115)
`United States Patent
`4,814,111
`[11] Patent Number:
`
`Kearnset al. [45] Date of Patent:~Mar. 21, 1989
`
` *
`
`[75]
`
`[52] U.S. Ch. weeceescssessescssescesseeeeees 260/403; 260/412.4
`[54] PROCESS FOR PURIFICATION OF
`[58] Field of Search ..............cccceeees 260/403, 412.4
`PHOSPHOLIPIDS
`[56]
`References Cited
`Inventors:
`John J. Kearns, Princeton; Paul A.
`U.S. PATENT DOCUMENTS
`Tremblay, Mercerville, bothof N.J.;
`Raymond J. Robey, Macungie;
`4,466,923
`8/1984 Friedrich oes 260/412.4
`Swaminathan Sunder, Allentown,
`Primary Examiner—J. E. Evans
`both of Pa.
`Attorney, Agent, or Firm—Richard A. Dannells, Jr.;
`[73] Assignee: Air Products and Chemicals, Inc.,
`William F. Marsh; James C. Simmons
`Allentown, Pa.
`[57]
` .
`ABSTRACT
`The portionofthetermofthis patent
`[*] Notice:
`subsequent to Dec. 22, 2004 has been—_A processfor the separation and purification of individ-
`disclaimed.
`ual phospholipids, especially phosphatidylcholine or
`taini
`bers
`of
`the
`sub-class
`of
`phosphatid
`Bil
`App
`21] Appl. No.: 928,508
`lecithin and phosphatidylethanolamine, from mixtures
`ay.
`containing members of
`the sub-class of phosphatides,
`[22] Filed:
`incorporating methods of solvent extraction appropri-
`Nov.7, 1986
`sgt
`ate to the scale of the sample and utilizing an acetoni-
`Related U.S. Application Data
`trile, acetonitrile-hydrocarbon, or acetonitrile-fluoro-
`Division of Ser. No. 698,668, Feb. 6, 1985, Pat. No.
`carbon solvent, which exhibit differential solubility
`9eaeenien 1984shundawed of Ser. No.
`properties towards the individual phospholipids.
`[52] Unt, C14 oneecesscscseseseccssesssseeseaeees C11C 1/00
`4 Claims, 3 Drawing Sheets
`
`[60]
`
`AcetonitrileHexane
`
`0.
`
`0.30.
`
`° oO
`
`2 6
`
`CoefficientPC 40
`PcPartition
`
`begend
`@ 1% PC in 2%. Phosphatides
`© 2% PC in 4% Phosphatides
`O 4% PC in 8% Phosphatides
`A 8% PC in 16% Phosphatides
`
`50
`
`60
`
`70
`
`Temperature °C
`
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`

`

`Coefficient 0.0
`
`AcetonitrileHexane
`Partition
`
`PC PC © nNoO
`
`0.10
`
`
`US. Patent—Mar. 21, 1989 Sheet1 of 3 4,814,111
`FIG.
`1
`
`
`
`Legend
`@ 1% PC in 2% Phosphatides
`O 2% PC in 4% Phosphatides
`O 4% PCin 8% Phosphatides
`A 8% PC in 16% Phosphatides
`
`0.
`
`0.30
`
`Oo
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`of
`
`Temperature °C
`
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`

`

`
`
`US. Patent—Mar. 21, 1989 Sheet 2 of 3 4,814,111
`
`
`
`FIG. 2
`
`1%
`0 2%
`O 4%
`
`Legend
`PE in 2% Phosphatides
`PE in 4% Phosphatides
`PE in 8% Phosphatides
`PE ini6% Phosphatides
`
`
`PE]Acetonitrile—
`Coefficient[PE]Hexane
`
`
`
`
`
`Partition
`
`A 8%
`
`20
`
`30
`
`40
`
`50
`
`60
`
`70
`
`Temperature Ke
`
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`

`

`
`
`US. Patent—Mar. 21, 1989 Sheet 3 of 3 4,814,111
`
`
`
`FIG. 3
`
`240.
`
`220
`
`Legend
`e 2% Phosphatides
`O 4% Phosphatides
`O 8% Phosphatides
`A 16% Phosphatides
`
`)Pc )PE
`Partition
`
`CoefficientPartition
`
`Selectivity
`
`
`
`oefficient
`
`(c(
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`70
`
`Temperature T
`
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`

`

`1
`PROCESS FOR PURIFICATION OF
`PHOSPHOLIPIDS
`
`4,814,111
`
`2
`teaches the novel use of a solvent selected from the
`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 advantageous in 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,
`knownas lecithin, are membersofthe 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 numerousapplications
`in the petroleum industry. Because of its widespread
`occurrencein 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 ofthe physical properties and
`functional characteristics of PC.
`Purified egg phospholipids are currently used as a
`starting material to synthesize other compounds such a8
`glycerophosphocholine; saturated, unsaturated, single
`and mixed fatty acids, phosphatidylcholines, phos-
`phatidylethanolamines, phosphatidylglycerols, phos-
`phatidylserines, phosphatidic acids, and diether lipids,
`etc.
`
`This application is a division of application Ser. No.
`698,668, filed Feb. 6,1985, now U.S. Pat. No. 4,714,571
`which in turn is a continuation-in-part of Ser. No.
`579,535, filed Feb. 13, 1984 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 Yoik 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
`6.2.3 Gram Scale Purification of Egg Yolk Derived
`PC and PE
`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 Plant Scale Purification of Egg Yolk
`Derived PC and PE by Extraction Using Coun-
`tercurrent Reciprocating Plate Karr Column
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`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 55
`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”), phosphatidyiserine (“PS”) and phos-
`phatidylglycerol (“PG”) groups.
`Particular embodiments ofthis invention incorporate
`various knownsolvent-based separation methods using
`the solvent systems here disclosed to be mosteffective
`in this novel application. Specific phospholipids can be
`extracted in high purity from mixtures of phospholipids 65
`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
`
`60
`
`2.2 Phospholipid Purification
`At present, high purity PC is typically obtained by
`time consuming, expensive methodssuch 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 form of chromatography is then used to sepa-
`rate the individual lipid components. HPLC and flash
`chromatography onsilica gel or alumina represent the
`state of the art in chromatography. For example, Jun-
`galwala et al. [Biochem. J. 155:55 (1976)] have de-
`scribed HPLCin 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 andpurification 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,
`
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`

`3
`and ethanol-water-hydrocarbon. These systems, how-
`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 CO. 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 Heuschens
`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 ofthe uniquedifferential 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 orthe 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 chromatographyfor the purification of PC and
`PE derived from egg yolks and for the partial purifica-
`tion of PC, PE, PS, PG andcardiolipids (““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 COQ2, chloroform-methanol and
`acetonitrile or acetonitrile-hydrocarbon solvents. These
`extractions take advantage ofthe solubility differentials
`of phospholipids in these solvents. By altering the num-
`ber.of the extraction steps, a purification scheme may be
`designed that is best suited to the phospholipid mixture
`
`20
`
`25
`
`35
`
`45
`
`50
`
`55
`
`65
`
`4,814,111
`
`4
`in question and the ultimate degree of separation and
`purity desired.
`Equipment such as extractors, filter units, evapora-
`tors, crystallization vessels or countercurrentliquid/liq-
`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 sequenceofsteps, 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 suchfactors 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 dependencyofthe partition coefficient of PC in an
`acetonitrile-hexane system;
`FIG.2 is a graphical representation of the tempera-
`ture dependencyofthe 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 areherein described. Egg
`yolk derived PC has a limited solubility in acetonitrile at
`room temperature (2 grams/liter), while egg yolk de-
`rived PEis 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 increasethediffi-
`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 uponthe solubil-
`ities and/or thepartition coefficients of the solutes. The
`
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`4,814,111
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`15
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`25
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`35
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`40
`
`45
`
`5
`countercurrent distribution method is the method of
`choice for achieving the highest purity in the separated
`phosphatides.
`Thestarting 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).
`Thestarting material of the present inventionis 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 importantto first remove substance
`from the source orstarting material whicheasily parti-
`tion into acetonitrile and, consequently, would raise the
`impurity level of the end-product. For instance, some
`NLscontained 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 sequenceof extractions which includes
`precipitation with cold acetone, extraction with a mix-
`ture of one ofthe paired solvents of the group methy-
`lene chloride-methanol, methylene chloride-ethanol,
`hexane-methanol, hexane-ethanol,
`trichlorofluorome-
`thane-ethanol, chloroform-methanol, or ethanol-diethy]
`ether, or ethanol or methylene chloride alone and pre-
`cipitation with a mixture of hexaneacetone. 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 purestate. Any
`NLs,if still present, can be removed byprecipitation
`with acetone to achieve even greater purity PC. The
`PC so preparedis 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 befreed of any remaining
`NLsby precipitation of the PC with acetone from a
`hexaneor chloroform solution ofthe residue containing
`the PC and NLs.
`
`60
`
`65
`
`6
`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 acetoneseveral times or extracted with
`supercritical CO2 to remove the NLs.
`To obtain PC and PEofgreaterpurity, the phospha-
`tide mixture (prepared accordingtothefirst 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 gramsofsolute per 100
`mL, of solvent, with the preferred embodimentcontain-
`ing an amountofsolute of 2 g/100 mLof 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
`pre-equilibrated in accordance with the present process
`to reduce the loss of the hexane phase. However, such
`pre-equilibration is not necessary to obtain satisfactory
`results.
`Any knownseparation procecuresutilizing the vari-
`ant partition coefficients of PC and PE in acetonitrile-
`hydrocarbon or acetonitrile-fluorocarbon are within
`the scope ofthe 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
`
`Thepartition coefficient of PC in acetonitrile-hexane
`solventis 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 temperatureis 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 lowerpartition
`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 thepartition coefficient for PE.
`This differencein partition coefficients enables one to
`calculate the selectivity of PC over PE in the acetoni-
`trile phase. FIG. 3 shows an inverse relationship be-
`tweenselectivity and temperature. Selectivity is highest
`at lower phospholipid concentrationsat a given temper-
`ature. An approximately 2% phosphatide system at 25°
`C. demonstrates a high selectivity, over 200, favoring
`the distribution of PC over PEin the acetonitrile phase.
`Once the partitioning of the phosphatides is com-
`plete, the acetonitrile can be readily removed by meth-
`ods knownto thoseskilled 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,orit
`can be further purified by redissolving 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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`7
`In apreferred 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 beginsto 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, althoughin either case the percentage of PC in
`the product exceeds 95%. Forthis 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 in partition coefficients are sufficient
`to yield high purity after one extraction. The absolute
`quantity of PC extracted into the acetonitrile per runis,
`however, low and multiple extractions and/or excess
`amounts of acetonitrile are normally required before a
`significant percentage(i.e., about 50% orgreater) of the
`PC originally present in the sample are transferred from
`the hexane phase. A numberof multiple extractions, on
`the orderof five to thirty, and an excess of acetonitrile
`of about ten to twenty times the volume of hexane are
`particularly advantageous embodiments of the proce-
`dure.
`The operating temperature may vary over a wide
`range, however,
`the most advantageous results are
`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
`amountof oxygen presentin the system, by maintaining
`the process in a dark environment, or by the addition of
`small amounts of antioxidants such as butylated hy-
`droxytoluene or tocopherols or a combination thereof.
`6. EXAMPLES
`
`10
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`20
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`25
`
`35
`
`45
`
`4,814,111
`
`8
`with a Soxhlet extractor (500 mL capacity) is suited for
`laboratory use.
`After one hour or moreofreflux and after the equiva-
`lent amountof acetonitrile cycling through the extrac-
`tor exceeds 20 liters, the unit is put in distilling mode
`with a vacuum ranging from 10 mm Hgto such reduced
`system pressure that allows the acetonitrile to boil to a
`temperature below 40° C. Theresidue of the evaporated
`acetonitrile, 50 g, 68% NLs, 30% PC, 1% PEis dis-
`solved in 100 mL of hexane by warming the mixture of
`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. Thesolutionis filtered with suction in a Buch-
`ner funnel. The filter cake is washed with cold fresh
`acetone (200 mL.) and pumpeddryin a dessicator under
`vacuum (10 mm Hg) yielding a residue of 15-20 g. The
`residue is redissolved in 50 mL of hexane and precipi-
`tated with 1 liter of cold acetone as above, to obtain a
`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 point4 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 funnel, and the filter cake is
`washed with 200 mL of cold acetone and dried in a
`dessicator under a 10 mm Hg vacuum. Theresult 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 ofthe acetonitrile in Section 6.1.2 below
`is also preferabie.
`
`The following examples serve to illustrate specific
`embodiments of the present invention but do notlimit
`the scope thereof.
`
`50
`
`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 g), 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
`
`35
`
`60
`
`65
`
`6.1.2 Direct Acetonitrile Extraction of Phospholipids
`from Acetone-Washed Egg Yolks
`Ten egg yolks (total weight approximately 200 g ona
`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 Nb! papers;thefiltrate is discarded. Thefilter
`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 1-50liters, preferably .
`20 liters, of acetonitrile for 1-20 hours at 10 mm of Hg
`and at a bath temperature of 40° C. Theresidue of evap-
`oration ofthe acetonitrile (12 g) is dissolved in 20 mL of
`hexane and precipitated with 400 mL of acetone, as
`
`AKER EXHIBIT 2016 Page 8
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`AKER EXHIBIT 2016 Page 8
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`4,814,111
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`5
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`15
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`25
`
`30
`
`9
`10
`described above,yielding a sample of PC weighing 10
`Twenty gramsof the oiled-out layer (which ina rep-
`grams, at greater than 95% purity.
`resentative run contained about 68% NLs, 30% PC and
`0.1-1% PE) was placed in a supercritical CO2 extract-
`6.1.3 Purification of Egg Yolk Derived PC with
`ing unit and extracted at a pressure above 1,071 pounds
`Acetonitrile After Initial Extraction of Phospholipids
`per square inch(psi), i.e. 5,000 psi and at a temperature
`by Conventional Methods
`above 31° C.,i.e. at 40° C., at which pointthe supercriti-
`Ten egg yolks (total weight approximately 200 g, wet
`cal CO? had a density of about 0.94 g/mL. The flow
`basis) or the dry powdertherefrom, (total weight ap-
`rate through the system was maintained at about 0.1-1
`proximately 100 g) are first washed twice with two 400
`liters/minute, with a total supercritical COto oiled-out
`mL portions of dry acetone, as described in Example 10
`sample ratio of from about 100 to 200, preferably 150.
`6.1.2. The phospholipids are then extracted from the
`Preferably, from about 1 to about 5 percentethanol may
`resulting pellet with two 250-mL portions of a mixture
`be added to the supercritical CO2. The added ethanol
`of chloroform-methanol solvent in a 1:1 volumetric
`renders the supercritical CO2 more polar, thereby im-
`ratio, at room temperature using mechanical mixing or
`proving cholesterol extraction. About 4.5 g of residue,
`homogenizing. The mixture is filtered in a Buchner
`which contained more than 95% pure PC, with about
`funnel with suction and thefiltrate is placed in a separa-
`0.2% PE and 1.2% cholesterol, was removed from the
`tory funnel to which 225 mL of water is added to form
`extractor once extraction was complete.
`a biphasic system. Theflask is gently swirled and the
`In another embodiment in which supercritical CO,
`phases allowed to separate. The lower chloroform
`extraction precedes acetonitrile extraction, 10 g of ly-
`phase is rotoevaporated to dryness under vacuum of1
`ophilized egg yolk powder was extracted with the CO
`mm Hg at 35° C., carefully avoiding foaming. Theresi-
`at a temperature above 31° C., ie. at 40° C. and ata
`due (approximately 25 g) can betreated in either of two
`pressure above 1,071 psi, i.e. at 5,000 psi, with a flow
`ways:
`rate of 75 g of COper minute. The eluant was moni-
`(1) By acetone precipitation, as above (ie., dissolved
`tored at 205 nm, and the extraction was stoppedafter
`in 40 mL hexane andprecipitated with 800-1000 mL of
`the absorbancefalls below 0.1 O.D. units.:The extract,
`cold acetone at from 0°~5° C.). This fraction contains
`containing about 3 g of oil (triglycerides and choles-
`the phosphatides. Its approximate composition is typi-
`terol), was discarded. The remaining residue which
`cally 70% PC; 15% PE; 5% NLs; 5% PS; andless than
`weighed about 5.3 g, was extracted with two 50-mL
`1% lyso-PC (if the eggs are fresh). These phosphatides
`portions of chioroform-methanol(1:1), at which point
`can be dissolved in a hydrocarbon and placed in a coun-
`45 mLof water were added to the combined extracts to
`tercurrent extraction device using acetonitrile as the
`form a biphasic system, and the lower chloroform layer
`counter-solvent, as described below. Alternately, the
`was evaporated to dryness.
`phosphatides may bedissolvedin hexane (30 g phospha-
`The resulting residue was dissolved in 100 mL of
`tides per