weed my
`
`[E-‘J
`Ijffi/\lE§ Yo QJEDSIHJUEE
`
`FRE SENIUS-KABI, Exh. 1012
`
`
`1 of 8
`
`FRESENIUS-KABI, Exh. 1012
`
`

`
`§lOTECHNOLOGYPRO€ESSES:
`Scale-up and Mixing
`
`Eolited by
`
`CHESTER 5. HO
`State University of New York at Buffalo
`Buffalo, N.Y.
`
`JAMES Y. OLDSHUE
`Mixing Equipment Company
`Rochester, New York
`
`American Institute of Chemical Engineers
`New York, New York
`
`
`2 of 8
`
`

`
`Copyright 1987
`American Institute. of Chemical Engineers
`;_j?34‘5‘§E_as’t\ 47tiij.S'tre_E2t'
`'
`'
`l
`-
`New Ylork, New York 10017
`
`AiChE shall not be responsible for statements or opinions advanced in papers
`or printed in its publications.
`
`Library 0? Cengress Cataloging-in—Publication Data
`
`Biolechnology processes.
`Includes index.
`2. Bio-
`1. Biochemical enginee1‘ing—-Congresses.
`technoI0gy—Meth0d0l0gy—-—Congresses.
`I. Ho, Chester 5.,
`1950-
`.
`ll. Oldshue, James Y.
`-Ill. American
`institute of Chemical -Engineers.
`TP24-8.3.-B'62
`19-87
`‘E 6'O’.'6
`ISBN 0-'8169—04i‘0—3
`
`874 4393
`
`’
`
`MANU-FACTURED IN THE UNITED STATES ‘OF AMERICA
`
`
`3 of 8
`
`

`
`Dispersal of insouble Fatty Acid Precursors
`in Stirred Reactors as a Mechanism
`
`to Control Antibiotic Factor Distribution
`
`FLOYD M. HUBER
`RICHARD L. PIEPER
`
`ANTHONY J. TIETZ
`fermentation Technology Department
`Eli Lilly and Company
`Indianapolis, lndiana 46285
`
`I
`
`_
`
`Biosynthesis of factors in the A—21973C antibiotic complex was controlled by
`addition of appropriate fatty acid precursors. Toxicity associated with higher fatty
`acids was avoided by continuous addition of the fatty acid at a rate nearly equal to
`the uptake by the producing organism. Inability of the producing organism to assimilate
`a solid, insoluble long-chain fatty acid was overcome bydissolution of the acid in
`another substrate.
`
`Substance A2l978C is a complex of antibiotics
`produced by Streptomyces roseosporus, having
`a common cyclic polypeptide nucleus and dif-
`ferent fatty acid side chains-(L)
`(Q)
`(Figure 1). Separation of the major factors
`C1. C2, and C3 revealed differences in both
`in vitro antibiotic activity and toxicology
`E6i the different naturally occurring com-
`pounds. Deacylation of the alkanoyl side
`chain,
`followed by reacylatiqn with a series
`of fatty acids indicated the nrdecanoyl‘sub—
`stitution at position R, resulted in the best
`therapeutic potential (§)
`(5)
`(E).
`
`permit isolation ,in sufficient quantity
`directly from fermentation broth {Table 1).
`
`_In an attempt to produce LYl46032 by
`fermentation,
`the addition of decanoic acid
`during the antibiotic production phase of
`the fermentation mas proposed.
`Initial
`efforts in shaken cultures were unsuccessful
`due to either the toxicity-or insolubility
`of the fatty acid; 'In‘this paper we will
`describe'efforts to'direct'the biosynthésis
`of AZ1978C factors in continuously stirred
`reactors operating in a fedebatch mode.
`
`The natural occurrence of the n—decanoyl
`factor, designated LY146032, was too low to
`
`MATERIALS 7' AND METHODS
`
`TABLE 1
`Distribution of Naturally Occurring Factors in A21978C_
`Fermentation
`
`A21978C
`Factor
`
`C1
`C2
`C3
`C5
`LY146032
`
`_
`
`I Concentration
`’ Halml
`
`Z of ‘Total
`
`A?.l978C_
`Complex -
`
`'
`
`77
`113
`72
`trace
`trace
`282
`
`'
`
`'
`
`27 . 3
`40. 1
`25 .5
`-
`—
`
`Eli Lilly and Company, Indianapolis,
`Indiana.
`'
`
`BIOTECHNOLOGY PROCESSES
`
`A mutant strain of Streptomyces
`roseosporus NRRL 11379 was used to inoculate
`50 ml of vegetative ‘medium of the following
`composition: Trypticase soy broth
`_ (Baltimore __lu3io,lo_gical Laboratories ,
`Baltimore-,
`i1aryland),- 30 .mg/'mll;;,potato '
`. dextrin,' 25_ mglml. _.
`'_]:he inoculated medium was
`incubated for 48 hours, at 30°C in a 250 ml
`Erlenmeyer -flask on -a ‘shaker rotating. through
`- an arc of two, inches in diameter at -250 ‘RPM.
`0ne—half- ml of the mature ,v_e.get:_ative culture
`was dispensed into multip1_e_ containers and
`stored in the vapor phase of liquid nitrogen.
`One ml of the stored culture was used to
`inoculate 800 ml _of the vegetative medium
`described above.
`-The inoculated Lvegetative
`medium was incubated in a 2000 ml Erlenmeyer
`‘I
`flask‘ at 32°C for 120 hours on a shaker
`
`--——- 249
`
`
`4 of 8
`
`

`
`Dispersal of Insoluble Fatty Acid Precursors
`
`rotating through an arc of two inches in 1-
`diameter at 250 RPM.
`The entire content§_of
`the two flasks (approximately 1400 ml after
`incubation) were used to inoculate.l900 liters
`of a secondary vegetative stage having the
`following composition (mgfml):
`soybean flour,
`5.0; yeast extract
`(Difeo Laboratories,
`Detroit, Michigan), 5.0; calcium gluconate,
`10.0; KCl, 0.2; MgS0n'7H20, 0.2; FeS0n'7Hz0,
`0.004; Sag 471 antifoam (Union Carbide,
`Danbury, Connecticut).
`The potassium,
`magnesium, and ferrous salts were prepared ’
`separately as follows:
`7.6 g FeS0n-7H20 was
`dissolved in 76 ml of concentrated H61.
`380 g
`of Mgsot-7HgD and 380 g of K01 and deionized
`water were added to bring the total volume to
`3800 ml.
`The inoculated medium was incubated
`2% hours in a stainless steel vessel at 30°C.
`The vessel was aerated at 0L85'vfw/m and
`stirred with conventional agitators.
`
`compound has very low solubility in water.
`In order to avoid the obvious problems aris-
`ing in supplying a limiting nutrient as a
`solid phase,
`the substrate was dispensed to
`the stirred reactor as a five percent solu-
`tion dissolved in a fifty percent ethanol!
`water mixture. There was an imediate
`
`response in oxygen uptake to the onset of
`the decanoic acid feed, as-illustrated in
`Figure 2. Also, a significant improvement
`in LY146032 concentration was immediately
`realized (Table 2).
`
`TABLE 2
`
`Distribution of A_21978C Factors with Decanoic Acid Feed’
`
`A21978C
`Factor‘
`
`Concentration
`Us /1111
`
`2 of Total
`A21978C
`Complex
`
`19.8
`29.9
`11.5
`5.2
`33.5
`
`The mature secondary seed (8.332 V/v)
`was used to inoculate a production medium of
`the following composition (mg/ml):
`soybean
`flour, 22.0; Fe(NHn)2SOn'6H2U, 0.66; glucose
`monohydrate, 8.25; Sag 471, 0.22; potato
`dextrin, 33.0; and molasses (blackstrap),
`2.75.
`'
`-
`
`c1
`oz
`c3
`c5
`
`LY146032
`
`72
`109
`42
`19
`
`Egg
`364
`
`Two types of stirred reactors were used.
`The smaller vessel, operated at 120 liters,
`- was agitated with two conventional flat
`Rushton type impellers at.relatively high
`power input.
`The larger vessel, operated at
`4550 liters, was equipped with impellers
`having curved paddles and was operated at
`relatively low power input. Air flow in both
`reactors was supplied at 0.5 v/v/m by_large
`open tubes which were estimated to contribute
`very little to the overall mixing. Respira-
`tion rates were estimated by difference in
`inlet and exhaust gas concentration via a
`Perkin—Elmer mass spectrometer. Distribution
`of A21978C factors was estimated by high
`performance liquid chromatography as
`described previously (2);
`
`I..
`
`Examination of the batch fermentation
`medium suggested that the-growth limiting
`nutrient was carbon in the form of carbohy-
`drate.
`It was then hypothesized that in a
`fed-batch operation a moderately toxic sub-
`strate, such as decanoic acid,’could‘be fed
`continuously to the fermentation if the
`metabolic consumption rate exceeded the
`addition rate.
`
`Delivery of decanoic acid to the
`culture presented a problem. With a melting
`point of 34°C the compound is a solid at the
`fermentation temperature of 30°C, and the
`
`(3) N-decanoic acid/ethanol/water 122:2 fed
`'50‘ml'per hour to 120 L'op§rating volume.
`
`Material balances suggested that only
`a small portion of the decanoic acid that
`was fed could be accounted for by incorpora-
`tion into the product. Thus; most of the
`fatty acid was apparently catabolized,
`presumably by the beta—oxidation pathway.
`In an attempt to increase the amount of
`deeanoic acid available for-the incorpora-
`tion,
`the concentration of fatty acid in the
`
`TABLE 3
`Distribution of A21978C Factors with Increased Decanoic Acid
`Feed“
`'
`
`Z of Total
`A2l978C
`Complex
`
`A21978C
`Factor
`
`C1
`C2
`C3
`C5
`LY146032
`
`Concentration
`
`pg/ml
`
`131
`189
`107
`52
`784
`
`1263
`
`(a)
`
`N—decanoic acid/ethanol/water 1:2:2 fed
`50 ml per hour to 120 L operating volume.
`
`B1OTECHNOLOGY PROCESSES
`
`
`5 of 8
`
`

`
`Floyd M. Huber, Richard L. Pieper, and Anthony J, Tietz
`
`TABLE 5
`
`Distribution of A21978C Factors with Decanoic Acid/Methyl
`Oleate Fed-to a Larger Reactori
`
`A21978C
`Factor
`
`C1
`C2
`C3
`C5
`,‘LY146032
`
`Concentration
`'
`us/ml
`
`260
`337
`270
`170
`489
`
`1527
`
`Z of Total
`A2l973C
`Complex
`
`17.0
`22.1
`17.7
`11.1
`32.0
`
`(3) N-decanoic acidfmethyl oleate 1:1 fed
`490 ml per hour to 4550 L operating
`' V0 lllthé
`
`the uptake of the fatty agid
`large vessel,
`was not equivalent throughout the population
`in the reactor (Table 6).
`'
`
`TABLE6
`
`,
`
`Mixing Time and Power input in Pilot-Scale Equipment Used in
`A2197BC Fermentation
`
`Vessel Size Mixing Time
`(1)
`(seconds) ’
`
`(a)
`
`Power Input
`(Hp/100 Gal.)
`
`150
`
`soon
`
`7
`
`43
`
`1.36
`0.82
`
`(a) Time required for pH to reach equilib-
`rium after addition of a sufficient
`quantity of 6M NaOH to increase pH by
`0.3 ‘
`'
`
`The first approach to achieve better
`incorporation of the fatty acid precursor in
`the poorly mixed larger vessel was to
`increase the feed rate of the decanoic acid]
`methyl oleate mixture.
`The percentage of
`LY146032 increased to nearly that achieved
`in the smaller equipment, but the total yield
`of A21978C was reduced to about two-thirds of
`the amount achieved at the lower feed rate
`(Table 7).
`
`feed was increased to twenty percent——the
`solubility limit of decanoic acid in aqueous
`ethanol.
`A significant increase in both the
`LY145032 and total yield was observed
`(Table 3);
`
`Although the fed-batch fermentation
`employing the fatty acid/ethanol/water addi—
`tion proved an effective method of directing
`the synthesis of the A21978C complex,
`the
`presence of the volatile alcohol presented
`both safety problems and uncertainties in
`quantitating the carbon balance.
`It was not
`known to what extent the producing organism
`could metabolize ethanol, since it was likely
`that much of the alcohol was escaping in the
`exit gases. Methyl oleate_was identified in
`batch shaken cultures-as a metabolizable,
`non—toxic and low volatile solvent. A mix-
`ture of equal volumes of methyl oleate and
`decanoic acid remained liquid at 30°C.
`The
`results of feeding the mixture of decanoic
`acid dissolved in methyl oleate on an equiva-
`lent basis to the previously used aqueous
`ethanol feed resulted in a slightly higher
`total yield with a similar concentration of
`the desited LY146032 (Table 4).
`
`"TABLE 4
`Distribution of A21 978C Factors in a Decanoic Acidr‘M¢thyi
`Oleate Fed Fermentation‘
`
`% of Total“
`A21978C
`Complex
`
`H.
`
`.
`
`.
`
`1
`_1
`
`A21978C
`Factor
`
`Concentration
`
`c1
`c2
`c3
`cs
`LY146032
`
`?-
`
`no .
`'
`.
`
`,
`
`,
`
`'
`
`pg/ml
`
`154
`191
`127-
`68
`913
`
`1453
`
`(3) N—decanoic acid/methyl 151 oleate fed
`13 ml per hour to 120 L'operating volume
`
`SCALEQUP CONSIDiE2RATIONS
`
`The initial scale—up of thetprocess to
`a larger 6000 L pilotescale stirred reactor
`did not produce-equivalent LY146032 factor
`distribution (Table §).
`
`The percentage'of LY146032 was approxi-
`mately one—half of that obtained in the
`smaller equipment.‘ It was hypothesized that
`the microbial population was oxidizing the
`decanoic acid preferentially over the methyl
`oleate, and due to the poorer mixing in the
`
`The reduction in overall yield associ«
`ated with the increased feed of the decanoic
`acid precursor was believed to be a result of
`"the feed rate of the fatty acid approaching
`the metabolic rate of consumption. The con-
`sequences of introducing the toxic fatty acid
`
`- BIOTECHNOLOGY PROCESSES
`
`p
`
`251
`
`
`6 of 8
`
`

`
`(A)(3
`
`ix:0
`
`
`
`
`
`OxygegjUptakeMmo|fL/Min 3.
`
`LID?
`
`GU
`
`D-ser
`
`\.
`3‘-Me_G|u (L-tI1lre9)
`L-K’3{n
`
`-- " ‘ O/II
`L-Thr/
`C’
`T
`L-Asp
`f
`
`FIOCOF
`
`»
`
`’
`
`A2197BCa
`A2197ac,
`A21978
`A2197B(c2:
`A2197ac4
`A21978C5
`‘- mix|ufg of mg isomers
`
`v
`
`-
`_ C1oalkanpyl"f _
`,
`ma-mgi}1h3lrI dr:ica_noYl_ I
`-m0
`If H“ 903F103‘
`10-methyldodecanoyl -
`C12 alkanuyl t
`C13 alkanoyl t
`1 structure uncertain
`
`FIGURE 1. Naturally occurring A21978C factors.
`
`Decanolc Acid
`I ‘ Overted
`
`
`
`DXYUptakel|IIml|.IlJIIn
`
`Dispersal of Insoluble Eattyficid Precursors-._.
`
`,
`
`.50
`
`J:O
`
`" 3; .s_t=.xr.t-..
`Déc'a‘ndic .-\‘cid__
`‘
`Feed
`
`L
`
`.
`'
`
`‘
`
`'
`
`'
`
`A
`
`'
`
`.
`
`15
`'
`
`""30
`'
`
`-' "45""
`' Hours
`’
`"
`
`‘
`
`..
`
`‘
`
`|.
`
`FIGURE 3. The toxic effect of_Q_ve‘n-‘eeding _d-ec..:noic_ acid.
`
`15.00"
`
`45.09
`30.00
`Hours
`‘
`FIGURE 2. The effect of n-decanoic acid feed upon oxygen uptake.
`
`60.00‘
`
`75.00
`
`_
`FIGURE 4. Feeding of mdecanoac acidfmethyl oleate to a large reactor
`Via [W0 em;-y poim5_
`
`‘ 252
`
`-
`
`BIOTECHNOLOGY PROCESSES
`
`
`7 of 8
`
`

`
`TABLE?
`Distribution of A2197BC Factors with Increased Decanoic Acid!
`Methyl Oleate Feda to a Larger Reactor
`
`A21978C
`Factor
`
`c1
`c2
`(:3
`C5
`LY146032
`
`Concentration
`ug/m1
`
`% of Total
`A21978C
`Complex
`
`.
`
`.
`
`1
`1
`
`122
`117
`33
`93 I
`607
`
`1022 '
`
`(a) N—decanoic acid/methyl oleate lzl fed 590
`ml per hour to.4550 L operating volume‘
`
`at a_rate greater than the microbial consump—'
`tion rate are illustrated in Figure 3;
`In
`this reactor the feeding system failed,
`resulting in significant overfeeding of the
`fatty acid; The resfilting accumulation of
`the fatty acid caused rapid*lysis of the
`culture as evidenced by-the rapid cessation-
`of respiration.
`‘
`'
`
`‘A second approach to solve the mixing
`problem in the large fermenter was.to-intro-
`duce the fatty acid/ester mixture at'two
`widely separated‘entry—points.
`One entry
`point was at the-top of the vessel and=au
`second was in between the two impellers -
`(Figure 4)- The.subsequent LYl460I2 concen-
`tration obtained (Table 8)
`indicated that7at
`greater incorporation of the n—decanoyl side
`
`-
`‘
`"
`:1'='
`vfi‘
`TABLE8
`Distribution of A21978C‘Factors-withDecanoic Acid/Methyl V.
`Oleate Fed to -a Larger Reactor via Two Entry Points“
`
`A2l9I8C K
`Factor-
`
`-; Concentration
`’V U3/ml
`
`-
`
`Z of Total-
`A21978C
`"C0mfilEXu
`--1..
`
`'
`
`3
`
`C1
`G2 .
`I C3.
`V
`C5
`LY146032
`
`,
`._.‘,,
`
`I
`
`._
`
`,:
`
`-
`':
`
`'
`
`7-5
`_/" U 119 ..
`._
`V
`103
`15
`'l090
`
`.
`
`q-1&4,”-:8’; t
`
`(é?”flfdecanoiciacid/methjl 5iEate‘Ia1;£éd'212
`V mlfpe Whour'to'eaph“9fltwo entry points;
`455otL“wbfk1ng-vo1hne:’*
`
`'
`
`-BIOTECHNOLOGY PRbCE5SE5
`
`chain had occurred as a result of the dual
`introduction.
`
`the following scaledup prob— _
`In summary,
`lems associated with the production of
`LYl46032 have been addressed and solved:
`
`A sound strategy was provided to
`1.
`feed the microbial culture a very toxic
`substance.
`9
`
`A mechanism was devised to supply
`2.
`the normally solid substrate to the reactor
`in a convenient Iiquid form.
`
`’
`
`Preferential substrate utilization
`'3.
`was‘identified_and the necessary feeding
`hardware was‘construEted to promote homogen-
`eous utilization‘of"lipoidal materials when
`the fermentation was scaled up to larger
`reactors.
`
`LITERATURE CITED
`
`Hamill, R.L. and M.M.1Hoehn, U.S, Patent
`'4,208,403.to Eli Lilly and Company
`(June 17,_l980).
`
`‘_Debono; Mandel, U.S. Patent 4,39fi,067 to
`_ Eli Lilly and_Company (August 16, 1983).
`
`_
`
`'
`
`'v;M.
`,B._.I. Abbott,
`Debono, Mgmué1;
`Krupinski, R.M{'Molloy, D.J. Berry, E.T.
`‘Counter, L.C._floward,_J,L. Ott and RLH.
`__ liamill, T'Sy_n't11e's:"s;|,and 'Stru'ct11:r_e Activity
`Relationships ofWflew_Analogs of the New
`Gram-Positiveflipopeptide Antibiotic
`A2l978C,W.Abstract 1077) Interscience
`Conference on antimicrobial Agents and
`H_Chemctherapj
`(ICAAC)
`(Qctober I984).
`
`. Eukuda, 1_3_.S.:',.__B_._J.'Abb_ott,, D._J. Berry,
`L.D,,Boeok, Manuel Debonog R.I._Hamill,
`V.M§ Krupinski and R.L, Holloy;_VDeacy-
`‘H;latiQn_and,Reacylation'of_A21978C,
`’ Acidic fiipopepfide Antibiotic:_Prepara—
`.tion of New,Analogs,H Abstract 1076,
`ulntersciepce Conference on Antimicrobial
`Agents _and 'Chemotherapy_ Crcgmc)
`. I0ctober 1QS§)3f_
`
`—
`
`Counter, F.T., P.J. Baker; L.D. Boecfi,
`Manuel Debono, P.W. Ensminger, R.L.
`'
`Hamill, V.M. Kruginski,.R.H. Molloy and
`_J}Lg__tr, “LYl46032>[N¥(n¥decanoy1)
`'A2lQ]8CwNucl”'
`],;a_Néw Acidic Lipopep—
`tide Antibiot;c::Synthesis and Biological
`h_
`' —, Evaluation,“ Abstract 1078, Interscience
`_ Conference on Antimicrobial Agents and
`Chemotherapy (ICAAG)
`(October 1984).
`
`
`8 of 8