United States Patent ~ [191
`Baker
`
`[11]
`[45]
`
`Patent Number:
`Date of Patent:
`
`4,874,843
`Oct. 17, 1989
`
`[54] CHROMATOGRAPHIC PURIFICATION
`PROCESS
`
`Patrick J. Baker, Greenwood, Ind.
`[75] Inventor:
`[73] Assignee: Eli Lilly and Company, Indianapolis,
`Ind.
`
`[21] App]. No.: 128,351
`
`[22] Filed:
`
`Dec. 3, 1987 "
`
`[51] Int. Cl.‘ .............................................. .. C07K 1/14
`[52] US. Cl. .................................. .. 530/317; 530/322;
`530/344
`[58] Field of Search ...................... .. 530/317, 322, 344
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`3,515,717 6/1970 Cha et a1. .......................... .. 260/210
`4,208,403 6/1980 Hamill et al.
`.. 424/115
`4,440,753 4/ 1984 McCormick et al.
`424/ 124
`4,537,717 8/1985 ' Abbott et al. ....... ..
`260/112 5
`4,719,287 l/1988 Login et a1. ....................... .. 530/317
`
`FOREIGN PATENT DOCUMENTS
`
`0145484 11/1984 European Pat. Off. .
`
`OTHER PUBLICATIONS
`Lea?et “Isolation of Antibiotics from Fermentation
`Broths by DIAION HP 20, A Synthetic Adsorben ”,
`Mitsubishi Chemical Industries Limited, pp. l-22.
`Grieser et al., “Liquid Chromatography on a Porous
`Polystyrene-Divinylbenzene Support”, Analytical
`Chemistry, vol. 45, No. 8, Jul. 1973, pp. 1348-1353.
`Primary Examiner—Lester L. Lee
`Attorney, Agent, or Firm-Mary Ann Tucker; Leroy
`Whitaker
`ABSTRACI‘
`[57]
`This invention relates to a new chromatographic pro
`cess for purifying fermentation products, particularly
`the antibiotic LY146032, from fermentation broths by
`use of a reverse phase non-functional resin.
`In the process of this invention the resin is loaded with
`the compound in the aqueous phase, the water then is
`removed from the resin and the resin converted to the
`organic phase for the resolution step. This process is
`applicable to purifying compounds which are adsorbed
`on reverse phase non-functional resins.
`
`11 Claims, No Drawings
`
`PETITIONERS
`
`EXHIBIT NO. 1007 Page 1 of 4
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`

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`1
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`CHROMATOGRAPHIC PURIFICATION PROCESS
`
`SUMMARY OF THE INVENTION
`This invention relates to a new chromatographic
`process for purifying fermentation products, particu
`larly the antibiotic LY146032, from fermentation broths
`by use of a non-functional resin in reverse mode.
`In the process of this invention the resin is loaded
`with the compound in the aqueous phase, the water
`then is physically removed from the resin and the resin
`rewetted with a polar organic solvent for the resolution
`step. This process is applicable to purifying compounds
`which are adsorbed on non-functional resins.
`
`15
`
`4,874,843
`2
`acetate buffer solvent and passed through a column
`containing HP-20ss non-furnnctional macroreticular
`resin. The column is developed with the same solvent
`and the puri?ed fractions containing LY146032 are
`combined, diluted with water and loaded on a column
`containing HP-20 resin. The column is washed with
`water to remove salts, eluted with acetonitrilezwater
`(about 60:40) and the LY146032 fractions collected.
`These steps must be repeated as often as necessary to
`give a product of the desired purity.
`Final resolution and separation of LY146032 from
`structurally similar compounds is impeded by the pres
`ence of impurities which are not identi?able by ultravi
`olet analysis of the fermentation broth. These so-called
`“non-uv” impurities are primarly saponins and other
`fragments. These compounds have solubility character
`istics similar to LY146032 and are dif?cult to separate
`from LY146032. The presence of these compounds
`causes foaming during concentration procedures and
`poor resolution during subsequent chromatographic
`sepration steps.
`Attempts to remove these impurities by various chro
`matographic methods, including reverse-phase chroma
`tography on silica gel/C18 (Quantum LP-l), normal
`phase chromatography over silica gel, and ion-ex
`change chromatography, failed to signi?cantly improve
`the purity of LY146032 over the use of HP-20 as de
`scribed above. All of these methods are plagued by low
`capacity, poor resolution and low recovery of
`LY146032.
`A novel isolation procedure had to be developed to
`overcome these problems. Thus, it was unexpectedly
`found that replacing the ?rst HP-20 step described
`above with a “reverse method” procedure, wherein
`adsorption is carried out with the non-functional resin
`in aqueous phase (polar) and resolution is carried out
`with the resin in organic phase (non-polar), achieves
`substantial improvements in both purity and yield of the
`product. The “reverse method” process of this inven
`tion improves the purity of LY146032 twofold and,
`since it removes impurities that interfere with the subse
`quent puri?cation steps, improves the ?nal purity from
`about 80% to about 93%. In addition, the overall yield
`is increased from about 5% to about 35%.
`An additional bene?t of the “reverse method” pro
`cess is that fewer subsequent separation steps are re
`quired to achieve the increased purity and yield.
`LY146032 is susceptible to transpeptidation degradation
`(U .S. patent application No. 07/060,148, ?led June 10,
`1987). Reducing the number of steps and the time re
`quired to purify the product greatly lessens the forma
`tion of transpeptidation by-products.
`The novel process of the present invention is useful
`for purifying fermentation products that are adsorbed
`onto non-functional resins. These fermentation prod
`ucts include lipopeptides, for example LY146032,
`echinocandin B and the like, and glycopeptides, for
`example vancomycin, actoplanin, teichoplanin, A82846
`and the like.
`The present invention begins with clari?ed, acidic
`aqueous broth containing the fermentation product or
`with partially puri?ed fermentation product in an acidic
`aqueous buffer solution. The process comprises:
`a. contacting an aqueous solution of a fermentation
`product with a non-functional resin in aqueous phase;
`b. physically removing the water from the charged
`resin;
`
`35
`
`BACKGROUND OF THE INVENTION
`The antibiotic LY146032 is the N-decanoyl deriva
`tive of the A-21978C antibiotics and has now been iden
`ti?ed as a member of factor A-2l978C0. The A-21978C
`20
`antibiotics are prepared by fermentation methods de
`scribed in U.S. Pat. No. 4,208,403. LY146032 methods
`for its preparation are speci?cally disclosed in U.S. Pat.
`No. 4,537,717. In this method A-2l978C is prepared by
`fermentation techniques. The fatty acid side chain then
`is removed with an enzyme to give the A-2l978C nu
`cleus and the nucleus is reacylated with the desired acyl
`group, for example the n-decanoyl group, to give the
`A-2l978C cyclic peptides as described in US. Pat. No.
`4,527,717.
`An improved method for preparing these cyclic pep
`tides is described by Floyd M. Huber, Richard L.
`Pieper and Anthony J. Tietz in copending US. patent
`application Ser. No. 773,762, ?led Sept. 9, 1985, entitled
`IMPROVED PROCESS FOR A-2l978C DERIVA
`TIVES.
`Because of the interest in LY146032 and other thera
`peutically useful fermentation products and their impor
`tance, new and more effective methods of isolating
`these useful compounds from fermentation mixtures
`continually are sought.
`The novel process of this invention was previously
`disclosed, but not claimed, in US. patent application
`No. 07/060,148, ?led June 10, 1987, for use in purifying
`the B-isomer of LY146032.
`DETAILED DESCRIPTION
`The present process provides a novel method for the
`separation and puri?cation of a wide variety of fermen
`tation products, including the antibiotic LY146032,
`from their fermentation broths or partially puri?ed
`process streams by adsorbing the antibiotic from aque
`ous medium onto a non-functional resin, followed by
`physically removing the water from the resin, rewetting
`the resin with a polar organic solvent and eluting the
`product by increasing the polarity of the solvent.
`Prior to the present inventionfthe partial puri?cation
`of LY146032 was accomplished by ?ltering the whole
`fermentation broth and passing the ?ltrate through a
`column containing HP-20 resin (Diaion High Polymer
`HP-Series, Mitsubishi Chemical Industries Limited,
`Tokyo, Japan). The column is washed with water and
`waterzacetonitrile (about 90:10 to about 80520), and the
`LY146032 eluted with waterzaceonitrile (about 60:40).
`Elution is monitored by paper chromatography or ul
`traviolet monitor and fractions containing LY146032
`65
`are collected, concentrated under vacuum freeze-dried.
`This procedure gives semi-pure LY146032 which
`then is dissolved in an acetonitrile-methanol-sodium
`
`45
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`PETITIONERS
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`EXHIBIT NO. 1007 Page 2 of 4
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`4,874, 843
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`3
`c. rewetting the charged resin with a polar organic
`solvent;
`d. washing the resin with a polar organic solvent to
`remove non-uv impurities;
`e. eluting the fermentation product from the non
`functional resin by increasing the polarity of the sol
`vent; and
`f. recovering the ferementation product.
`When the process begins with whole broth, the broth
`is ?ltered and the pH adjusted to about pH 4.0 to about
`pH 5.0, preferably about pH 4.5, using aqueous acetic,
`hydrochloric sulfuric, phosphoric and like acids.
`When the process begins with partially puri?ed fer
`mentation product, the product is dissolved in an aque
`ous buffer solution of about pH 40 to about pH 5.0,
`preferably about pH 4.5. Suitable buffer solutions are
`aqueous solutions of sodium phosphate, ammonium
`phosphate, ammonium acetate, sodium acetate and the
`like. Preferably, the buffer is sodium acetate.
`The fermentation product containing solution is
`passed over a column containing the non-functional
`resin in aqueous phase. Non-functional resins useful in
`the process of this invention can be described generally
`as macroporous copolymers of styrene and divinylben
`zene. Non-functional resins are a known class of resins
`and information concerning these resins, their sources
`and their characteristics appears in J. Chromatography
`201, 287-292 (1980). Typical non-functional resins in
`clude Diaion HP-20, Doulite ES-861, Amberlite XAD
`16, Amberlite XAD-4 and the like. Diaion HP-20 is a
`preferred non-functional resin.
`The resin is removed from the column and ?ltered or
`dried to remove the water. The resin may be dried in a
`vacuum oven or air dried, for example in a Handy
`35
`Dandy Filter (Sharples Filter Co.). The charged resin
`then is rewetted with a polar organic solvent. If the
`resin is substantially dry, rewetting may take place ei
`ther by adding the resin to the polar organic solvent or
`by adding the polar organic solvent to the resin. If ap
`preciable amounts of water remain in the resin, the resin
`must be added slowly to the polar organic solvent to
`avoid stripping the fermentation product from the resin.
`The resin then is repacked into a column and is washed
`with a polar organic solvent to remove non-uv impuri
`45
`ties. The fermentation product is eluted from the resin
`by increasing the polarity of the solvent.
`In the above process, the term “polar organic sol
`vent” includes methanol, ethanol, acetone, n-propyl
`alcohol, isobutyl alcohol, n-butyl alcohol, methyl ethyl
`ketone, acetonitrile, tetrahydrofuran, and like solvents
`which have appreciable water solubility or are miscible
`with water. Acetonitrile is a preferred polar organic
`solvent. The polar organic solvent may be acidi?ed
`with from about 1% to about 10% aqueous acid, prefer
`ably about 5% aqueous acid. Aqueous acetic, hydro
`chloric, sulfuric, phosphoric and like acids may be used.
`Acidi?cation to about pH 4.0 to about pH 5.0 improves
`the resolution of the process and, when the fermenta
`tion product is LY146032, reduces the amount of tran
`speptidation by-product formed.
`In the elution step, the polarity of the solvent is in
`creased by diluting the polar organic solvent with a
`more polar solvent, for example water or methanol. For
`example, the fermentation product may be eluted with
`65
`an acetonitrilezwater solvent in the ratio of from about
`95:5 to about 40:60, preferably from about 85:15 to
`about 80:20.
`
`4
`The fermentation product is recovered from the elu
`ate by methods known in the art, for example crystalli
`zation, concentration and lyophilization.
`The invention is illustrated by the following exam
`ples, which are not to be considered as limiting.
`
`EXAMPLE 1
`Puri?cation of LY146032
`Crude LY146032 (137.5 g) was dissolved in 3.5 l of
`1% sodium acetate buffer at pH 4.5. The solution was
`passed through a column containing 3 l of HP-20 resin
`(Diaion High Porous Polymer HP-series, Mitsubishi
`Chemical Industries Limited, Tokyo, Japan). The ef?u
`ent was discarded. The resin was washed with 30 l of
`chilled deionized water and the wash water was dis
`carded.
`The resin, charged with LY146032, was removed
`from the column and ?ltered to remove residual water.
`The charged, semi-dry resin then was added slowly to
`32 l of acetonitrile. The charged resin in acetonitrile was
`repacked into the column.
`The column was washed with 48 l of acetonitrile
`water solution (95:5) to remove the non-uv impurities,
`e.g., the saponins and tripeptide fragments. The column
`was eluted with 17 l acetonitrile-water solution (85:15).
`One liter fractions were collected. The elution was
`monitored by analytical HPLC and fractions containing
`LY146032 were combined.
`The LY146032 containing fractions were diluted
`with ?ve volumes of chilled deionized water and passed
`through a column containing 500 ml of HP-2O resin.
`The column was washed with 500 ml deionized water
`to remove salts. The column was eluted with acetoni
`trile~water solution (60:40). Fractions shown by HPLC
`to contain LY146032 were combined, concentrated and
`lyophilized to provide 53.3 g (60% yield) of 89.3%
`purity LY146032.
`
`EXAMPLE 2
`Puri?cation of LY146032
`Crude concentrate (157 1) containing 1914.2 g of
`LY146032 activity (purity 24.4%) was diluted to 600 l
`with chilled puri?ed water. HP-20 resin (60 l) was
`added to the diluted crude concentrate. The pH was
`adjusted to 4.7 with glacial acetic acid, n-butanol (6 l)
`was added to control foaming, and the mixture was
`stirred for 20 hours. The resin then was washed with
`300 l of water.
`The charged resin was put in a Handy Dandy Filter
`and blown dry. The dried, charged resin was rewetted
`with acetonitrile (6001) and loaded into a column. The
`column was washed with acetonitrile (60 l).
`The resin was washed with a mixture of acetonitrile:
`methanol:0.25% phosphoric acid (80:l5:5) to remove
`non-uv impurities. The column was eluted with 1201 of
`acetonitrilezwater solution (80:20). Fractions (25 l) were
`collected and the fractions containing LY146032 were
`combined. The combined fractions were concentrated
`and freeze-dried to provide 993.1 g (51.9% yield) of
`LY146032 (purity 50.5%).
`EXAMPLE 3
`Puri?cation of Echinocandin B
`Fermentation whole broth (200 L ml) containing 22.75
`mg of Echinocandin B was diluted with two volumes
`(400 ml) of methanol. The insoluble mycelial solids
`
`PETITIONERS
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`EXHIBIT NO. 1007 Page 3 of 4
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`5
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`15
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`4,874,843
`6
`5
`e. eluting the fermentation product from the non
`were removed by ?ltration. The pH was adjusted to 5.0
`functional resin by increasing the polarity of the
`with 5N hydrochloric acid and the ?ltrate was added to
`solvent; and
`HP-20 resin (10 ml). The mixture was diluted with an
`f. recovering the fermentation product.
`equal volume of water and stirred for 60 minutes to
`2. The process of claim 1 wherein the fermentation
`effect adsorption of the Echinocandin B onto the resin.
`product is a lipopeptide.
`The charged resin was separated by ?ltration and dried
`3. The process of claim 2 wherein the fermentation
`for 16 hours at 25° C. in a vacuum oven. The dry,
`product is LY146032.
`charged resin was rewetted with acetonitrile and
`4. The process of claim 2 wherein the fermentation
`packed into a column. The column was washed with
`product is Echinocandin B.
`three column volumes of acetonitrile. Elution with
`5. The process of claim 1 wherein the fermentation
`three column volumes of 90% acetonitrile/10% water
`product is a glycopeptide.
`resulted in recovery of 26% yield with a purity equiva
`-6. The process of claim 4 wherein the fermentation
`lent to the reference sample. Further elution with three
`product is vancomycin.
`column volumes of 80% acetonitrile/20% water re
`7. The process of claim 1 wherein the resin is Diaion
`sulted in the ‘additional recovery of 38% yield with a
`purity of 41.2% of the reference sample.
`HP-20.
`"
`8. The process of claim 1 wherein the resin is amber
`I claim:
`1. A process for purifying fermentation products
`lite XAD-4.
`which comprises:
`9. The process of claim 1 wherein the polar organic
`solvent is acetonitrile.
`a. contacting an aqueous solution of a fermentation
`20
`10. The process of claim 1 wherein the fermentation
`product with a non-functionalized macroporous
`copolymer of styrene and divinylbenzene in aque
`product is eluted from the non functionalized macropo
`rous copolymer of styrene and divinylbenzene with
`ous phase;
`b. physically removing the water from the charged
`acetonitrile containing from about 5% to about 60%
`water.
`resin;
`11. The process of claim 9 wherein the fermentation
`0. rewetting the charged resin with a polar organic
`product is eluted from the non-functionalized macropo
`solvent;
`rous copolymer of styrene and divinylbenzene with
`d. washing the resin with a polar organic solvent that
`acetonitrile containing fromabout 10% to about 20%
`is the same as or different than the polar organic
`solvent used in step (C) to remove non-uv impuri
`water.
`ties; '
`
`a:
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`1:
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`* =1:
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`*
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`PETITIONERS
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`EXHIBIT NO. 1007 Page 4 of 4
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