United States Patent rlel
`Tremblay et al.
`
`[1 1]
`
`[45]
`
`Patent Number:
`Date of Patent:
`
`4,714,571
`Dec.22,1987
`
`[54] PROCESS FOR PURTFTCATTON OF
`PHOSPHOLIPIDS
`[75] Inventors: Paul A. Tremblay, Mercerville; Jof
`J. Kearns, Princeton, both of N.J,
`[73] Assignee: Ihe Liposome Company, Inc.,
`Princeton, N.J.
`Appl. No.: 698,668
`Filed:
`
`12tl
`
`l22l
`
`Feb.6,1985
`
`[63]
`
`[5 1]
`[52]
`t58l
`
`[56]
`
`CltC l/00
`260/N3i260/412.4
`......2û/403, 412.4
`
`Related U.S. Application Data
`Continuation-in-part ofSer. No. 579,535, Feb. 13, 1984,
`abandoned.
`Irt. Cl.4
`u.s. cl.
`FÍeld of Search
`References Cited
`U,S. PATENT DOCUMENTS
`12/1945
`2,390,528
`t2/1955
`2,727,U6
`7/1957
`2,801,255
`7/1962
`3,ù17,597
`3/1975
`3,869,482
`4,235,793
`11,/1980
`1/1983
`4,367,178
`1/t984
`4,425,276
`6/1984
`4,452,743
`
`Gunther
`Gunther
`
`2û/ß3
`2û/403
`zû/&3
`2fi/4{.3
`
`2ffi/403
`
`FOREIGN PATENT DOCUMENTS
`612t0 5/1979 Japan
`OTHER PUBLICATIONS
`Jungalwala et al., "Biochem. J.", 155, 55-60 (19?6).
`Bott et al., Some Extractions and Separations with Car-
`bon Dioxide with Carbon Dioxide at Near-Critical
`Conditions, in Inte¡national Solvent Extraction Confer-
`ence, Denver, Colorado, Aug. 26-Sept. 2, 1983, pp.
`55G557.
`Brunner et al., Selection of Solvents for Supercritical
`Extraction in l¡rternational Solvent Extraction Confer-
`encq Denver, Co.; Aug. 2GSept. 2, 1983, pp. 558-559.
`Mangold, 1983, JAOCC @ Q)z 226-228.
`himary Examiner-J. E. Evans
`Attorney, Agent, ot Fírm-Pennie & Edmonds
`[57]
`,{BSTRACT
`A process for the separation and purification ofindivid-
`ual phospholipids, especially phosphatidylcholine or
`lecithin and phosphatidylethanolamine, from mixtures
`contâini¡g members of the sub-class of phosphatides,
`incorporating methods of solvent extraction appropri-
`ate to the scale of the sample and utilizing an acetoni-
`trile, acetonitrile-hydrocarbon, or acetonitrile-fluoro-
`carbon solvent, which exhibit differential solubility
`properties towards the individual phospholipids.
`
`44 Claims, 3 Drawing Figures
`
`Leqend
`. t!"h PC ¡n 27o Phospholides
`O Z"/" FC in 47ô Phosphol¡des
`É 4% PC in 89¿ Phosphot¡des
`A 8% PC ìn 157" Phosphot¡des
`
`?o
`
`<-
`
`31 o2o
`
`c
`
`orozo
`
`40 50 60 70
`Tenperolure oc
`
`
`
`NEPN Ex. 2036
`Aker v. Neptune
`IPR2014-00003
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`U.S. Patent Dec.22,l9ï7 Sheet I of 3
`
`4r7l4rí7l
`
`FIG. I
`
`o
`
`Le gend
`PC in 2"/" Phospholides
`PC in 47" Phospholides
`PC in 8% Phosphotides
`PC in 157" Phosphotides
`
`a loA
`O2%
`E4%
`A B%
`
`L
`
`:
`'Ë o¡
`qC
`+g(¡)x
`(Jq)<=
`(JO
`t!I
`
`o.30
`
`o.20
`
`o
`
`c
`.9
`.9
`
`s3
`
`C)
`c
`.9
`
` o.ro
`
`Lo
`o_
`
`o.
`
`010
`
`20 30 40 50 60
`Te mpero lure oC
`
`70
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`U.S, Patent Dec.22,t9r7
`
`Sheet 2 of 3
`
`4,714,571
`
`oD
`
`o
`
`F I G. 2
`Legend
`. 1"/" PE in 2"/" phospholides
`Q Z"t" PZ in 4"/" phospholides
`tr +"¡ Pt in 8% Phosphotides
`A 8% PE in 16% Phospholides
`
`XIOI
`
`90
`
`80
`
`70
`
`o
`'L-
`
`q,c
`ox
`c)I
`o- H50
`
`ooO
`
`UJ
`
`60
`
`(¡)
`
`:e 40
`c)o
`O
`
`30
`
`co =ä
`
`o_
`
`20
`
`lo
`
`o
`
`20
`
`o
`
`Te mperolure
`
`o
`
`C
`
`60
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`U.S. Patent Dec. 22, t987
`Sheet 3 of 3
`FIG.3
`
`4r7l4r57l
`
`Legend
`o 2"/. Phospholides
`O 4% Phospholides
`tr 8"/" Phospholides
`A le % Pnosphotides
`
`o A
`
`o
`
`to 20 30 40 50 60 70
`Te mperolure oC
`
`24
`
`22
`
`180
`
`t60
`
`4
`
`o(L
`
`EC
`.9.9
`.=.9fs
`^o q¡
`IIO ()
`
`l-
`lal^cc
`
`.9.9
`.=.9
`l+
`3E
`(J
`
`:=
`C)
`o)
`c)(t
`
`6
`
`NEPTUNE EX. 2036
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`4,714,57I
`12
`pRocEss FoR prrRrFïcÀrroN oF
`:i"J: ".:it'.'#'.Tf i# ni,ii;äåiil'å"ïn?t;::i:;
`PHOSPHOLIPIDS
`consisting of pentane, hexane, isohexane, heptane and
`octane, and mixtures of hydrocarbons sucb as petro-
`The present application is a continuation-in-part of 5 leum ether or mixtures ol acetonitrile and fluoiocar-
`prior copending application Ser. No. 579,535, filed Feb. bons.
`13, 1984, now abandoned.
`The present invention is advantageous in that it is
`both less time consuning and less costly than other
`,
`TÀBLE OF CONTENTS
`*o*o methods'
`l. Field of the Invention
`2. Background of the Inventio¡
`2. BACKGROUND OF THE INVENTION
`2.1 Phospholipids
`2.1 phospholipids
`2.2 Phospholipid Purification
`Phospholipids, including PC, which is commonly
`3. Su--ary of the Invention
`rs HoII3s lecithin, ar9 m.embers of the class of phospha-
`4. Srief D;cription of the Figures
`- tides' They are of signiltcant commercial importance
`5, Detailed Description of thJ Invention
`5.1 partition Cocffrcient of pC and pE in Mixed because of thejr wetting. and emulsifying properties.
`They are widely used as ingredients in food products,
`Acetonitrile-Hexane Solvent
`6. Examples
`cosmetics, pharmaceuticals, insecticides, paints, plaslics
`6.1 Phäpholipid Puriflrcation Using Acetonitrile Sol- ,O and,textiles, and have also found numerous applications
`-
`in the Petroleum industry. Because of its widespread
`vent
`6.1.1 Di¡ect Extraction of phospholipids from Egg occturence il"".t."3 PC is known colloquially as "na-
`yolk Using Acetonitrile
`ture's emulsifier." The occutrence of PC as a compo-
`ospholip nent of cell membraneq hq been the subject of much
`6.1.2
`ids
`25 recent scientific research, Emphasis in this research has
`6.1.3
`pC with beenonthedetenninationofthephysicalpropertiesand
`Acetonitrile After Initial Extraction of Phospho ful:tioml- cha¡acteristics of PC.
`lipids by Convcnrional Methods
`Pu¡ified egg phospholipids are currently used as a
`6.1.i Rcmoval of Egg Yolk Neutral Lipids by Su- slarting material to synthesize other compounds such as
`-With
`30 gly-cerolhosphocholine; saturated, unsaturated, single
`percritical COz ñIore or after Extraition
`and mixed fatty acids, phosphatidylcholines, phos-
`Acetonitrile
`6.2 Phospholipid Purification at Different Scale Lev- phatidylethanolamines, phosphatidylglycerols, phos-
`els and Using Countercurrent Extraction
`phatidylserines, phosphatidic acids, and diether lipids,
`6.2.1 Large Scale PurifÌcation of Phosphatides etc.
`Egg Yolks
`35
`2.2 phospholipid purilication
`- -frog.chicken
`6.2.2 Microgram and Milligram Scale Countercu¡-
`. At present, high purity PC is typically obtained by
`rent Pu¡ification of Egg Yolk Derived PC and
`PE
`time consuming, expensive methods such as high pres-
`6.2.3 Gram Scale Purification of Egg Yolk Derived sure liquid chromatography (IIPLC) solid-liquid col-
`PC and PE
`û umn ch¡omatography (SLCC), flash chromatography,
`6,2.4 Gram to Kilogram Scale Purification of Egg and lhin layer chromatography (ILC).
`Yolt Derived PC and PE by Extraction UsinÈ
`These methods involve the separation of the lipids,
`Packed Column Method
`typically by solvent extraction or by other solvent-
`6.2.5 Pilot Scale Purification of Egg Yolk Derived based techniques. Neutral lipids can be separated from
`PC and PE by Extraction Using Countercurrenl 45 the phospholipid class by precipitation with cold ace-
`Reciprocating Plate Karr Column
`tone. A forrn of chromatography is then used to sepa-
`rate the individual lipid components. HpLC and flash
`l. FIELD OF THE IIWENTION
`chromatography on silica gel or alumina represent the
`invention relates to a process for the
`The pres€nt
`state of the art in chromatography. For example, Jun-
`production of high-purit¡ individual phospholipids 50 galwala et al. [Biochem. J. 155:55 (1976)] have de-
`from mixtures thereof, by means of separation tèch- scribed HPLC in silica-gel, using a mixture of acetoni-
`oiques utüizing solvents novel for this purpose. More
`eluant, to separate phos-
`specifically, this invention concerns a process for sepa-
`omyelin, These methods,
`rating and purifymg phospholipids, especially those,of
`faster than conventional
`the sub'class of phosphatides, including but not limited 55 column chromatography, permit higher solvent flow-
`to
`phos- rates through the column (throughprit) than are attain-
`ph
`tetha- able with slow conventional column chromatography.
`no
`phos- Chromatographic means are, however, generally slow
`ph
`
`this invention incorporate 60
`separation methods using
`losed to be most effective
`in this novel_a.pplication. Specifrc phospholipids can be most valuable and expensive compounds.
`e¡tractcdinhighpurityfrommixturesofphospholipids U.S. Pat. No.2,651,646, issueå ro Goldsmith, dis-
`derived from egg yolks, soya beans or other sources 65 closes a method of purifying monoglycerides from d!
`because of the different degrees of solubility of the glycerides, using riuttipie iolvent-systems including
`phospholipids in the solvent used. This invention methanol-hydrocarbon, methanol-waier-hydrocarbonl
`teaches the novel use of a solvent selected from the and ethanoi-water-hydrocarbon. These systems, how-
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`4,714,57r
`34
`ever, do not exhibit highly selective solubjlization char- in question and the ultimate degree of separation and
`acteristics towârds phospholipids, as do the acetonitrile purity desired.
`and acetonitrile-hydrocarbon mixtures taught in this
`Equipment such as extractors, filter units, evapora-
`invention. Other solvent systems, such as ethylene gly-
`tors, crystallization vessels or countercurrent liquid,4iq-
`col-hydrocarbon, tested in the course of research lead- 5 uid extraction devices can be employed herein, These
`ing to this invention, also lacked the discovered selec- are state of the art unit operations currently used
`tive solubilization cha¡acteristics ofthe acetonitrile and throughout the industry. Estimations show that the
`acetonitrile-hydrocarbon solvents.
`capital costs for these methods are less than for chro-
`Another purificatioa approach that has been taken, malographic methods for the purification of egg yolk
`and one wñich avoids the toxicity and flammability l0 derived PC an{ PE.
`problems associated with many oiganic solvents, in- Q" P9 obtained is as pure as that obtained by con-
`volves the use of supercritical fluids, especially super- ventional column chromatography' The system operat-
`critical COz. A supeicritical fluid is produced-by iub-
`ing parameters, such as the sequence ofsteps, the quan'
`tity ofsolvents used, and the tempelature, can be varied
`jecting certain gasies to pressure andìemperaturè "on-
`-ditionã
`such thaithe gas exists in a high density state and l5 to obtain a ra¡ge of PC purities. The desired degree of
`has in general the flq-w properties oíg rer, úut cannot purity depends on such factors as the ultimate use ofthe
`Í$sume-a true liquid ro'rm. t" an aplropriate system, PC-and cost ltmitations'
`,. T3" T:,h9S here taught ar-e suitable for phospho-
`supercritical fluiás such * rop"r"titì"i coz can bé
`lipid purif^tcation at all scales-from the milligram level
`*èd fo, extraction and purificàtioo prrrpor"r. For * ^^
`emple, in u.s. pat. No. #alna Heigel and Hueschens æ
`19,r!L.,91K9: f.:1hoo., that is, from laboratory scale to
`asôtoie the use of supercritical co; in an extraction mclustnal quantlty'
`system to remove oily components from crude soy
`4. BRIEF DESCRIpTION OF THE FIGURES
`lecithin preparations, thereby obtaining partially puri-
`,*_ ______r :__--
`The present inventio¡r may be more readily under-
`fied lecithin.
`25 stood by reference to the following figures, wherein
`3. SUMMARY OF TIIE INVENTION
`FIG. 1 is a graphical representation of the tempera-
`Alrernate means of phospholipid purification have :ï:::f*fl:v^-"-f the partition coeflicient of PC in an
`been investigared, and methó¿s dåvelóped based on the *:t"åT'ilti:::,-J^tl,ti:-^-^-¿^+:^-
`^r .r^ ,
`discovery ofthe unique differentiar sorublitv prop".'-î ,o ,".:i:'o!"äåöTi,i3r'triÏåi:it:ü.lil"åïËtf'fi
`of acetonitrils acetonitrile-hydrocarbon m¡xtures and '-
`acetonitrile-fluorocarbon mixtures towards phospholip- -J{d:'il
`;;io-ni..|i;-ü"iåri.'r'rt..; -¿
`"Ë.0ü"¿ representation of the selectivity
`ids, especially as to the separation of PC and PE from
`põ;;;; ÞËìÃ-tire ace^ænirrile phase of an acetoni-
`"f
`."p'osed as a iunction of tempera-
`;:li'",:Tr.n,ktffi#rt"iîïî: illiï;:ffiJ","r'i ,, :il:¡#; 'v't.à,
`laboratory or the industrial scale. It represents a simple,
`efficient, rapid and economical means of producing
`5. DETAILED DESCRIPTION OF THE
`II'MNTION
`purilied phospholipids, particularly PC and PE. The
`process of this invention readily permits the achieve- This invention is based on the discovery of the unex_
`ment of greater than 9OVo Pure coûPonents. PuÌitiï ,{t pected selective solubility differences of ihospholipids
`greater than 98/o can also be attained without signifi- i""f, us ÞC *¿ pf, tt" major groups of pìospiolijids,
`cant additional difliculty. This invention enables the
`in acetonitrile. foi purpoies -of iiustáion, emliodi-
`production oflarge quantitie_s ofhigh purity phospho- ments ofthe invention aô applied to the purifrcation of
`lipids which are needed for the preparation of pharma- egg yolk derived pC and pÈ-are herein dìscribed. Egg
`ceuticals. The large volume of high purity phospholie ¿S yãIf ¿erived pC has a limited solubility in acetonitrileã
`ids that can te easily and cheaply produced via the
`ioom temperature (2 grams,4iter), wlile egg yolk de-
`proc€ss of this invention will also permit their broader rived pE is practicaily-insoluble il this solvãit at room
`use in other commercial applications where less pure temperature:
`preparations have heretofore sufficed, thus resulting in
`In accordance with the present invention, pC and pE,
`concommitant improvements in the quality of those SO of highest purity are readily and economically isolated
`products'
`fromã mixìure-of phosphaiides using acetoniirile and-
`The process of this i¡vention eliminates the use of
`/or an acetonitrile-hydiocarbon solJent system or an
`column ch¡omatography for the purification of PC and acetonitrile-fluorocarbon solvent system. Tire preferred
`P€ derived from egg yolks and forthe partial purifica- sou¡ce material for mixtures of phosphatidês in the
`tionofPC,PE,PS,PGandcardiolipids("Cl's")from 55 present i¡vention is egg yolks oi.oy" beans. Such
`other-sources. Separation and purification ofphospha- õource material proviðes a mixture óf phosphatides
`tides from sources other than egg yolks or soya beans consisting primarily of pE and pC. The
`-use ól other
`may be less complete because other source materials source mãtèrial, suèh as plant or animal tissue extracts,
`nay contain more co'nplex mixtures of phosphatides is within the scope of this invention; however, some
`which increases the dillïculty of separation.
`60 sources yield comþlex mixtures which increase the difli-
`The process of this invention is based on multiple culties of purifying the individual phosphatides. It has
`solvent extractions performed sequentially using ace- now bee¡r discovèred that acetonitrilè preferentially
`tone or supercritical CO2, chloroform-methanol and extråcts PC, whereas PE, being insoluble in acetonitrile,
`acetonitrile or acetonitrile-hydrocarbon solvents. These remains in the hydrocarbon phase of the two-solvent
`extractions take advantage ofthe solubility differentials 65 system. Extraction wjth acetonitrìle or an acetonitrile-
`of phospholipids in these solvents. By altering the num- hydrocarbon solvent system can be employed in various
`ber ofthe extraction steps, a purification scheme can be
`separation techniques which a¡e based upon the solubil-
`designed that is best suited to the phospholipid mixture ities andlor the partition coefficients ofthe solutes. The
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`4,714,571
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`countercurrent distribution method is the method of
`Alternatively, the egg yolk, preferably in dried form,
`choice for achieving the highest purity in the separated may be directly exhaustively extracted with acetoni-
`phosphatides.
`trile. The acetonitrile extract is then evaporated to dry-
`The starting material of the process herein is a mix- ness and the residue, consisting mostly of PC and NLs,
`ture containing PC and one or more other phospbatides 5 is washed with acetone several times or extracted with
`of the group PE, PS, PG, CL, sphingomyelins (SPLs) supercritical Cz to remove the NLs.
`and phosphatidic acid (free form).
`To obtain PC and PE ofgreater purit¡ the phospha-
`The starting material of the present invention is pref- tide mixture þrepared according to the first protocol) is
`erably obtained from egg yolks or soya beans since, in
`first dissolved in a suitable hydrocarbon solvent, prefer-
`addition to the reasons indicated earlier, they are l0 ably hexane. The solute concentration can vary over a
`readily available and inexpensive. The source materials broad range, but the most advantageous results a¡e
`can bc treated with any known methods to remove achieved when the solute concentration is within the
`extraneous substances which might hinder the purihca- range of about 0,5 to about 20 grams of solute per 1@
`tion of the phosphatides, such as proteins, carbohy- -- mlofsolvent,withthepreferredembodimentcontain-
`lr ing an añount of solute of 2 g/lffi mL of solvent when
`drates, and neutral lipids (NLs).
`In a typical composition of hen's egg yolk, the dry
`operating between 2O'J5" C. A higher solute concen-
`weight consists of lSVoPC,4O7a NIx, and 5/o PE, and tration can 6" ¡¡¿i¡t¡ined at higher temperatures.
`therestofthecomponentscompriseproteinsandcarbo- Before partitioning the PC from a phosphatide mix-
`hydrates.
`ture, the acetonitrile and hydrocarbon a¡e desirably
`^^
`.Although this ürvention discloses several possible zu preequilibrated i¡ accordance with the present process
`protocols for the purilication of PC and/or PE from
`to reduce the loss ofthe hexane phase. Howeveì, such
`egg yolk, all methods a¡e based on the discovery of
`preiquilibration is not necessÍuy to obtain satisfactory
`acetonitrile's unique solubility properties toward phos- results.
`pholipids.
`.1 Aay known separation procedures utilizing the va¡i-
`It is especially important to first remove substances " ant partition coefficients of PC and PE in acetonitrile-
`from the source or starting material which easily parti- hydrocarbon or acetonitrile-fluorocarbon are within
`tion into acetonitrile and, consequently, would raise tbe
`the scope of the present invention, It is, however, pref-
`impurity level of the end-product. For instance, some erable to use multiple extraction techniques to obtain
`NLs containcd in cgg yolls easily partition into acetoni- ,O signifrcant quanliliss qf pQ.
`trile. Therefore, NLs and pigments, such as color carot- --
`5'l Partitio¡ CoefÏicient of PC and PE in Mixed
`etroids and choiestc¡ol, -ãy u" firit removed from the
`source material in accordance with the present inven-
`AcetOnitrile-Hexane Solvent
`tion tårough â sequence of extractions which includes The partition coeflicient of PC in acetonitrile-hexarie
`precipitation with
`,, solvent is a function ofboth temperature and total phos-
`-- phatide concertration. The linear temperature dèpen-
`ture of one of the
`lene chloride-met
`dcncy of the partition coeflicient is ciearly shown in
`hexane-methanol, hexane-ethanol, trichlorofluorome- FIG. 1. As the temperature is increased, the coellicient
`ldiethyl increascs, indicating an increased amount of PC in the
`and pre- 40 acetonitrile phase, The partition coefficient decreases,
`Alterna- however, as total phosphatide concentration increases,
`!ívcly, a dry egg yolk preparation or the oily residue indicating the PC:ÞC molecular interaction in the hex-
`from the extraction of egg yolks with chloroform- ane layer.
`mcthanol or with one of the other solvents could be
`PE, on the
`partition
`extracted with supercritical Coz instead of acetone to 45 coeflicient in
`shown in
`rcmove the NLs and pigments.
`FIG. 2. Altho
`ows a di-
`According to the invention, the phosphatides can be
`rect linear relationship with temperature, as in the case
`firstdissolved in an organic solvent, such ashexane, and ofPC, there is very little, ifany, measurable concentra-
`the PE prccipitated with acetonitrilg or they can be
`tion dependence on the partitiãn coeffrcient for PE.
`directly extracted with acetonitrile, After evaporation 5 ¡ This difference in partilion coefficients enables one to
`ofthe acetonitrile solvent, the remaining phospholipid calculate the selectivity ofPC over PE in the acetoni-
`residue contâins the Pc in a sulficiently pure state. Ary
`lationship be-
`Nf f if still present, can be ¡emoved by precipitation
`vity is hþhest
`with acetone to achieve ever greater purity PC. The
`given teñper-
`PC so prepared is greater than' 95Vo pure with less than 55 ature. An approximately 2Vo phosphatideiystem ai25'
`ZVoPE prcacnt.
`C. demonstiãtes a high seteciitity, over 2õ0, favoring
`The egg yolk can be extracted or first dried and
`rPEintheacetonitrilephasel
`wlsled with cold dry acetone or extracted with super-
`of the phosphatides is com-
`critical Co¿. Thesc e¡traction steps are well known
`be readily removed by meth-
`procedures which removc a large part of the NLs from ) ods known to thos€ skilled in the art, such as by evapo-
`thc egg.yolk solids, leaving most ofthe phospholipids. ration under vacuum to reduce the boiling temþerature
`According to the invcntion, the residue can then be andminimizethermaldecompositionofth-ePC,-ThepC
`exhaustively washed rvitb acetonitrile which will ex- so obtained is sufliciently pure to be used as such, or it
`tract thc PC, leaving insoluble PE. The preparation can be further purified'by.rediisolving in a mininum
`containing the PC can then be freed of any remaining 6s amountofhexa¡¡eandp.eðipitut"dwith-coldacetoneto
`]qlt by precipitation of the PC with acetone from a
`remove any traces of Nr,i. In accordance with this
`hexane or chloroforrn solution ofthe residue containing process, a product having a purity greater than 98Za can
`the PC and NLs.
`be obtaineã.
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`4,714,571
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`In a preferred embodiment, the evaporation of the with a Soxilet extractor (50o mL capacity) is suited for
`acetonitrile is stopped before alì ofthe solvent has been laboratory use.
`removed, As evaporation proceeds, the limited solubil- After one hour or more of reflux and after the equiva-
`ity capacity of the diminishirg volume of acetonitrile lent amount of acetonitrile cycling through the eitrac-
`for PC causes PC to oil out. It has been found that a PC 5 tor exceeds 20 lirers, the unii is pìt in distilling mode
`preparation containing less contaminating PE can be with a vacuum ranging from l0 mm Hg to such reduced
`obtained by iy'lating the oiled-out fraction before the syste' pressure thãt allows the acetonitrile to boil at a
`evaporation of the acetonitrile is complete..Th. *9lnl
`iÉÀp.rut*" u"row ¿lo. c. The residue of the evaporated
`of acetonitrile that must be evaporated is dependent u*.o,¡;rí", SO-si;, 6gVo NLs, 30Zo pC, lVo pE is dis_
`upon the quantity of PC present but typically ranges ¡o ;k;ñ-100;i'of hexanebywarmingthemixtureto
`from about 75 to about 95vo of the initial volumc' 4öõ:.õ;i;;dissolution,whereupon,2litersofdry
`-"'-1.--,
`Where more PC is present, less acetonitrile need be
`removed before the pb begins to oil out, and rri""-r,Ërä ]"^1t91?:::9H' fr" mixture is warmed until a solu-
`lbî.tu Íbl"d; The solution is then filtered and cooled
`In any evenr, the pc-ultimately oLtained by ;hil
`-rc
`,, to. +'-a' C and maintained at 4"-8" c' for 12 hours, with
`method lontains less contami¡ating re than doäs
`from the process wherein all of the acetonitrile is ie- '- stirring. The solution is filtered with suction in a Buch-
`ner funne-I. 'The.hlter cake is washed with cold fresh
`moved, alihough in either case the percentage of PC in
`tbe product exãeeds 95Vo. For thisìeason, i-he workup acetone (200 mL) and pumped dry in a dessicator under
`any of the pro- vacuum (10 mm Hg) yielding a residue of 15-20 g. The
`of an oiled-o
`cesses herein
`ess step invoives 26 residue is redissolved in 50 mL of hexane and precipi-
`tated with I liter of cold acetone as above, to obtain
`PC solution.
`the evaporati
`The differences in partition coellicients are sufficient sample of egg derived PC weighing 10 grams, at greater
`to yield high purity PC after one extraction. The abso- than 957o purity.
`lute quantity of PC extracted into the acetonit.ile per
`Alternatively and preferably, the purification proce-
`nrn is, however, low and multiple extractions atd/or 25 dure may be stopped before all ofthe acetonitrile has
`excess amouûts of acetonitrile are nor¡nally required been removed, and an oiled-out fraction may be recov-
`before.a signiflcant percentâge (i.e,, about 5O/o ot
`ered and processed to obtain an ever more pure PC
`greater) of the PC originally present in the sample are preparation (see Section 5). In a typical run, whãn about
`transferred from the hexane phase-. A numter of multi- -SOO .f of àcetonitrile remain in the rotoevaporator
`ple extractions, on the order of five to thirty, and an 39 unit, an oiled-out fraction weighing about 45 g may be
`-excess of acetonitrile of about ten to twenty times the presetrt.
`yolume of hexane are particularly advantageous em-
`Tïis fraction may be removed and dissolved in 100
`bodimenæ of the procedure.
`rhe operatingiemperarure may vary over a wide iffi r,::Jii:,,tirüä1g""l::J:l:t:åå"Ïfr"Î;#
`range, however, the most advantageous results
`^*-". :s r"irüåãltin"i itì"red úot and cooled slowly to 4'-8"
`achieved when the temperature is \+,ithin the range of - ï'lîl__-,^;-î_';
`at that temperature for 12 hours with
`about 0"-65" c., preferably 25"-q" c. The pro""s, å"1 9g1'*?*^tÍ.
`is tben filtered under reduced
`- be utilized at a'higher táperature, when'under pä-
`ÌlTï^--9: T"- Tt_o]"o
`fT:i," i." å"chner funnel, and the fllter cake is
`sure in a continuorñ extraction modé. At higher reá;;-
`'' a.tures, however, care must be taken to mirimize'tie ,^ Y*l"q with 200 mL of cold acetone and dried in a
`:, decompositioo oî pc by limiting the time that it is e¡- { des{:ator under a 10 mm Hg vacuum. The result is a
`' posed io temperatures'greater than about 50. C, This 15-20gresiduewhichisredissolvedin50mLofhexane
`-with I lite¡ of cold acetone as de-
`õan be achieved in seveial ways, either by limiting the and precipitated
`amoutrt of oxygen present in the iyrt"-, by maintalning scribed above, A PC sample weighing about 9 g with a
`the process ¡i ã ¿ait environrnentl or Ui túe addition o-f ,. Pu.ity of greater tban 95Vo may thus be obtained.
`smail amounts of antioxidants such as butylated hy- +r Similartreatmentofanoiled-outfractionbeforecom-
`droxytoluene or tocopherols or a combinatiõn thereoî plete removal ofthe acetonitrile in Section 6.1.2 below
`is also Preferable'
`6. ExAMpLEs
`6'1'2 Direct Àcetonitrile Extraction of Phospholipids
`The following examples serve to illustrate spe<'-
`embodiments of the present invenrion but do not fåi: to
`from Acetone'washed Egg Yolks
`the scope thereof,
`Ten egg yolks (total weight approximately 200 g on a
`6.r phosphoripid pu¡incation using Acetonitrle as ;í:ljä'i'i'#lfl?"riåï:i;iff:ål1]. ifJì"ï:
`6.r.r Direct Exrraction orphosphoripids rrom Egg t' äi:L"ä:fftrfi.!?ff*täïflr"'å?:.ï"råi;
`Yolk Using Acetonitrile
`and frltered wi¡h suction in a Buchner funnel with 2
`Ten egg yolks from Grade A large hen eggs (total W.attTan N.bl.P-apers; the filtrate is discarded. The filter
`weight 200 g), after lyophilization orãitrogen--<lî-¡¡ing to
`cake is again blended or homogenized, as above, with
`remóve o,utãi -" rotjécted to extraction futh oã" ñt". 56 500 mL of dry acetone, the mixture is cooled to from
`of acetonitrile in a Soxhlet extractor to extract the con-
`-10" to -5' C. for 2-24 hours, filtered as before and
`tained phospholipids, Because the normal boiling point the filter cake dried in vacuo. The residue is placed, as
`of acetonitrile is 80' C,, and since the extracted phos- . above, in a Soxhlet extractor and the PC is extracted
`pholipids should not be subjected to such high tempera- under vacuum with an equivalent l-50 liters, preferably
`tures to avoid thermal decomposition, the system 65 20 liters,ofacetonitrileforl-20hoursattOmmofHg
`should be under vacuum so that acetonitrile can be andatabathtemperatureof40"C.Theresidueofevap-
`vaporized at a temperature below 50' C., preferably 4O" oration ofthe acetonitrile (12 g) is dissolved in 20 mL of
`C. A Buchi Rotoevaporator Model EL-130 equipped hexane and precipitated with 400 mL of acetone, as
`
`Solvent
`
`NEPTUNE EX. 2036
`
`

`
`Twenty grams of the oiledout layer (which in a rep-
`resentative ¡un contained zboltíS%o NLs, 307¿ PC and
`
`-
`4,714,571
`910
`described above, yielding a sample of PC weighing 10
`grems, at greater than 95Vo punty.
`ó.r.3 purincation orEgg york Derived pc with ,_ Î,1; í:,1T*:åtå""i"Tå,:åt""ï:lti,fr?ïiiå:i:i
`Acetonitrile After Initial Extracrion of Phospholipids 5 ö'*îilö;t;ireferaUty at 5,0@ psi and at a temper-
`by conventional Methods
`ature above 3l' c., preferably at 40'c., at which point
`Terr egg yolks (total weight approximately 20O g, wet
`the supercritical CO2 has a density of about 0.94 grtmL,
`basis) or the dry powder therefrom, (total weight ap- The flow rate through the system is rnaintained afabout
`proximately 100 g) are first washed twice with two 400 0.1-1 liters/mínute, with a total supercritical COz to
`rnl- portions of dry acetone, as described in Example l0 oiled-outsampleratioof fromabout lOOto20O,prefera-
`6.1.2. The phospholipids are then extracted from the bly 150. Preferably, from about I to about 5 percent
`resulting pellet with two 25GmL portions of a mixture ethanol may be added to the supercritical COz. The
`of chloroform-methanol solvent in a l:1 volumetric addedethanolrcndersthesupercriticalCO2morepolar,
`ratio, at room temperature using mechanical mixing or , _ thereby improving cholesterol extraction, About 4.5 g
`homogenizing. The mixture is filtered in a Buchner 15 of residue, which typically contains morc thar. 95Vo
`funnel with suction and the filtrate is placed in a separa- pure PC, with about O.2Vo PE and l,2Vo cholestrol, can
`tory funnel to which 225 mL of water is added to form be removed from the extractor once extraction is com-
`a biphasic system. The flask is gently swirled and the plete.
`phases allowed to scparate. The lower chloroform ^_ In another
`cal COz
`phasc is rotoevaporatd to dryness under vacuum of 1 20 extraction pre
`g of ly-
`mm Hg at 35' C., carefully av'oiding foaming. The resi- ophilized egg
`the COz
`at a temPerature above 3l' C., preferably at ,10" C. and
`due (approximately 25 g) can be treated in either of two
`ways:
`t 5,000 psi,
`(1) BV
`The eluant
`in ao mL
`is stopped
`cold acet
`units. The
`the phosphatides, Its approximate composition is typi- extract, typically containing about 3 g of oil (triglycer-
`cally TOVo PC; l5Volfi Slo NLs; 5% P-S; and less t-tran ides and cholesterol)' is discarded. The remaining resi-
`IVo lysr:-PC (if the eggs are fresh). These phosphatides ,^ due_ whicb_generally. weighs about 5.3 g, is extracted
`canbedissolvedinahydrocarbonandplacedirtacoun- r'witå_tw-o50-mLportionsofchloroform-methanol(l:l),
`tercurrert extraction device using acitonitrile as the at wlich point 45 mL of water are added to the com-
`counter-solvent, as described belõw. Alternarel¡ the :ü:î.iåHïråi#v;rt"'1"ïïiji'Fff:. the lower
`
`2s
`
`The resulting residue is dissolved in 100 mL of hex-
`ane, placed in a separatory funnel and extfacted with
`fifteen l0O-mL portions of acetonitrile that has been
`preequilibrated with hexane. The combined acetonitrile
`extracts are then evaporared o.*r r".if;t"Iî:.å",i
`a
`yields about 1.6 g
`o
`svoPC.co¡tz¡i'
`nants generally consist of less than LVo PF,, 0.5Vo tri-
`glycerides a¡¡d O.5Vo cholesterol.
`ftrile (a0 g in zm0 mL) with homogenization, or it may 45 6.2 phospholipid purification at Different Scale Levels
`-'- - --';ã-i;;;-Countercu¡rent
`be redissolved in hexane and extracted with 4 liters of
`Extraction
`acetonitrile, as described above, and further purified bv
`'
`acetone precipitation,
`6'2.1 Large Scale Purilication of Phosphatides from
`Chicken Egg Yolks
`6.1.4 Removal of Egg Yolk Neutral Lipids by
`Supercritical coz Befãå or After Extraction With 50 The fresh yolks of 6o jumbo eggs (total weight ap-
`proximately 3600 g) are thorouglrly blended with 2
`Acetonitrile
`ro avoid the use oracerone a¡i a means orremoving l'jÏir'"riffi:*ff ii 3J;å":?Tffi"i.t;iÏ9ld.t:
`neutral lipids, either of the followiag methods based and thei alternately centrifuged at 0.-4. C. for lO min.
`upon the use of supercritical CO2 may be employed. 55 utes at g000 rpm (Sb0OX g) o-r frltered with suction in a
`'
`In one embodimcnt of the invention in which acetoni- Buchner funnel.
`trile extraction is carried out fust, 12 yolks from Grade
`The resulting p€llet or filter cake is re-suspended in 2
`l^lryqç hen- eggs are lyophiliz_ ed to produce about litersof dryu"ãtoo" at2" C.andtheaboveôoolingand
`llGl3O g of dried material. This material is then ex- centrifugation or filtering rt.pr *.;õ;"t.d.
`tracted with 2 liters of acetonitrile in a Buchi Model eo The rãsultant mixture is theì filtered with suction and
`El-130RotoevaporatorequippcdwithaSoxhletextrac-
`the precipitate dried in a rotary
`under re_
`!9.-Ih" system is operatcd under reduced pressure at
`"u"por"io.
`ducåd pråssure to remove any iesidual acetoûe