the mixing step, thereby minimizing the duration of time the RKO is at the second higher temperature.

The aqueous portion may be substantially free of salt when the second mixture is mixed with the aqueous portion to provide the third mixture. In any of the embodiments herein, the aqueous portion can be substantially free of base. Exemplary bases include ammonia, basic amino acids (e.g., arginine, lysine, and ornithine), carbonates (e.g., K)₂CO₃、Na₂CO₃、(NH₄)₂CO₃) Bicarbonate (e.g., KHCO)₃、NaHCO₃) A hydroxide salt (e.g., KOH, NaOH), or a combination of any two or more thereof. For example, in any of the embodiments herein, the aqueous portion can be substantially free of carbonate, bicarbonate, or a combination thereof; in any of the embodiments herein, the aqueous portion can be substantially free of KHCO₃And KOH. In any of the embodiments herein, the aqueous portion may be substantially free of acid. Exemplary acids include inorganic acids (such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginates, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid), acidic amino acids (such as aspartic acid and glutamic acid), or a combination of any two or more thereof. In any of the embodiments herein, the aqueous portion can be demineralized, deionized, or distilled water. For example, deionized water exhibits a resistivity of at least 0.2M Ω -cm, preferably at least about 1M Ω -cm, at 25 ℃ and may be of class IV, III, II or I according to ASTM D1193-91.

As used herein, when a composition is "substantially free of" a specified component, this means that the component is present in an amount of less than 0.1 wt.% of the total related composition (preferably less than 0.01 wt.%).

The method can include mixing the second mixture with the aqueous portion in units of volumetric aqueous portion kg RKO at a ratio of about 0.10 to about 0.40. kg RKO is understood to be kg based on RKO in the first mixing step. This decision is made based on the nature of the method. Aqueous partial volume: kg RKO may be about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.22, about 0.24, about 0.26, about 0.28, about 0.30, about 0.32, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, or any range including and/or between any two of these values. The second mixing step may be carried out at a temperature as follows: about 6 ℃, about 8 ℃, about 10 ℃, about 12 ℃, about 14 ℃, about 16 ℃, about 18 ℃, about 20 ℃, about 22 ℃, about 24 ℃, about 26 ℃, about 28 ℃, about 30 ℃, about 32 ℃, about 34 ℃, about 36 ℃, about 38 ℃, or any range including and/or between any two of these values.

The third mixing step of the method can include mixing the first PCL with the second organic solvent in a volume ratio of the first PCL to the second organic solvent of about 2 to about 10; the volume ratio of the second organic solvent to the first PCL can be about 2, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, about 3.2, about 3.4, about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.2, about 8.4, about 8.6, about 8.8, about 9.0, about 9.2, about 9.4, about 9.6, about 9.8, about 10.0, or a range including any two or more of these values. The third mixing step may be carried out at a temperature as follows: about 15 ℃ to about 40 ℃, such as about 15 ℃, about 16 ℃, about 17 ℃, about 18 ℃, about 19 ℃, about 20 ℃, about 22 ℃, about 24 ℃, about 26 ℃, about 28 ℃, about 30 ℃, about 32 ℃, about 34 ℃, about 36 ℃, about 38 ℃, about 40 ℃, or any range including and/or between any two of these values.

In any of the embodiments herein, the method can further comprise combining the first PCL or the second PCL with a stabilizer, a viscosity reducer, or both, to produce a phospholipid-enriched fraction ("PLEF"). Stabilizers include, but are not limited to, antioxidants. Exemplary antioxidants include vitamin a, vitamin E, astaxanthin, canthaxanthin, beta-carotene, all-trans retinol, and flavonoids such as naringin, naringenin, hesperetin/kaempferol, rutin, luteolin, neohesperidin, and quercetin (quercertin). In any of the embodiments herein, the antioxidant can comprise vitamin E, wherein the vitamin E is present in an amount of about 3g/kg of the second PCL to about 5g/kg of the second PCL. The amount of vitamin E (in grams) that can be included per kg of the second PCL (based on the dry weight of the second PCL) can be about 3, about 3.2, about 3.4, about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5 grams, or any range including and/or between any two of these values. Viscosity reducing agents include, but are not limited to, ethanol, acetone, glycerol, propylene glycol, polyethylene glycol (e.g., PEG 300, PEG 400), peanut oil, coconut oil, castor oil, cottonseed oil, corn oil, sesame oil, soybean oil, sunflower oil, caprylic/capric triglycerides, propylene glycol diesters of caprylic/capric acid, propylene glycol monolaurate, propylene glycol monocaprylate, caprylic/capric/diglyceryl succinate, medium chain fatty acid esters of propylene glycol, aerosol, cetostearyl alcohol (cetostearyl alcohol), cetyl alcohol, glyceryl behenate (glyceryl behenate), glyceryl tricaprylate, glyceryl palmitostearate, caprylic/capric/stearic triglycerides, di-diglyceryl/caprylate/caprate/stearate/adipate, stearic acid, sterols, lauric acid, propylene glycol, polyethylene glycol, coconut oil, castor oil, cottonseed oil, corn oil, sesame oil, soybean oil, sunflower, Oleic acid, polyglycolized glycerides, polyethylene glycol-40 hydrogenated castor oil, glyceryl monocaprylate, coco/citric/lactic glycerides, glyceryl mono-dicaprylate/caprate, diglyceryl isostearate succinate, coco glycerides, glyceryl caprylate, oleoyl polyethylene glycol-8 glycerides, linoleoyl polyethylene glycol glycerides, PEG-8 caprylic/capric glycerides, propylene glycol laurate, polyglycerol dioleate, polyoxyethylene-polyoxypropylene copolymer, PEG-6 caprylic/capric glycerides, polyoxyethylene triolein, and polyoxyethylene (20) sorbitan monooleate, as well as those described in U.S. patent publication nos. 2017/0182073 and 2017/0020928 (incorporated herein by reference). The volume ratio of the viscosity reducer (or the sum of two or more viscosity reducers) to the first PCL can be about 0.1 to about 0.3, such as about 0.1, about 0.12, about 0.14, about 0.16, about 0.18, about 0.20, about 0.22, about 0.24, about 0.26, about 0.28, about 0.3, or any range including and/or between any two of these values. The volume ratio of the viscosity reducer (or the sum of two or more viscosity reducers) to the second PCL can be about 0.1 to about 0.3, such as about 0.1, about 0.12, about 0.14, about 0.16, about 0.18, about 0.20, about 0.22, about 0.24, about 0.26, about 0.28, about 0.3, or any range including and/or between any two of these values.

In any of the embodiments herein, wherein the process is a continuous process, the continuous process may proceed from the RKO to the second PCL and/or the PLEF at a rate of at least about 4 kg/hour of raw krill oil; thus, the rate may be about 4 kg/hour, about 5 kg/hour, about 6 kg/hour, about 7 kg/hour, about 8 kg/hour, about 9 kg/hour, about 10 kg/hour, about 15 kg/hour, about 20 kg/hour, about 25 kg/hour, about 30 kg/hour, about 35 kg/hour, about 40 kg/hour, about 45 kg/hour, about 50 kg/hour, about 60 kg/hour, about 70 kg/hour, about 80 kg/hour, about 90 kg/hour, about 100 kg/hour, about 110 kg/hour, about 120 kg/hour, about 130 kg/hour, about 140 kg/hour, about 150 kg/hour, about 160 kg/hour, about 170 kg/hour, about 180 kg/hour, about 100 kg/hour, about 110 kg/hour, about 120 kg/hour, about 130 kg/hour, about 140 kg/hour, about 150 kg/hour, about 160 kg/hour, about 170, About 190 kg/hour, about 200 kg/hour, about 220 kg/hour, about 240 kg/hour, about 260 kg/hour, about 280 kg/hour, about 300 kg/hour, about 320 kg/hour, about 340 kg/hour, about 360 kg/hour, about 380 kg/hour, about 400 kg/hour, about 420 kg/hour, about 440 kg/hour, about 460 kg/hour, about 480 kg/hour, about 500 kg/hour, or any range including and/or between any two or more of these values, or any range including and greater than any one of these values.

In a related aspect, a method is provided that includes substantially removing the first organic solvent, the second organic solvent, and water of the PLEF produced by any embodiment of the method to produce a phospholipid-enriched composition. For example, such a removal step may be carried out by a thin film evaporator in which a thin layer of the PLEF is heated under vacuum with or without a purge of an inert gas (e.g. nitrogen). The method may further comprise combining one or more free fatty acids with the PLEF produced by any embodiment of the method, either before or after the removing step. The method may further comprise combining the free fatty acid-enriched mixture with the PLEF produced by any embodiment of the method, either before or after the removing step.

In other related aspects, a phospholipid-enriched composition produced by any such embodiment of the method is provided. The phospholipid-enriched composition may be used to treat hypertriglyceridemia in a subject. The phospholipid-enriched composition may also include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" generally includes both carriers and excipients, and is further described herein.

In other related aspects, a unit dosage form is provided that includes the phospholipid-enriched composition of any of the embodiments herein. The unit dosage form may be a liquid unit dose contained in a liquid or contained within a capsule. The capsules may comprise one or more of gelatin, carrageenan and hypromellose. The unit dosage form of any of the embodiments herein can include an effective amount of a phospholipid-rich composition.

By "effective amount" is meant the amount of the composition required to produce the desired effect in a subject. One example of an effective amount includes an amount or dose that results in acceptable levels of toxicity and bioavailability for therapeutic (e.g., pharmaceutical) use. As used herein, a "subject" or "patient" is a mammal. Typically, the subject is a human, such as a human having or suspected of having arthritic pain or hypertriglyceridemia. The terms "subject" and "patient" are used interchangeably.

Thus, the present technology provides compositions and medicaments comprising any of the phospholipid-enriched compositions disclosed herein and optionally a pharmaceutically acceptable carrier or one or more excipients or fillers. The compositions may be used in the methods and treatments described herein. Such compositions and medicaments include a therapeutically effective amount of any of the phospholipid-enriched compositions as described herein. The compositions may be packaged in unit dosage form.

The examples given herein are intended to more fully illustrate various aspects of the disclosure. The examples should in no way be construed as limiting the scope of the disclosure.

Example I

Batch process

The following representative batch process utilized 5kg RKO as the starting material. RKO was weighed with balance and transferred with stirring to a container already containing about 10L of acetone. Additional acetone was then added to the vessel for a total of 50L of acetone. The mixture was then stirred at a temperature of 30 ℃. The RKO/acetone mixture was then pumped into the vessel through a solvent resistant filtration system (filtration train) comprising a pre-filter with a porosity of 10 μm and a final filter with a porosity of 0.45 μm to provide a filtered mixture of RKO/acetone.

To the filtered mixture of RKO/acetone 1L of water was added between 20 ℃ and 25 ℃ under continuous stirring for at least 5 minutes. The stirring was stopped and the mixture was allowed to settle in the treatment vessel until a higher light phase (i.e. low density layer) and a lower heavy phase (i.e. high density layer) were observed, wherein the light phase was clear and a clear interface was observed between the light and heavy phases. After this phase was reached, the heavy phase was transferred to another vessel and the volume was calculated.

Half of the volume of the heavy phase was then added to the vessel with constant stirring, followed by seven (7) volumes of acetone per total volume of the heavy phase, and then the remaining half of the heavy phase was transferred to the same vessel. Stirring was continued for at least 5 minutes between 20 ℃ and 25 ℃. The stirring was then stopped and the resulting mixture was allowed to settle in the treatment vessel until two phases (a low density light phase and a high density heavy phase) were present, with the light phase being clear and a clear interface being observed between the light and heavy phases. After this settling has been achieved, the heavy phase (which contains the refined phospholipids) is then transferred to a vessel.

The weight of the heavy phase is then determined. Absolute ethanol was added as a viscosity reducer to a glass vessel at a ratio of 0.175L ethanol/L heavy phase with continuous stirring. Samples of the mixture were taken and analyzed to determine the dry weight of the phase. According to the results of the determination, a vitamin E preparation (. alpha. -tocopherol) was added manually as an antioxidant to the heavy phase at a rate of 4g vitamin E/kg dry heavy phase. The mixture is then stirred between 15 ℃ and 25 ℃ for at least 5 minutes to provide a phospholipid-enriched fraction. The total duration of the batch process from RKO to the phospholipid-rich fraction is about 24 hours or less, but can be increased if desired to provide a longer duration for phase separation and/or to allow for larger sized vessels.

For storage, the phospholipid-rich fraction was transferred to a vessel under a nitrogen blanket, sealed, and stored at 2-8 ℃. Starting from 5kg RKO, the average yield of the phospholipid-rich fraction on a dry weight basis (i.e. after drying the phospholipid-rich fraction) was 30%.

Example II

Continuous process

A continuous process was carried out in which RKO was preheated (between 30 ℃ and 65 ℃) and acetone was fed using 2 calibrated pumps, in which the mixing of RKO and acetone was carried out using a mixing pump. The flow rate of acetone was 56-64L/h and the flow rate of RKO was 5.75-6.25kg/h over the duration of the process run. The resulting RKO/acetone mixture feed was maintained at a temperature of 32.5. + -. 2.5 ℃ and filtered in-line through a filter of 10 μm and 0.45 μm pore size.

The filtered RKO/acetone feed was directed into a static mixer along with a demineralized water feed, wherein each feed utilized two calibrated pumps to ensure a water flow rate of 1.0-1.4L/h and a filtered RKO/acetone feed flow rate of 62-68L/h. The static mixer feeds a horizontal settler, where the light and heavy phases (containing phospholipids) are each collected continuously at the end of the settler. The horizontal settler is maintained at a temperature of 20-25 ℃.

The heavy phase is then directed to a second static mixer, to which acetone feed is also directed. Two calibration pumps ensure that the flow rate of the heavy phase into the static mixer is 2.2-2.6L/h and the flow rate of acetone is 16-18L/h. The resulting mixture flows from the static mixer to a vertical settler maintained at a temperature of 20-25 ℃, wherein the vertical settler provides a light phase and a heavy phase comprising phospholipids. Each phase is discharged continuously from the vertical settler.

The addition of ethanol and vitamin E, as well as the general storage conditions involving the heavy (phospholipid-containing) phase, may be carried out as described in a batch process, or may be carried out by a continuous procedure. The total duration of the continuous process from RKO to the phospholipid-rich fraction was about 8 hours.

Compared to the batch process described above, the continuous process has been found to produce 3-5 times more material (similarly high quality) with the same equipment footprint, is easier to automate, and requires fewer operators.

While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure, including such departures from the present disclosure as come within known or customary practice in the art to which the essential features hereinbefore set forth may be applied and fall within the scope of the appended claims.