`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1036
`
`
`
`U.S. Patent
`
`Dec. 22, 1987
`
`Sheet 1 of3
`
`4,714,571
`
`F|G.I
`
`04
`
`Legend
`
`0 1 °/o PC in 2% Phosphotides
`
`O 2% PC in 4°/o Phosphotides
`
`D 4% PC in 8% Phosphatides
`A 80/0 PC in 16% Phosphatides
`
`0.30
`
`AcetonitrileHexane
`
`
`
`PartitionCoefficient
`
`0.10
`
`0.0
`
`O
`
`10
`
`2O
`
`30
`
`4O
`
`5O
`
`60
`
`70
`
`0
`
`Temperature
`
`C
`
`000002
`
`000002
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`
`
`U.S. Patent
`
`Dec. 22, 1987
`
`Sheet 2 of3
`
`4,714,571
`
`F I G. 2
`
`Legend
`
`' 1% PE in 2% Phosphotides
`O 2% PE in 4% Phosphatides
`U 4% PE in 8% Phosphotides
`
`A 8% PE in16°/o Phosphotides
`
`x1o’4
`
`90
`
`80
`
`
`
`[PE]Acetonitrile
`
`
`
`
`
`
`
`PartitionCoefficient[PE]Hexane
`
`TO
`
`60
`
`50
`
`1O
`
`40
`
`30
`
`20
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`70
`
`0
`
`Te mpercnure
`
`C
`
`000003
`
`000003
`
`
`
`U.S.Patent
`
`Dec.22,1937
`
`Sheet3of3
`
`F|G.3
`
`4,714,571
`
`240
`
`Legend
`
`. 2% Phosphotides
`
`Q 4% Phosphutides
`
`[1 8% Phosphotides
`
`A 16% Phosphotides
`
`10
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`20
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`50
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`40
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`50
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`60
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`70
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`Temperature
`
`°C
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`000004
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`220
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`200
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`65 0
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`(Coefficient)PE5R33‘En’oOoO
`
`(Coefficient)PC Partition
`Partition
`Selectivity
`
`000004
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`
`
`1
`
`PROCESS FOR PURIFICATION OF
`PHOSPHOLIPIDS
`
`4,714,571
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`2
`group consisting of acetonitrile, and mixtures of aceto-
`nitrile and one or more hydrocarbons of the group
`consisting of pentane, hexane, isohexane, heptane and
`octane, and mixtures of hydrocarbons such as petro-
`leum ether or mixtures of acetonitrile and fluorocar-
`bons
`
`The present invention is advantageousin that it is
`both less time consuming and less costly than other
`known methods.
`
`2. BACKGROUND OF THE INVENTION
`
`2.1 Phospholipids
`
`including PC, which is commonly
`Phospholipids,
`known as lecithin, are members of the class of phospha-
`tides. They are of significant commercial importance
`because of their wetting and emulsifying properties.
`They are widely used as ingredients in food products,
`cosmetics, pharmaceuticals, insecticides, paints, plastics
`and textiles, and have also found numerous applications
`in the petroleum industry. Because of its widespread
`occurrence in nature, PC is known colloquially as “na-
`ture’s emulsifier.” The occurrence of PC as a compo-
`nent of cell membranes has been the subject of much
`recent scientific research. Emphasis in this research has
`been on the determination of the physical properties and
`functional characteristics of PC.
`Purified egg phospholipids are currently used as a
`starting material to synthesize other compounds such as
`glycerophosphocholine; saturated, unsaturated, single
`and mixed fatty acids, phosphatidylcholines, phos-
`phatidylethanolarnines, phosphatidylglycerols, phos-
`phatidylserines, phosphatidic acids, and diether lipids,
`etc.
`
`2.2 Phospholipid Purification
`
`At present, high purity PC is typically obtained by
`time consuming, expensive methods such as high pres-
`sure liquid chromatography (HPLC), solid-liquid col-
`umn chromatography (SLCC), flash chromatography,
`and thin layer chromatography (TLC).
`These methods involve the separation of the lipids,
`typically by solvent extraction or by other solvent-
`based techniques. Neutral lipids can be separated from
`the phospholipid class by precipitation with cold ace-
`tone. A forrn of chromatography is then used to sepa-
`rate the individual lipid components. HPLC and flash
`chromatography on silica gel or alumina represent the
`state of the art in chromatography. For example, Jun-
`galwala et al. [Biochem. J.
`l55:55 (l976)] have de-
`scribed HPLC in silica-gel, using a mixture of acetoni-
`trile, methanol and water as eluant, to separate phos-
`phatidylcholine from sphingomyelin. These methods,
`because they are relatively faster than conventional
`column chromatography, permit higher solvent flow-
`rates through the column (throughput) than are attain-
`able with slow conventional column chromatography.
`Chromatographic means are, however, generally slow
`and costly. On a large scale, especially, the large quan-
`tity of column packing required and the high associated
`instrumentation costs limit the use of column chroma-
`tography to the separation and purification of only the
`most valuable and expensive compounds.
`U.S. Pat. No. 2,651,646, issued to Goldsmith, dis-
`closes a method of purifying monoglycerides from di-
`glycerides, using multiple solvent systems including
`methanol-hydrocarbon, methanol-water-hydrocarbon,
`and ethanol-water-hydrocarbon. These systems, how-
`
`The present application is a continuation-in-part of 5
`prior copending application Ser. No. 579,535, filed Feb.
`13, 1934, now abandoned.
`TABLE OF CONTENTS
`
`1. Field of the Invention
`2. Background of the Invention
`2.1 Phospholipids
`2.2 Phospholipid Purification
`3. Summary of the Invention
`4. Brief Description of the Figures
`5. Detailed Description of the Invention
`5.1 Partition Coefficient of PC and PE in Mixed
`Acetonitrile-Hexane Solvent
`6. Examples
`6.1 Phospholipid Purification Using Acetonitrile Sol-
`vent
`
`6.1.1 Direct Extraction of Phospholipids from Egg
`Yolk Using Acetonitrile
`6.1.2 Direct Acetonitrile Extraction of Phospholip-
`ids from Acetone-Washed Egg Yolks
`6.1.3 Purification of Egg Yolk Derived PC with
`Acetonitrile After Initial Extraction of Phospho-
`lipids by Conventional Methods
`6.1.4 Removal of Egg Yolk Neutral Lipids by Su-
`percritical CO2 Before or after Extraction With
`Acetonitrile
`6.2 Phospholipid Purification at Different Scale Lev-
`els and Using Countercurrent Extraction
`6.2.1 Large Scale Purification of Phosphatides
`from Chicken Egg Yolks
`6.2.2 Microgram and Milligram Scale Countercur-
`rent Purification of Egg Yolk Derived PC and
`PE
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`6.2.3 Gram Scale Purification of Egg Yolk Derived
`PC and PE
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`
`6.2.4 Gram to Kilogram Scale Purification of Egg
`Yolk Derived PC and PE by Extraction Using
`Packed Column Method
`6.2.5 Pilot Scale Purification of Egg Yolk Derived
`PC and PE by Extraction Using Countercurrent
`Reciprocating Plate Karr Column
`1. FIELD OF THE INVENTION
`
`The present invention relates to a process for the
`production of high-purity,
`individual phospholipids
`from mixtures thereof, by means of separation tech-
`niques utilizing solvents novel for this purpose. More
`specifically, this invention concerns a process for sepa-
`rating and purifying phospholipids, especially those of
`the sub-class of phosphatides, including, but not limited
`to the variant fatty acid chain members of the phos-
`phatidylcholine (“PC”) or lecithin, phosphatidyletha-
`nolamine (“PE”), phosphatidylserine (“PS”) and phos-
`phatidylglycerol (“PG”) groups.
`Particular embodiments of this invention incorporate
`various known solvent-based separation methods using
`the solvent systems here disclosed to be most effective
`' in this novel application. Specific phospholipids can be
`extracted in high purity from mixtures of phospholipids
`derived from egg yolks, soya beans or other sources
`because of the different degrees of solubility of the
`phospholipids in the solvent used. This invention
`teaches the novel use of a solvent selected from the
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`ever, do not exhibit highly selective solubilization char-
`acteristics towards phospholipids, as do the acetonitrile
`and acetonitrile-hydrocarbon mixtures taught in this
`invention. Other solvent systems, such as ethylene gly-
`col-hydrocarbon, tested in the course of research lead-
`ing to this invention, also lacked the discovered selec-
`tive solubilization characteristics of the acetonitrile and
`acetonitrile-hydrocarbon solvents.
`Another purification approach that has been taken,
`and one which avoids the toxicity and flammability
`problems associated with many organic solvents,
`in-
`volves the use of supercritical fluids, especially super-
`critical CO2. A supercritical fluid is produced by sub-
`jecting certain gasses to pressure and temperature con-
`ditions such that the gas exists in a high density state and
`has in general the flow properties of gasses, but cannot
`assume a true liquid form. In an appropriate system,
`supercritical fluids such as supercritical CO2 can be
`used for extraction and purification purposes. For ex-
`ample, in U.S. Pat. No. 4,367,178 Heigel and Hueschens
`disclose the use of supercritical CO2 in an extraction
`system to remove oily components from crude soy
`lecithin preparations, thereby obtaining partially puri-
`fied lecithin.
`
`3. SUMMARY OF THE INVENTION
`
`Alternate means of phospholipid purification have
`been investigated, and methods developed based on the
`discovery of the unique differential solubility properties
`of acetonitrile, acetonitrile-hydrocarbon mixtures and
`acetonitrile-fluorocarbon mixtures towards phospholip-
`ids, especially as to the separation of PC and PE from
`other phosphatides and from one another. The novel
`process of this invention may be used on either the
`laboratory or the industrial scale. It represents a simple,
`efficient, rapid and economical means of producing
`purified phospholipids, particularly PC and PE. The
`process of this invention readily permits the achieve-
`ment of greater than 90% pure components. Purities
`greater than 98% can also be attained without signifi-
`cant additional difficulty. This invention enables the
`production of large quantities of high purity phospho-
`lipids which are needed for the preparation of pharma-
`ceuticals. The large volume of high purity phospholip-
`ids that can be easily and cheaply produced via the
`process of this invention will also permit their broader
`use in other commercial applications where less pure
`preparations have heretofore sufficed, thus resulting in
`concommitant improvements in the quality of those
`products.
`The process of this invention eliminates the use of
`column chromatography for the purification of PC and
`PE derived from egg yolks and for the partial purifica-
`tion of PC, PE, PS, PG and cardiolipids (“CL’s”) from
`other sources. Separation and purification of phospha-
`tides from sources other than egg yolks or soya beans
`may be less complete because other source materials
`may contain more complex mixtures of phosphatides
`which increases the difficulty of separation.
`The process of this invention is based on multiple
`solvent extractions performed sequentially using ace-
`tone or supercritical CO2, chloroforrn-methanol and
`acetonitrile or acetonitrile-hydrocarbon solvents. These
`extractions take advantage of the solubility differentials
`of phospholipids in these solvents. By altering the num-
`ber of the extraction steps, a purification scheme can be
`designed that is best suited to the phospholipid mixture
`
`4,714,571
`
`4
`in question and the ultimate degree of separation and
`purity desired.
`Equipment such as extractors, filter units, evapora-
`tors, crystallization vessels or countercurrent liquid/1iq-
`uid extraction devices can be employed herein. These
`are state of the art unit operations currently used
`throughout the industry. Estimations show that
`the
`capital costs for these methods are less than for chro-
`matographic methods for the purification of egg yolk
`derived PC and PE.
`The PC obtained is as pure as that obtained by con-
`ventional column chromatography. The system operat-
`ing parameters, such as the sequence of steps, the quan-
`tity of solvents used, and the temperature, can be varied
`to obtain a range of PC purities. The desired degree of
`purity depends on such factors as the ultimate use of the
`PC and cost limitations.
`The methods here taught are suitable for phospho-
`lipid purification at all scales——from the milligram level
`to tens-of-Kgs per hour, that is, from laboratory scale to
`industrial quantity.
`4. BRIEF DESCRIPTION OF THE FIGURES
`
`The present invention may be more readily under-
`stood by reference to the following figures, wherein
`FIG. 1 is a graphical representation of the tempera-
`ture dependency of the partition coefficient of PC in an
`acetonitrile-hexane system;
`FIG. 2 is a graphical representation of the tempera-
`ture dependency of the partition coefficient of PE in an
`acetonitrile-hexane system; and
`FIG. 3 is a graphical representation of the selectivity
`of PC over PE in the acetonitrile phase of an acetoni-
`trile-hexane system, expressed as a function of tempera-
`ture.
`
`5. DETAILED DESCRIPTION OF THE
`INVENTION
`
`This invention is based on the discovery of the unex-
`pected selective solubility differences of phospholipids
`, such as PC and PE, the major groups of phospholipids,
`in acetonitrile. For purposes of illustration, embodi-
`ments of the invention as applied to the purification of
`egg yolk derived PC and PE are herein described. Egg
`yolk derived PC has a limited solubility in acetonitrile at
`room temperature (2 grams/liter), while egg yolk de-
`rived PE is practically insoluble in this solvent at room
`temperature.
`In accordance with the present invention, PC and PE
`of highest purity are readily and economically isolated
`from a mixture of phosphatides using acetonitrile and-
`/or an acetonitrile-hydrocarbon solvent system or an
`acetonitrile-fluorocarbon solvent system. The preferred
`source material for mixtures of phosphatides in the
`present invention is egg yolks or soya beans. Such
`source material provides a mixture of phosphatides
`consisting primarily of PE and PC. The use of other
`source material, such as plant or animal tissue extracts,
`is within the scope of this invention; however, some
`sources yield complex mixtures which increase the diffi-
`culties of purifying the individual phosphatides. It has
`now been discovered that acetonitrile preferentially
`extracts PC, whereas PE, being insoluble in acetonitrile,
`remains in the hydrocarbon phase of the two-solvent
`system. Extraction with acetonitrile or an acetonitrile-
`hydrocarbon solvent system can be employed in various
`separation techniques which are based upon the solubil-
`ities and/or the partition coefficients of the solutes. The
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`countercurrent distribution method is the method of
`choice for achieving the highest purity in the separated
`phosphatides.
`The starting material of the process herein is a mix-
`ture containing PC and one or more other phosphatides
`of the group PE, PS, PG, CL, sphingomyelins (SPLS)
`and phosphatidic acid (free form).
`The starting material of the present invention is pref-
`erably obtained from egg yolks or soya beans since, in
`addition to the reasons indicated earlier,
`they are
`readily available and inexpensive. The source materials
`can be treated with any known methods to remove
`extraneous substances which might hinder the purifica-
`tion of the phosphatides, such as proteins, carbohy-
`drates, and neutral lipids (NLs).
`In a typical composition of hen’s egg yolk, the dry
`weight consists of 15% PC, 40% NLs, and 5% PE, and
`the rest of the components comprise proteins and carbo-
`hydrates.
`.Although this invention discloses several possible
`protocols for the purification of PC and/or PE from
`egg yolk, all methods are based on the discovery of
`acetonitrile’s unique solubility properties toward phos-
`pholipids.
`It is especially important to first remove substances
`from the source or starting material which easily parti-
`tion into acetonitrile and, consequently, would raise the
`impurity level of the end-product. For instance, some
`NLs contained in egg yolks easily partition into acetoni-
`trile. Therefore, NLs and pigments, such as color carot-
`enoids and cholesterol, may be first removed from the
`source material in accordance with the present inven-
`tion through a sequence of extractions which includes
`precipitation with cold acetone, extraction with a mix-
`ture of one of the paired solvents of the group methy-
`lene chloride-methanol, methylene chloride-ethanol,
`hexane-methanol, hexane-ethanol,
`tn'chlorofluorome-
`thane-ethanol, chloroform-methanol, or ethanol-diethyl
`ether, or ethanol or methylene chloride alone and pre-
`cipitation with a mixture of hexane-acetone. Alterna-
`tively, a dry egg yolk preparation or the oily residue
`from the extraction of egg yolks with chloroform-
`methanol or with one of the other solvents could be
`extracted with supercritical CO2 instead of acetone to
`remove the NLs and pigments.
`According to the invention, the phosphatides can be
`first dissolved in an organic solvent, such as hexane, and
`the PE precipitated with acetonitrile, or they can be
`directly extracted with acetonitrile. After evaporation
`of the acetonitrile solvent, the remaining phospholipid
`residue contains the PC in a sufficiently pure state. Any
`NLs, if still present, can be removed by precipitation
`with acetone to achieve even greater purity PC. The
`PC so prepared is greater than 95% pure with less than
`2% PE present.
`The egg yolk can be extracted or first dried and
`washed with cold dry acetone or extracted with super-
`critical CO2. These extraction steps are well known
`procedures which remove a large part of the NLs from
`the egg yolk solids, leaving most of the phospholipids.
`According to the invention, the residue can then be
`exhaustively washed with acetonitrile which will ex-
`tract the PC, leaving insoluble PE. The preparation
`containing the PC can then be freed of any remaining
`NLs by precipitation of the PC with acetone from a
`hexane or chloroform solution of the residue containing
`the PC and NLs.
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`Alternatively, the egg yolk, preferably in dried form,
`may be directly exhaustively extracted with acetoni-
`trile. The acetonitrile extract is then evaporated to dry-
`ness and the residue, consisting mostly of PC and NLs,
`is washed with acetone several times or extracted with
`supercritical C2 to remove the NLs.
`To obtain PC and PE of greater purity, the phospha-
`tide mixture (prepared according to the first protocol) is
`first dissolved in a suitable hydrocarbon solvent, prefer-
`ably hexane. The solute concentration can vary over a
`broad range, but the most advantageous results are
`achieved when the solute concentration is within the
`range of about 0.5 to about 20 grams of solute per 100
`mL of solvent, with the preferred embodiment contain-
`ing an amount of solute of 2 g/ 100 mL of solvent when
`operating between 20"—25° C. A higher solute concen-
`tration can be maintained at higher temperatures.
`Before partitioning the PC from a phosphatide mix-
`ture, the acetonitrile and hydrocarbon are desirably
`preequilibrated in accordance with the present process
`to reduce the loss of the hexane phase. However, such
`preequilibration is not necessary to obtain satisfactory
`results.
`‘
`
`Any known separation procedures utilizing the vari-
`ant partition coefficients of PC and PE in acetonitrile-
`hydrocarbon or acetonitrile-fluorocarbon are within
`the scope of the present invention. It is, however, pref-
`erable to use multiple extraction techniques to obtain
`significant quantities of PC.
`5.1 Partition Coefficient of PC and PE in Mixed
`Acetonitrile-Hexane Solvent
`
`The partition coefficient of PC in acetonitrile-hexane
`solvent is a function of both temperature and total phos-
`phatide concentration. The linear temperature depen-
`dency of the partition coefficient is clearly shown in
`FIG. 1. As the temperature is increased, the coefficient
`increases, indicating an increased amount of PC in the
`acetonitrile phase. The partition coefficient decreases,
`however, as total phosphatide concentration increases,
`indicating the PC:PC molecular interaction in the hex-
`ane layer.
`PE, on the other hand, has a much lower partition
`coefficient in acetonitrile-hexane solvent, as shown in
`FIG. 2. Although the partition coefficient shows a di-
`rect linear relationship with temperature, as in the case
`of PC, there is very little, if any, measurable concentra-
`tion dependence on the partition coefficient for PE.
`This difference in partition coefficients enables one to
`calculate the selectivity of PC over PE in the acetoni-
`trile phase. FIG. 3 shows an inverse relationship be-
`tween selectivity and temperature. Selectivity is highest
`at lower phospholipid concentrations at a given temper-
`ature. An approximately 2% phosphatide system at 25°
`C. demonstrates a high selectivity, over 200, favoring
`the distribution of PC over PE in the acetonitrile phase.
`Once the partitioning of the phosphatides is com-
`plete, the acetonitrile can be readily removed by meth-
`ods known to those skilled in the art, such as by evapo-
`ration under vacuum to reduce the boiling temperature
`and minimize thermal decomposition of the PC. The PC
`so obtained is sufficiently pure to be used as such, or it
`can be further purified byredissolving in a minimum
`amount of hexane and precipitated with cold acetone to
`remove any traces of NLs. In accordance with this
`process, a product having a purity greater than 98% can
`be obtained.
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`In a preferred embodiment, the evaporation of the
`acetonitrile is stopped before all of the solvent has been
`removed. As evaporation proceeds, the limited solubil-
`ity capacity of the diminishing volume of acetonitrile
`for PC causes PC to oil out. It has been found that a PC
`preparation containing less contaminating PE can be
`obtained by isolating the oiled-out fraction before the
`evaporation of the acetonitrile is complete. The amount
`of acetonitrile that must be evaporated is dependent
`upon the quantity of PC present but typically ranges
`from about 75 to about 95% of the initial volume.
`Where more PC is present, less acetonitrile need be
`removed before the PC begins to oil out, and vice-versa.
`In any event,
`the PC ultimately obtained by this
`method contains less contaminating PE than does PC
`from the process wherein all of the acetonitrile is re-
`moved, although in either case the percentage of PC in
`the product exceeds 95%. For this reason, the workup
`of an oiled-out phase is preferred for any of the pro-
`cesses herein described in which a process step involves
`the evaporation of acetonitrile from a PC solution.
`The differences inpartition coefficients are sufficient
`to yield high purity PC after one extraction. The abso-
`lute quantity of PC extracted into the acetonitrile per
`run is, however, low and multiple extractions and/or
`excess amounts of acetonitrile are normally required
`before a significant percentage (i.e., about 50% or
`greater) of the PC originally present in the sample are
`transferred from the hexane phase. A number of multi-
`‘ple extractions, on the order of five to thirty, and an
`' excess of acetonitrile of about ten to twenty times the
`volume of hexane are particularly advantageous em-
`bodiments of the procedure.
`The operating temperature may vary over a wide
`range, however,
`the most advantageous results are 35
`achieved when the temperature is within the range of
`about 0°—65° C., preferably 25°—40° C. The process may
`be utilized at a higher temperature, when under pres-
`" sure in a continuous extraction mode. At higher temper-
`atures, however, care must be taken to minimize the
`':. decomposition of PC by limiting the time that it is ex-
`’ posed to temperatures greater than about 50° C. This
`can be achieved in several ways, either by limiting the
`amount of oxygen present in the system, by maintaining
`the process in a dark environment, or by the addition of 45
`small amounts of antioxidants such as butylated hy-
`droxytoluene or tocopherols or a combination thereof.
`6. EXAMPLES
`
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`with a Soxhlet extractor (500 mL capacity) is suited for
`laboratory use.
`After one hour or more of reflux and after the equiva-
`lent amount of acetonitrile cycling through the extrac-
`tor exceeds 20 liters, the unit is put in distilling mode
`with a vacuum ranging from 10 mm Hg to such reduced
`system pressure that allows the acetonitrile to boil at a
`temperature below 40° C. The residue of the evaporated
`acetonitrile, 50 g, 68% NLs, 30% PC, 1% PE is dis-
`solved in 100 mL of hexane by warming the mixture to
`40° C. to expedite dissolution, whereupon, 2 liters of dry
`acetone are added. The mixture is warmed until a solu-
`tion is formed. The solution is then filtered and cooled
`to 4°-8° C. and maintained at 4°—8° C. for 12 hours, with
`stirring. The solution is filtered with suction in a Buch-
`ner funnel. The filter cake is washed with cold fresh
`acetone (200 mL) and pumped dry in a dessicator under
`vacuum (10 mm Hg) yielding a residue of l5—20 g. The
`residue is redissolved in 50 mL of hexane and precipi-
`tated with 1 liter of cold acetone as above, to obtain
`sample of egg derived PC weighing 10 grams, at greater
`than 95% purity.
`Alternatively and preferably, the purification proce-
`dure may be stopped before all of the acetonitrile has
`been removed, and an oiled-out fraction may be recov-
`ered and processed to obtain an even more pure PC
`preparation (see Section 5). In a typical run, when about
`500 mL of acetonitrile remain in the rotoevaporator
`unit, an oiled-out fraction weighing about 45 g may be
`present.
`This fraction may be removed and dissolved in 100
`mL of hexane by warming the solution to 40° C., at
`which point 4 liters of dry acetone are added. The warm
`solution is then filtered hot and cooled slowly to 4°—8°
`C. and maintained at that temperature for 12 hours with
`stirring. The solution is then filtered under reduced
`pressure in a Buchner furmel, and the filter cake is
`washed with 200 mL of cold acetone and dried in a
`
`dessicator under a 10 mm Hg vacuum. The result is a
`15-20 g residue which is redissolved in 50 mL of hexane
`and precipitated with 1
`liter of cold acetone as de-
`scribed above. A PC sample weighing about 9 g with a
`purity of greater than 95% may thus be obtained.
`Similar treatment of an oiled-out fraction before com-
`plete removal of the acetonitrile in Section 6.1.2 below
`is also preferable.
`
`The following examples serve to illustrate specific
`embodiments of the present invention but do not limit
`the scope thereof.
`
`6.1 Phospholipid Purification Using Acetonitrile as
`Solvent
`
`6.1.1 Direct Extraction of Phospholipids from Egg
`Yolk Using Acetonitrile
`
`Ten egg yolks from Grade A large hen eggs (total
`weight 200 3). after lyophilization or nitrogen-drying to
`remove water, are subjected to extraction with one liter
`of acetonitrile in a Soxhlet extractor to extract the con-
`tained phospholipids. Because the normal boiling point
`of acetonitrile is 80° C., and since the extracted phos-
`pholipids should not be subjected to such high tempera-
`tures to avoid thermal decomposition,
`the system
`should be under vacuum so that acetonitrile can be
`vaporized at a temperature below 50° C., preferably 40°
`C. A Buchi Rotoevaporator Model EL-130 equipped
`
`50
`
`6.1.2 Direct Acetonitrile Extraction of Phospholipids
`from Acetone-Washed Egg Yolks
`
`55
`
`65
`
`Ten egg yolks (total weight approximately 200 g on a
`wet basis or, approximately 100 g on a dry basis) are
`blended in a Waring blender or homogenized in a Poly-
`tron homogenizer (Brinkman) with 400 mL of dry ace-
`tone at room temperature, cooled to 0°-5‘ C. for 4 hours
`and filtered with suction in a Buchner funnel with 2
`Wattman Nbl papers; the filtrate is discarded. The filter
`cake is again blended or homogenized, as above, with
`500 mL of dry acetone, the mixture is cooled to from
`— 10° to —5° C. for 2-24 hours, filtered as before and
`the filter cake dried in vacuo. The residue is placed, as
`. above, in a Soxhlet extractor and the PC is extracted
`under vacuum with an equivalent 1-50 liters, preferably
`20 liters, of acetonitrile for 1-20 hours at 10 mm of Hg
`and at a bath temperature of 40° C. The residue of evap-
`oration of the acetonitrile (12 g) is dissolved in 20 mL of
`hexane and precipitated with 400 mL of acetone, as
`
`000008
`
`000008
`
`
`
`4,714,571
`
`9
`described above, yielding a sample of PC weighing 10
`grams, at greater than 95% purity.
`
`6.1.3 Purification of Egg Yolk Derived PC with
`Acetonitrile After Initial Extraction of Phospholipids
`by Conventional Methods
`
`5
`
`10
`Twenty grams of the oiled-out layer (which in a rep-
`resentative run contained about 68% NLs, 30% PC and
`0.1—1% PE) is placed in a supercritical CO2 extracting
`unit and extracted at a presure above 1,071 pounds per
`square inch (psi), preferably at 5,000 psi and at a temper-
`ature above 31° C., preferably at 40° C., at which point
`the supercritical CO2 has a density of about 0.94 g/mL.
`The flow rate through the system is maintained at about
`0.1-1 liters/minute, with a total supercritical CO2 to
`oiled-out sample ratio of from about 100 to 200, prefera-
`bly 150. Preferably, from about 1 to about 5 percent
`ethanol may be added to the supercritical C02. The
`added ethanol renders the supercritical CO2 more polar,
`thereby improving cholesterol extraction. About 4.5 g
`of residue, which typically contains more than 95 %
`pure PC, with about 0.2% PE and 1.2% cholestrol, can
`be removed from the extractor once extraction is com-
`plete.
`In another embodiment in which supercritical CO2
`extraction precedes acetonitrile extraction, 10 g of ly-
`ophilized egg yolk powder are extracted with the CO2
`at a temperature above 31° C., preferably at 40° C. and
`at a pressure above 1,071 psi, preferably at 5,0()0 psi,
`with a flow rate of 75 g of CO2 per minute. The eluant
`is monitored at 205 nm, and the extraction is stopped
`after the absorbance falls below 0.1 0.D. units. The
`extract, typically containing about 3 g of oil (triglycer-
`ides and cholesterol), is discarded. The remaining resi-
`due which generally weighs about 5.3 g, is extracted
`with two 50-mL portions of chloroform-methanol (1:1),
`at which point 45 mL of water are added to the com-
`bined extracts to form a biphasic system, and the lower
`chloroform layer is evaporated to dryness.
`The resulting residue is dissolved in 100 mL of hex-
`ane, placed in a separatory funnel and extracted with
`fifteen 100-mL portions of acetonitrile that has been
`preequilibrated with hexane. The combined acetonitrile
`extracts are then evaporated down to a 25-50 mL vol-
`ume, and the oiled-out fraction is removed and dried in
`a dessicator. This procedure typically yields about 1.6 g
`of residue which contains more than 95% PC. Contami-
`nants generally consist of less than 1% PE, 0.5% tri-
`glycerides and 0.5% cholesterol.
`
`6.2 Phospholipid Purification at Different Scale Levels
`and Using Countercurrent Extraction
`
`6.2.1 Large Scale Purification of Phosphatides from
`Chicken Egg Yolks
`
`The fresh yolks of 60 jumbo eggs (total weight ap-
`proximately 3600 g) are thoroughly blended with 2
`liters of dry acetone at 2° C. to form a suspension. The
`resulting suspension is cooled for 2 hours at 4°-8° C.,
`and then alternately centrifuged at 0°-4" C. for 10 min-
`utes at 8000 rpm (9000>< g) or filtered with suction in a
`Buchner funnel.
`
`The resulting pellet or filter cake is re-suspended in 2
`liters of dry acetone at 2° C. and the above cooling and
`centrifugation or filtering steps are repeated.
`The resultant mixture is then filtered with suction and
`the precipitate dried in a rotary evaporator under re-
`duced pressure to remove any residual acetone.
`The egg yolk derived phospholipids are further ex-
`tracted in three stages with a total of 2 liters of chloro-
`form and methanol (1:1 volumetric ratio) divided into 3
`equal portions. To the combined extracts, 900 mL of
`water are added to form a biphasic system. After allow-
`ing this extraction mixture to stand for 15 hours, the
`
`10
`
`15
`
`Ten egg yolks (total weight approximately 200 g, wet
`basis) or the dry powder therefrom, (total weight ap-
`proximately 100 g) are first washed twice with two 400
`mL portions of dry acetone, as described in Example
`6.1.2. The phospholipids are then extracted from the
`resulting pellet with two 250-mL portions of a mixture
`of chloroform-methanol solvent in a 1:1 volumetric
`ratio, at room temperature using mechanical mixing or
`homogenizing. The mixture is filtered in a Buchner
`funnel with suction and the filtrate is placed in a separa-
`tory funnel to which 225 mL of water is added to form
`a biphasic system. The flask is gently swirled and the
`phases allowed to separate. The lower chloroform
`phase is rotoevaporated to dryness under vacuum of 1
`mm Hg at 35° C., carefully avoiding foaming. The resi-
`due (approximately 25 g) can be treated in either of two
`ways:
`(1) By acetone precipitation, as above (i.e., dissolved
`in 40 mL hexane and precipitated with 800-1000 mL of
`cold acetone at from 0°-5° C.). This fraction contains
`the phosphatides. Its approximate composition is typi-
`cally 70% PC; 15% PE; 5% NLs; 5% PS; and less than
`1% lyso-PC (if the eggs are fresh). These phosphatides
`can be dissolved in a hydrocarbon and placed in a coun-
`tercurrent extraction device using acetonitrile as the
`counter-solvent, as described below. Altemately, the
`phosphatides may be dissolved in hexane (30 g phospha-
`tides per 100 mL solvent). This solution is then mixed
`with 4 liters of acetonitrile. The PE, and approximately
`5 of the PC separates out of the solution as an oil