BE545660A - - Google Patents

Description

       

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   The present invention relates to novel processes for the preparation of antiobiotic compounds and more particularly to processes for preparing antibiotic agents made by a microorganism not heretofore described.



   The Applicant has isolated from soils originating from the East Indies and a microorganism whose morphological / culture characteristics agree with the description of the genus Streptomyces of the order Actinomycetes, as described in the manual "Determinative Bacteriology".

 <Desc / Clms Page number 2>

 
 EMI2.1
 ae cergey \ sixth edition) page ce'38. The new microorganism is related by its morphology under the microscope, its culture characteristics and its physiology to some of the groups belonging to the S. ¯] 'bus and S Fia groups seen, but does not agree with the description of any known species.



  The new r'zcroor: .nisu, thus received so far the name of SreptoLC8S Oriental ': .. It has been discovered that several suuc-hes of the organism, which are called S.Orientalis M lJ-05èib5, 1 w5-lk215 and H5-12C, produce the subsziG.:these antiobiotics of the present invention, and these strains were deposited in the culture collection of tfHor'tf
Régional thern / Kesearch Laboratories "
 EMI2.2
 du -, - lord) at Pearia, lllinoi--, where they received culture numbers NRRL 2450, NERL 2451, and HRRL 2452 respectively, and were added to the permanent collection of microoranisms.



  The microorganism was isolated from a soil sample brought back from Tengeng, Indonesia, the isolation procedure being as follows: suspended a soil sample in sterile distilled water, diluted strongly. suspension and a small sample was placed on a nutrient agar plate. The plate is incubated at about 30 ° C for one week. We take the P.
 EMI2.3
 A few scattered and discontinuous colonies of Orientalis with a platinum spiral and used to inoculate obliquely the agar to prepare larger amounts of the microorganism.

   One of the strains of this microorganism, obtained as indi-
 EMI2.4
 as above, has been called 3Orientalis, strain 1-143-05665, and the preparation process according to the invention will be described with particular reference to this strain of the organism. It is however of course understood that the fermentation processes of the present invention
 EMI2.5
 include the use of other antibiotic-producing strains of S.4riantalis, such strains being easily produced and isolated by the methods of isolation and modification, of strain commonly used, which include the selection of cultured organisms.
 EMI2.6
 ves, and the exposure of organisms to e.gef.ts- modifiers., such

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 than X-rays, ultra-violet light and chemical agents,

     for example nitrogenous nutrients.



   The Applicant has found that when S.Orientalis is cultivated on an artificial nutrient medium containing a nitrogen source, a carbohydrate source, and appropriate mineral salts of the type recognized as being necessary for growth. microorganisms and in order to buffer the nutrient, a new antibiotic substance is formed (hereinafter referred to as "vancomycin") which is characterized by its broad spectrum of activity against Gram-positive organisms.



   Accordingly, the present invention relates to a process for preparing an antibiotic substance from the group comprising vancomycin and its addition salts with an acid, said vancomycin being characterized as follows: a white amorphous substance, soluble in water, insoluble in most organic solvents, stable in aqueous solution at a pH between approximately 2.5 and 9, having a molecular weight between approximately 3,000 and 3,200, containing approximately 7.17% nitrogen and of which an emulsion in mineral oil has a maximum absorption spectrum in the infrared region at the following wavelengths, expressed in microns:

   3.10- 3.41- 6.05 - 6.30 - 6.72 - 6.84- 7.26 - 7.64 - 8.13- 8.50- 8.66 - 8.87- 9, 42 - 9.76 - 10.10 - 10.34- and 11.24.



   The present invention further relates to a process for the preparation of an antibiotic agent which comprises culturing, under aerobic conditions, a strain of Streptomyces orientalis producing vancomycin in a culture medium containing sources of vancomycin. assimilable carbohydrate, nitrogen and mineral salts until these microorganisms produce significant antibiotic activity in this culture medium.



   The term "artificial nutrient culture medium" as used herein refers to a culture medium which is prepared by combining sources of nitrogen, carbohydrates, inorganic salts and water, e.g. opposition to strata found in nature.

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 EMI4.1
 



  Nitrogen sources suitable for preparing a medium
 EMI4.2
 ¯e nutrient culture for use in the production process of the new antibiotic vancouycin loux, r, z ', ¯sc.ité e. by soy flour, casein, peanut flour, corn steep liquor in 11 solid state, corn steep "-iquor in liquid state, .r.ixio acids, soluble distillery products, the pe: 1j () ues. etc ...



  Examples of carbohydrates which from the Carbohydrates used by Criantalis fear the crisss of ruicroor & anism, piir1: .2.culièrer.: Sl. t when they are:. t, i l.:. e; Ç ::;. 0., ::, '1., medium {culture e: 0ser-lencé by oblique drag on' é -:, -.: '- é <.'-:' :, r -have 3.a glucose, adonitol, arabinose, xylose, trehalose, r¯.utnitol, i-inositol, cellobiose, lactose, etc ... The 3: S qu 'is incorporated into the artificial nutrient medium obtained cGnfvrt.tr:.ent z the present invention are the mineral salts orliliIi: d :::' us C01LUS in the art to be necessary for the growth of ricroor; aniszr: es and / or adjusting the acidity of the medium, 1.::-=-1 "example sodium chloride, sodium nitrate, sulphate of.: rōriurrrs calcium carbonate, etc ...
 EMI4.3
 



  The minimal quantities of the elements in the state of "traces" necessary for the living tissue are provided by the above documents in which this
 EMI4.4
 items are found in iLlpur \ 8s state.
 EMI4.5
 



  S. Orientalis is characterized by the numerous physical, morphological and culture tests described below. We use the
 EMI4.6
 system of Rid-: Yav, tColor Standards and; .or; .0811cla'CureTt (1912) for the naming of some-u: ¯es of colors, and when Oi- uses this
 EMI4.7
 system, the initial color letter is capitalized. he
 EMI4.8
 It is of course understood that the present inventio-i does not relate to the products which peruet to obtain the process which is the subject thereof as pharmaceutical products and that all the properties, methods due-
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 valuation and possible applications are only mentioned for the purpose of
 EMI4.10
 better in terms of results obtained thanks to the process ue preparation
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 ration object of the invention.

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 EMI5.1
 



  .! 'O.L.l} microscopic olo ic;, p; # -, synthetic gar y; â-r-s. -ar of Czapek Modified by Waksraan- 11>): i - :, ycelium substra-cal typical very ratified, lying, and right aerial mycelium to ra: ifications bearing straight or irregularly ratified chains of cylindrical or ovoid conidios measuring 0 , 7-
 EMI5.2
 1.0 x z, - 1, - microns. No conidial chain in separate spirals. Conidia usually scattered after 14 day incubation.



  After twenty-eight days one strain exhibits moderately abundant spores; those other strains produce very few spores.
 EMI5.3
 nP; ar-éJ.E: etr, lucose spar7, Fine: "- microscopic orpiio -Io analogous to that observed on the! 8.1?; synthetic ar-elgar. colony morphology # gar-synthetic agar (fourteen days to 30 C): Colonies 6 mf.1 this diameter, in relief \; '1. <. Slightly convex, with a very thin envelope of aerial mycelium of a slightly tinted white, colorless reverse.
 EMI5.4
 igar-é.J.,. u.r with glucose asûru;: in.e (fourteen days at d0 C):

   Colonies of b to a mm of wt .: .r; ietre, protuberance with asporogenous or sporulated central papillae. powdery aerial mycelium of a slightly tinted white. Intense creamy yellow reverse.



   Culture characteristics
 EMI5.5
 A / "ar-synthetic agar-: Readily to moderate amount of substrate mycelium, pale crest in color inside out. Trace of slightly stained white aerial mycelium. No soluble base. Other strains produce slightly. more aerial mycelium.When starch replaces sucrose as a carbon source, all strains show good vegetative and aerial growth, and all produce tur-
 EMI5.6
 characteristic bidity in the agar-agar around. the seeding strip. The color of the substatal mycelium, upside down, ranges from pale cream to creamy buff or intense brown.

   The color of the aerial mycelium usually ranges from a slightly tinted white to a very slightly grayish tint, and a soluble pigment is formed, the color of which ranges from pale yellowish brown to light brown.

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 EMI6.1
 



  Glucose asparagine on a.rar-aar: Moderate or clear growth of substrate r -ycelium, cream in color becoming pale ocher buff on the reverse. Moderate amount of powdery, pale cream-colored aerial uycelium which turns to pale ocher-buff after twenty-eight days incubation. Trace of pale greenish yellow soluble pigment. From at-
 EMI6.2
 very strains appear identical to the standard culture on the same medium.



  #b. rar-agar aoez starch (i: faksr: 1yr (1.:. page ,, b2, medium 15 with soluble ar..idon): Moderate amount of su7ostratulated mycelium whose underside is cream colored when new , and becomes buff cream
 EMI6.3
 me or brown Buckthorn at - end of observation. Moderate amount of smooth powdery aerial illycelium, white in young crops, becoming pale cream in color and eventually slightly gray during ripening. Trace of soluble pigment of pale creamy yellow color in young cultures, darkening to pale brown after 28 days Weak starch hydrolysis on plates (1-3 mm from edge of culture) after 14 days ; unchanged after twenty-eight days.

   Other strains show slight variations in the amount and color of the aerial mycelium and soluble pigment formed. Differences which appear to be quantitative. with
 EMI6.4
 Calcium agar-agar / glycerol-malate: Moderate amount of L1y: substrate celium which rises from a pale cream color when young to intense creamy yellow after twenty-eight days. Moderate amount of powdery smooth aerial mycelium, showing a faint, faint-tinted white color. No soluble pigment. Growth
 EMI6.5
 restricted to the dfinoculatio4 line. Insoluble sweep thinned out in lragar-sgar around the crop.



  : nutritive ar-aar de -.aksrnan: Moderate amount of substratal mycelium, cream colored inside out. Trace of white aerial mycelium. No soluble pepper. Growth restricted to the dinoculation line.
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  Agé! .R-a8r d'.1rerson: Abundant subratal mycelium, the underside of which is colored / cream (close to Lars' yellow) becoming Ner- brown

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 EMI7.1
 prun, after twenty-eight days. Moderate amount of powdery aerial mycelium, slightly tinted white, becoming pale buff when ripe.



  Rough and deeply cracked growth surface; transient droplets of clear exudate near the tip of the sloping stump. Trace of soluble light brown pilent, the quantity of which gradually increases
 EMI7.2
 with advancement cs 1.tincubat: 'OY1. The amount of ..: ß'C t. : .7.1: 'i, aerial l produced by different c strains varies 1: ..; and: -lr ,, rul1 des Jlre (; 1iu: .1 being speckled with i.' .: luS C; .. ¯ s sectors having a very gle color. The quantity of â w., ¯ :: x: t soluble brown also varies: uri x.i. = "Li'G'rt'i". greenish brown is cultivated.



  G.r: xan de ro :: ^ e d c5 earth: moderate amount of layctliura substrs-
 EMI7.3
 tal and airy, restricted and with a slightly rough surface (but
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 no ..¯1. '.: IL1L'Fe) - <VC ::.' t.iLilrt air white - Mild or moderate modification
 EMI7.5
 from the staining of the buffer to the gold.
 EMI7.6
 Gelatin: quantity r.:odirE> of culture El 'surface:' .. OC.C7..i..L, 4.'JCl'Éi'G 'ru- .'02';: r.li. no film i1r <.. & cto. scanty aerial .ycÉ-liunl,
 EMI7.7
 White. Scanty submerged growth. No soluble pigment. Vi-
 EMI7.8
 tess of moderate liquefaction to nothing in seven days, about halfway: .5lete (w5 - 35 rai) in fourteen days, and complete e, itre- fourteen
 EMI7.9
 and seventeen days according to the strain.
 EMI7.10
 



  Broth l - '. 8 synthetic culture (nutrient formula el' af '.' 2I.ra;: r: .... synthetic): Scanty growth of an ellicule: C ".aCGL1..E1'ItE: without aerial riyc,:; o! :: br.3J, its submerged colonies- attached to the walls of the tube. No soluble pl.-mei..t. ill :: '- = -.' "011 screw t'F '1OEill: P <.. 1..S growth.



  3oui2.1ori of ¯.: ..: t.CFSe 1 .. 't: i'? "? É.t..i: y: .i.; 'F: th pleated film, with itlyC <, .. ¯üt: = sparse white acricn, .. 1 "" "", ". {-. R GiCiCE.r CiO Crü = .S SI1C e sub- nier .- '. Ce ..,. ... see. Small quantity r:. 'S pi. ; - .. have ooluble brown below 1.- film.



  L-.i Ci: <: "Y. (;: -" "50l 13O C) -: .20: r.0 c r ',, :::': 3:;,: r: CE: of all the zouc strains ¯ ... ¯ ..,: t; :: -.; ro3 =;, r ..: LC: L.:. C; 1¯is:; & airec aerial uycelium:; ri. dull-, r ± -.?. from coā: on.:. '"... ,,'. n 1 ... (.J partial in eleven to fourteen

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 EMI8.1
 days and complete in fourteen to twenty-one days of breed.
 EMI8.2
 



  Pil = .icnr, very dark soluble hides the sunflower color after fourteen days; but before this moment, the neutral reaction becomes alkaline .. final pi (after twenty-eight days, on sample 1.10Y011): 7, tu at S, 00 - No differences;:. t, r-ju.aiite3 of the strains .



  Lait de tourna soi (J; .7 C) -: l: li :. the Ctltn; ¯; tI :: crit: i are identical to those occurring 3'-00. p final: 7, -0.
 EMI8.3
 



  Generally speaking, a preferred method of producing
 EMI8.4
 vancor.ycin is as follows: we inoculate =: xe claque ci r sterile nutrient agar-agar with spores of r.1icroor ",: ar.isn (J.:I''3.$iî'Gë .... I had incubated for several days at about 30 C to obtain G.8S spores
 EMI8.5
 in abundance. The spores are harvested and inoculated on a medium
 EMI8.6
 liquid nutrient which is used to prepare a vegetative inoculation seaence after fermentation. The inoculated medium is ... under aerobic conditions for forty-eight hours at about 26 ° C, during which time the spores develop into a semen form.
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 this vegetative.

   The vegetative seed thus obtained is used to inoculate a liquid artificial nutrient culture medium with a view to
 EMI8.8
 the production of large quantities of fermented rillon containing
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 vancomycin.
 EMI8.10
 



  0àn wants it, or. can omit the t & SS of spore formation and production of the vegetative form in the microorganism, and one can directly inoculate ..: ent the medium of r3-¯ ,. ¯t: n with the 'spores. One such method is. ) ricip8: 'c:!; - :; 2-c, linked to PrDr-lact4on small amounts of ¯ '.. tihioz.e.

   For 1 <roucion on a large scale, we prefer the inoc'iltion: nas, sÍ -r- = 3 au:. \ - '; e:, '(' 0 (, uC "ti: -J: l: -, u Koyen of a secierxj T, it C- ü '..:. sT2, to avoid the \' -". tc ?: nr.T, .c the growth of the micro and in CO-'15,; ē¯C2: .t. -: '..- 3 :: 7', J'Jj, of the apparatus3 .. 'â.¯-e; é.¯ de scale. arr s inoculation, we wiit- le' .li.L''L liquid production, under aerobic conditions-, 2.; e ';',: - 8r, re between about 15 and 37 C;: êY'.t.:, ..: -. t about -in.:> i:.:

   days..-. at the end of the incubation period, r "l..i.. ¯.r.! '- i :. 1 .quelle yïG ... ti'ii a substantial quantity

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 antibiotic, the mycelium is removed from the fermented broth
 EMI9.1
 by any suitable means, such as filtration or scion, and the antibiotic substances are then collected from the broth by suitable methods, such as, for example, absorption methods. using suitable absorbents, for example charcoal, ion exchange resins, etc. In this process, one
 EMI9.2
 uses the complexes of varzcorycin with organic reagents or its salts, for recovery.

   Purification can be achieved by countercurrent extraction using systems containing organic solvent, etc.



   The following examples illustrate the process for preparing vancomycin.



  Example 1,
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 A plate of agur-a ,, ar is prepared containing the following products:
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<tb> starch <SEP> 20 <SEP> gr
<tb>
 
 EMI9.5
 as-paragine 1 gr
 EMI9.6
 
<tb> extract <SEP> from <SEP> beef <SEP> 3 <SEP> gr
<tb>
<tb> agar-agar <SEP> 20 <SEP> gr
<tb>
<tb> water <SEP> 1 <SEP> liter
<tb>
 
 EMI9.7
 In the gold plating. inoculates Orientali8 spores, strain ': 1r3-05à6 ,. and incubated for about 10 days at 30 ° C. The medium is then covered with sterile distilled water and scraped to loosen the spores. The resulting spore suspension
 EMI9.8
 is kept for further use during the process.

   A liquid nutrient culture medium is prepared as follows:
 EMI9.9
 
<tb> glucose <SEP> 15 <SEP> gr
<tb>
<tb> <SEP> soy flour. <SEP> 15 <SEP> gr <SEP>
<tb>
 
 EMI9.10
 ::: a: -; Í ::; solid corn-steep res I.i.ca.Gr 'Su Chloride de 50di.um si * carbonate G6 :: calciua 2: "... 1:
 EMI9.11
 
<tb> water <SEP> 1 <SEP> liter
<tb>
 

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 EMI10.1
 vil sterilizes the medium at 10 G for 30 minutes in uu ± le.¯ cc ¯ ¯ rrié, and or: cools it. A cm 2 of the spore suspension prepared as indicated above is used to inoculate the medium.



  The inoculated medium laying t. <: - hours at o Q was stirred with an alternating firing re-agitator having a stroke of 5 cases, at 11 turns per minute. uses the .. '. place of cultivation feri .. =. nt'. containing a vegetative SClliè11Ce for ens, .. encry a T: Z3.C:., t of nu & ritive culture costing the following products:
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 i- .:

  blackstrap slices 20 gr
 EMI10.3
 
<tb> Peptone <SEP> from <SEP> soy <SEP> 5 <SEP> gr
<tb>
<tb> Glucose <SEP> 10 <SEP> gr
<tb>
<tb> Sucrose <SEP> 20 <SEP> gr
<tb>
<tb> Carbonate <SEP> of <SEP> calcium <SEP> 2.5 <SEP> r
<tb>
<tb> Water <SEP> 1 <SEP> binding
<tb>
 
The medium is placed in a container having a suitable excess capacity so as to ensure the presence of oxygen in sufficient quantity.
 EMI10.4
 sufficient, and sterilized by heating at 120 C for about 30 minutes. when it is cold, the medium is inoculated with about 25 cm3 of the vegetative seed described above and then the culture is shaken.
 EMI10.5
 ture for about 0 hours at 6 G.

   The pH of the medium is at the start of fermentation between about 5 and about z0 and the final pH is from about 7.0 to about 8.0. A fermentation broth
 EMI10.6
 tion thus obtained contains approximately 1G racg of vancomycin by this. Comte is prepared following an adc culture medium. snel for 1. vanconycin production at c.:>ra>Ae scale. n mixes the following products:

   
 EMI10.7
 
<tb> starch <SEP> 30 <SEP> parts
<tb>
<tb> molasses <SEP> 20 <SEP> parts
<tb>
<tb> Peptone <SEP> from <SEP> soybean <SEP> 7.5 <SEP> parts
<tb>
 
 EMI10.8
 Vébétale protein hvdrolyzate and acid (so-called quality: n5ta.ino type ri., 3tleyn}, 5 parts 2À} x! OGO parts

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 EMI11.1
 The pH of the medium is adjusted to approximately 7-7.) And then -pusve the solution to .rLl'liOC.ûv2 for 30 minutes under a Prussian steam: ': \: 1J.viroll 1.05:. .



  Another suitable culture medium is prepared for the pro- c: lctic ':::.:: ¯: slç: Y:., 8.! L :. 1: .s c ..1'rJl: .., .... W ... s1: ¯i V6.r: ts:
 EMI11.2
 Ariidon 30 reaches 7o.tz.ta of oil .. 13 parts hydrolyzed soybeans Products of- f -: - -.,; Tation solbics obtained from the .. 'retc'-ticn of als pc. r le ur- 2.C'3 -: :) "jc :: tylicum pe-ries i'au 1000 parts
 EMI11.3
 The medium is adjusted to pH 7.0-7.2 and passed to autoclave under 1.05 pressure at ::: ', 200 kls for 30 minutes, before u tz xi y and t 2 For the production of V211cof.1ycin using the Turkish culture media indicated above, these media are placed in reservoirs fitted with devices for the introduction of pressurized air, they are inoculated with a vegetative cultivation of microorgeü1Ísme .LT'lGitc: lS¯ and they are fermented for;

   about L - 6 days at temperatures between 3 and 33, while stirring and introducing about 0.4 volume of air per volume of culture per minute. then we
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 remove the mycelium from the culture medium and treat the broth
 EMI11.5
 clear to collect vancomycin, known shown in the examples
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 following.
 EMI11.7
 Example 2 ...



  A ci 'eU5er., STIC8 ..: 0.1J broth having the composition
 EMI11.8
 next:
 EMI11.9
 Glucose 6 seen gr Tryptone i, Cu r "Hstv..inbaet" (Ttio-cides produced ¯ CC'¯ ". '" 1.?'awi.W e: Z - ".- ¯c ii;: Li' tE 2Tït.Y 'prayer) ICC gr iiau 0 free
 EMI11.10
 We place free wine from the nilieu in a fermentation tank

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 of 40 liters capacity and sterilized by heating with steam under pressure at about 120 ° C for about 20 minutes.

   A
 EMI12.1
 the sterilized broth is cooled and inoculated aseptically, lu-, r ,, 4olit with spores of S Grientalis, LRRL 24 $ 0, obtained by scraping ala culture of this organism on an oblique streak of agar-agar, retort described above -above. The organism grows in the broth at about 32 C for a period of about 24 hours. During the growing period
 EMI12.2
 sance, 3sboui..lon is stirred at about 25 ovrs / rr: inutes and aerated with sterile T & ir -. rate of approximately gas volume of air per volume of culture broth per minute. After fermentation, broth is used to inoculate the production medium described below.



   In a 1.325 liter iron fermentation tank, a fermentation medium is placed, suitable for the production of vancomycin on a large scale and having the following composition:
 EMI12.3
 
<tb> Dextrin <SEP> white <SEP> 18.9 <SEP> kg
<tb>
<tb> Glucose <SEP> 14.2 <SEP> kg
<tb>
<tb> Hydrolysol <SEP> acid <SEP> from <SEP> vegetable <SEP> protein
<tb>
 
 EMI12.4
 (quality known as: n 3t.a-Kir.o type  3taleylt) 2, - kg Canone of meat ("Peptone of lîlson:

    159 ", wet) li., 2 kg Soluble products obtained by fenieritation of ai: 5 with iostridium acetobutylicum (Soluble iron # ntables ls-1) 1y, 73 kg Silicon anti-foaming agent (" àTItifoam tif
 EMI12.5
 
<tb> from <SEP> the <SEP> Dow-Corning: <SEP> to <SEP> the <SEP> request
<tb>
<tb> Water <SEP> for <SEP> to make <SEP> 945 <SEP> liters
<tb>
 The pH of the sulphide medium is found to be about 6.7
 EMI12.6
 (Ji '"and the culture medium is then sterilized by: heating with steam under pressure for about 120 G for about 120 G for approx. about 20 minutes Then cool 1 $ broth, and add a3epti ..- to about 16 liters of a seed obtained from the outlet described above. :

  , in the place for about 6 .nF? Tp a temperature of about * ± # 3O01 C.GÎ.Sr rC r'w'.f of growth, we have the

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 broth at about 150 revolutions / minute and sterile air is blown into the broth at the rate of 340 liters per minute, or about 0.4 volume of air per volume of broth per minute. From time to time, additional amounts of anti-foaming agent are added as required. At the end of the growth period, a microbiological test of the broth indicates that it has an antibiotic power equivalent to about 200 mcg of active vancomycin per cm3 of broth.

   The pH of the broth after fermentation is about 7.8. The broth is filtered to remove mycelium, and the clear filtrate is subjected to the treatments described below to remove vancomycin therefrom.



   The pH of 66 liters of filter is adjusted. culture broth at level 7 with concentrated hydrochloric acid and passed through 2 liters of an aqueous suspension of "Permutit DR" (decolorizing ion exchanger sold by the Permutit company, New York) at a pH 6.5 contained in a glass column of. 10 mm. The resin was previously reduced to the hydroxyl form from the sulphate form by passing through the bed of the column a volume of 4% MaOH at a rate of 3 cm3 / cm2 of cross-sectional area per minute. .



  The resin is washed for about an hour with distilled water at a rate of about 6 carbons per cm2 of cross-sectional area per minute. The resin is then washed with distilled water at a rate of about 0.5 cm3 / cm2 of cross-sectional area per minute until the pH of the effluent falls below 9.

   The effluent is discarded and the container containing the antibiotic substance absorbed is washed with 10 liters of water. after which the washes are rejected: with water. The antibiotic substance was eluted from the resin with 20 liters ci 1 a mixture comprising 1 part of glacial acetic acid, 30 parts of acetone and 69 parts of water. The eluate, whose activity is about 85 of that of the initial antibiotic in the broth, is concentrated in vacuo to a volume of 15 liters to remove acetone.

     The aqueous solution is adjusted to pH 7 with a HaOH solution and stirred pestantly for 80 minutes with 860 g of activated carbon ("Norit SG" sold by the "American Norit Company" in dacksonville, Florida). We separate

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 charcoal by vacuum filtration through Whatman N 4 filter paper, using a pad of "Hyflo Super-Cel" (diatomaceous earth sold by the "Johns-Manville Company") as filter aid. The filtrate is discarded and the charcoal is washed with 10 liters of water, then filtered again. The washing water is discarded.

   The antibiotic substance is eluted from the charcoal with three 10 liter portions each of 30% aqueous acetone acidified to pH 2 with concentrated sulfuric acid. The mixed uats, containing about 75% of the initial active product of the broth, are concentrated in vacuo to a volume of about 10 liters and the concentrate is neutralized to pH 7 with caustic soda solution. The neutralized aqueous solution is again concentrated to about 730 cm3 by evaporation in vacuo, with gradual acidification to a pH of about 2.1 by addition of sulfuric acid. The antibiotic substance is precipitated from the acidic solution by adding an equal volume of saturated picric acid solution, followed by refrigeration at about 5 ° C for about 16 hours.

   The cold suspension is centrifuged and the supernatant liquid is removed. The wet solid product is washed with 500 cm3 of ice water, the washing water is discarded and the solid product is dissolved in a mixture of 100 cm3 of methanol and 20 cm3 of hydrochloric acid in aqueous solution at 10 %. The acid solution is poured into 2 liters of acetone, which precipitates a light tan solid product.



  After standing at low temperature for about 2 hours, the suspension is separated by centrifugation and the liquid, supernatant, is discarded.



  The remaining solid product is washed with 2.8 liters of acetone and 200 µm3 of diethyl ether. The washings are separated by decantation and the solid product is dried overnight in vacuo. The vancomycin hydrochloride thus obtained is an amorphous powder of a whitish tan color which weighs approximately 10.88 g and which titers approximately 900 mcg / mg.



  Example 3.



     16.25 liters of filtered culture broth, obtained by the method of Example 2, are adjusted to pH 8.5 with caustic soda.

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 10% aqueous solution and are passed through about 6.5 liters of a cation exchange resin ("IRC-50", cation exchange resin of the carboxylic acid type sold by the company Rohm & Haas Coupany "). found in the sodium state at a pH of about 8.5. The resin was previously regenerated from the hydrogenated state with aqueous caustic soda at 4, up to a pH value greater than about
8.5, and then washed with water to pH 8.5.

   After having circulated the slurry, filtered through the resin, the latter is washed with water - until the effluent becomes colorless. The active antibiotic absorbed on the resin is eluted by passing 0.1 N sulfuric acid over the resin, and the effluent is collected until its pH reaches 2. (In this elution process, a dilute solution of ammonium hydroxide can also be used, as a variant of the method of separating the antibiotic agent from the resin). The eluate is neutralized by bringing its pH to 7 with a hot aqueous solution of barium hydroxide, the precipitated barium sulphate is filtered off and the filtrate is concentrated to a volume of about 2.25 liters. , by vacuum evaporation.

   This residue is stirred for about 60 minutes with 250 g of activated charcoal washed with acetic acid ("Norit SG"). The carbon suspension is filtered and the filtrate is discarded. The cake is washed with two liters of water, the washings discarded and the antibiotic product eluted from the charcoal with three 3 liter portions of 50% ethanol, acidified to a pH of about 2 with hydrochloric acid. The mixed eluates are concentrated in vacuo to a volume of about 20 µm and treated with an equal volume of saturated picric acid solution. The resulting antibiotic precipitate is separated by centrifugation. The supernatant liquid is discarded and the solid product is dissolved in 20 cm3 of methanol saturated with hydrochloric acid.

   About 200 cc of ether is added, after which a precipitate formed of vancomycin hydrochloride in the crude state is formed. The precipitated product is collected by filtration. The supernatant liquid is discarded, the crude hydrochloride residue is dissolved in methanol and reprecipitated by the addition of acetic acid.

 <Desc / Clms Page number 16>

 tone. The vancomycin hydrochloride thus prepared is a heavy white amorphous powder; about 440 mg and having an antibiotic activity of about 900 mc / mg.



  Example 4.



   Nineteen liters of a culture broth filtrate, obtained by the method of Example 2, are adjusted to a pH of about 7 with 10% aqueous sodium hydroxide, and the nelane filtered. neutral.



  The filtr @ t is passed through a column containing a bed of an ion exchange resin ("Permutit DR") 5 cm in diameter and 32 cm high, which has previously been brought to the hydroxylated state. according to the method described in Example 2. after passing all of the filtrate through the column, the ion exchange resin is washed with water until the influent is substantially colorless. .

   The antibiotic substance, which is retained in the column, is eluted from the resin using 5 liters of a mixture comprising 1 part of glacial acetic acid, 30 parts of acetone and 69 parts of water. The eluate is concentrated to about 800 cm3 by evaporation in vacuo. The resulting cloudy aqueous residue was adjusted to pH 7.0 by addition of 10% aqueous sodium hydroxide, whereupon a clear solution formed. 2200 g of activated carbon ("Korit SG") are added to the neutralized solution. After having mixed vigorously, the charcoal is separated by filtering and washing vigorously with water.

   The filtrate and the washings are discarded and the vancomycin on the charcoal is eluted with 6 liters of 50% ethanol, the pH of which has previously been adjusted to 2 with sulfuric acid. The eluate is concentrated by evaporation in vacuo to a volume of about 500 cm 3, and the residue is filtered. The filtrate is adjusted to a pH of about 3 by the addition of a heated aqueous barium hydroxide solution, then concentrated again to a volume of about 80 cm3. To the concentrated solution is added 150 cm of a saturated aqueous solution of picric acid. The above-mentioned vancomycin picrate forms and is separated by centrifugation.

   Discard the supernatant and add

 <Desc / Clms Page number 17>

 to the solid residue a mixture containing 10 cm3 of 6N sulfuric acid in 100 cm3 of aqueous acetone, whereupon the vancomycin picrate is dissolved and converted into vancomycin sulfate which remains in solution. 800 cm3 of a-
 EMI17.1
 ketone, which precipitates vancornycin sulfate. The precipitate is separated by filtration. , washed with acetone and dried. The vancomyci sulphate thus obtained weighs about 1.125 g and is an amorphous, salt-white powder which, in the microbiological test, yields about 00 n.cg / ml.



    Example 5.



   64 grams of a crude preparation of vanccmycin hydrochloride obtained by the method of Example 2 were dissolved in 13 liters of water, giving the test about 820 mog / mg and containing a considerable amount of brown pigment. The solution is adjusted to pH about 7.0 with solid sodium hydroxide, and becomes cloudy as it is neutralized. The cloudy solution is acidified to a pH of about 5.0 with concentrated hydrochloric acid, whereupon the solution becomes clear.

   The clear acid solution is stirred for
 EMI17.2
 approximately one hour with 1420 g of activated carbon ("JMorit SG" ', which has been washed successively with acetic acid and water and dried in an oven). The charcoal is filtered off and washed vigorously with water. The washed charcoal cake is eluted with 3 successive portions of 50% ethanol adjusted to a pH of about 3 with hydrochloric acid, the volume of the first eluate being 1/5 liters and that of the two. following eluates being 12.5 liters.
 EMI17.3
 dn concentrates the eluates combined with ē <v: a. 3 crr, under vacuum, the pH being L.ain'G8nu at about 3.

   The concentrated solution is poured into 4 liters <¯. cold acetone, after which a white precipitate of vanconycin hydrochloride sol is formed. The acetone containing the precipitate of va: c0..yci ::: le hydrochloride is cooled for about 2 days and then filtered. This filter is washed with acetone and with ether and vacuum dried for about 2 hours. We end-

 <Desc / Clms Page number 18>

 undermines the drying of the solid product by allowing it to stand overnight at room temperature. A total amount of 39 g of white amorphous vancoi.ycin hydrochloride is obtained and it is observed,
 EMI18.1
 per microbiolonic test, that it has an antibiotic activity of about 9C0 mcg / mg.



  Example 6.



  One dissolves a -tnirie of vancorivcin hydrochloride (prepared according to the procedure '-' "- of Example 5) in 20 ml of 75% aqueous methanol and the pH of the solution is just about, by addition of Dilute aqueous sodium hydroxide A precipitate of free vancomycin forms and is allowed to stand for about 18 hours at 5 ° C. The resulting white suspension is subjected to centrifugation, the solid product washed with successive portions of. cold methanol and ether, and dried in vacuo.



   The prepared vancomycin weighs approximately 815 mg and is an amorphous, slightly tinted white powder, yielding 950 mcg / mg euso.



   Electrometric titration of v ancomycin in solution
 EMI18.2
 allyl-formamide and water (2: 1 parts by volume) ;. whose starting pH is 7.75, reveals the presence of ionizable groups of pK'Ó at 5.2 - 6.1 - 7.5 - 9.3 - 10.1 and 11.5.



  Example 7.



   A solution is prepared containing 43 g of vacomy sulphate.
 EMI18.3
 cine, giving the test about 0CO mcgÍmg, in one liter of water. The clear solution, which has a pH of about 2.2, is adjusted to a pH of about.
 EMI18.4
 ron o, 1 by the addition of a branded anion exchange resin ("IR-45") in basic or hydroxylated form. Gn repairs the cloudy supernatant liquid by decantation and the resin is washed with 500 cm3 of water.



  Wash water is added to the supernatant liquid and the pH of the solution is lowered to about 1.6 by addition of a sodium exchange resin.
 EMI18.5
 cations mark (": -: - 45") under the acid orne, The resin is separated from the liu;: ide cl ¯r by filtration and the filtrate is treated again with pane root chét. ,,:} se d ' basic anions mark ("IR -45") until

 <Desc / Clms Page number 19>

 that the pH is about 7.2. The surna- liquid is decanted again.
 EMI19.1
 #: ar.t, or- acidified it to a pH of about 1.8 with Acic Acidic Acid Resin and filtered.

   We net suitcase:>;: .. t; e the filter with union exchange residence b &6L; L: i ;; at pH 7.0 the supernatant liquid is decanted and ::. f e.judte 2 :. a ph ¯¯ 'ùI; V: .. 2' ± 1i..H: y with hydrochloric acid v ::: solution & (:: Ii v.6e to IL .. ', &. solution result- aunt, containing C! ....: hydrate of ve ....:, ço ... ycÍw :: ', (; tj concentrated under reduced pressure at ur.' urn of about 150 c nz,. ¯ Now add the concentrated solution to 2 liters of cold acetone. The mixture is refrigerated for about 4 days, after which the white precipitate is filtered off. Vancomycin hydrochloride which is formed and washed successively with portions of acetone and ether.

   The wet solid product was subjected to drying under reduced pressure for 2 days, and then allowed to stand in air to complete the drying. We obtain a total yield
 EMI19.2
 of 39) 4 gr of vancor hydrochloride: amorphous white 1ycin which, in the microbiological test, contains approximately 925 Iacg / mg ..



   As a guide, the inhibitory action of the antibiotic vancomycin hydrochloride against representative microorganisms is listed in Table I. Antibacterial activities were determined by the broth dilution test method. In this method, the organisms to be tested are cultivated in a nutritive broth con-
 EMI19.3
 holding various concentrations of vancomycin. The concentrations shown in the table are the minimum concentrations of vancomycin, in mog / cm3 of substrate, which inhibit the growth of the test organisms during incubation periods of 24 and 48 hours, respectively, at 37 C.

 <Desc / Clms Page number 20>

 



  TABLE I
 EMI20.1
 
<tb> Inhibitory <SEP> concentration
<tb>
<tb> Body <SEP> to <SEP> try <SEP> mcg / cm3
<tb>
<tb>
<tb> 24 <SEP> hours <SEP> 48 <SEP> hours
<tb>
<tb>
<tb>
<tb>
<tb> Bacillus <SEP> brevis <SEP> 3.13 <SEP> 3.13
<tb>
<tb>
<tb>
<tb>
<tb> Bacillus <SEP> cereus <SEP> 3.13 <SEP> 3.13
<tb>
 
 EMI20.2
 Bacillus licheni.cr..is 0.7 o78 Bacillus .eatler: ¯. gas 0.2 Eacillus polymyxa 0.39 0.39 Bacillus subtilis 0.9 0.9 Gorynebacterium diphteriae at 0178 1 micrococcus pyogones var.aureus 0.7 oe78 wepyogenes var4aureus (resistant to erythroniy-cine) 1.56 1.56 .Pvogenes var.aureus (resistant to - #### penicillin) o78 0278 1;

  i'.p7ogenes var.aureus (streptomycin resistant) 1.56 1.56 1'l.PYOP, Ones var.aureus H,, iîycobacterium phlei 12.5 25
 EMI20.3
 
<tb> Mycobacterium <SEP> smegmatis <SEP> 3.13
<tb>
<tb> Sarcina <SEP> lutea <SEP> 0.78 <SEP> 0.78
<tb>
<tb> Achromobacter <SEP> fischeri <SEP>> 100 <SEP>> 100
<tb>
<tb> Aerobacter <SEP> aerogene <SEP>> 100 <SEP> 100
<tb>
<tb> Brucella <SEP> bronchiseptica <SEP> 100 <SEP>> 100
<tb>
<tb> Escherichia <SEP> coli <SEP>;

  > 100 <SEP>> 100
<tb>
 
 EMI20.4
 Klebsiella pneumoniae 1GG / -2iQ0 Kycobacteriua avium 100 100
 EMI20.5
 
<tb> Proteus <SEP> vulgaris <SEP>> 100 <SEP> 100
<tb>
<tb> Pseudomonas <SEP> aeruginosa <SEP>> 100 <SEP>> 100
<tb>
 
 EMI20.6
 Shigella paradysenteriae .1GG> 1E70
 EMI20.7
 
<tb> Candida <SEP> albicans <SEP>> 100 <SEP>> 100
<tb>
<tb>
<tb> Saccharomyces <SEP> carlsbergensis <SEP>> 100 <SEP> 100
<tb>
<tb>
<tb> Aspergillus <SEP> niger <SEP>> 100 <SEP>> 100
<tb>
<tb>
<tb> Trichophyton <SEP> rubrum <SEP>> 100 <SEP>> 100
<tb>
 

 <Desc / Clms Page number 21>

 substances known according to the classification system of
Waksman, but the comparison shows that vancomycin is clearly different from other antibiotics.



   Netropsin: active against gram-negative and toxic bacteria, its toxicity being acute (LD50), when injected aveinously into a white mouse ;, 17 mg / kg; comycin hydrochloride, 453.7 mg / kg.



   Amiketin: extractable by a solvent and having a stronger activity against mycobacteria than against gram-positive bacteria; vancomycin is not solvent extractable and is active against gram-positive bacteria.



     Griseoflavin: soluble in ethyl acetate; vancomycin is insoluble in ethyl acetate.



   Sulfactin: soluble in chloroform, vancomycin is insoluble in chloroform.



     Vinacetin: soluble in organic solvents, such as chloroform, ethyl acetate, butyl acetate and acetone; vancomycin is insoluble in these solvents.



   Cinnamycin: inactive against gram-positive rod-shaped bacteria; vancomycin is active against these bacteria.



     Viomycin; mainly active against mycobacteria; vancomycin is active against gram-positive bacteria ... ¯
Nocardine: active against E.Coli; vancomycin is not active against E. Coli.



   Erythromycin: soluble in ethyl acetate; vancomycin is insoluble in ethyl acetate;
Carbomycin: soluble in ethyl acetate; vancomycin is insoluble in ethyl acetate.



   Micromonosporin: a protein; vancomycin is not a protein.

 <Desc / Clms Page number 22>

 



  Neonocardine: active against E.Coli; vancomycin is inactive against this microorganism.



     Cariicin: soluble in butanol and stimulates against fungi and bacteriophages; vancomycin is insoluble in butanol and active against gram-positive microorganisms.



     Proactinomycin: soluble in ethyl acetate; vancomyein is insoluble in ethyl acetate.



     Picromycin: can be extracted with ether; vancomycin is insoluble in ether.



   The accompanying drawing shows chromatograms on paper obtained by chromatography, with different solvent systems, vancomycin and other representative antibiotic agents. In each of the figures, the baseline of the paper chromatogram is indicated for each antibiotic by a short straight line.



   Figure 1 shows the chromatogram obtained when vancomycin (VA), viomycin (VI), streptothrici. ne (ST), cinnamycin (CI), neomycin (NE), dihydrostreptomycin (DI), hydroxystreptomycin (HY), mannosidostreptomycin (MA) and catenulin (CA) are deposited on ahatman filter paper No. 4 and are developed with an aqueous solvent comprising 80% ethanol and 1.5% sodium chloride, buffered with 0.95 M sodium sulfate and sodium acid sulfate monohydrate (0.05 M).



   FIG. 2 represents the chromatogram obtained when the antibiotic substances listed above are deposited on a Chatman n 1 filter paper, and that they are developed with a solvent consisting of a mixture of 2 parts of n-Butanol, of 1 part acetic acid and 1 part water.


    

Claims (1)

CLAIMS 1. Process for the preparation of an antibiotic agent, characterized in that a strain ofStreptomyces Orientalis producing vancomycin is cultivated under aerobic conditions in a culture medium containing assimilable sources of carbohydrate, d nitrogen and mineral salts until these microorganisms show in this culture medium an activity - antibiotic note @@@. 2. Method according to claim 1, characterized in that the strain of Stroptomyces Orientalis producing vancomycin is cultivated in a culture medium containing carbohydrate, nitrogen, and mineral salts assimilated. bles, under submerged aerobic conditions, and vancomycin is collected in this culture medium. 3. Method according to either of claims 1 and 2, characterized in that raaitient the culture medium at a temperature of about 25 to 37 C, and one continues to grow the microorganism for a period about 1 to 5 days. 4. Method according to any one of the preceding claims, characterized in that a strain of Streptomyces "Orientalis, strain NRRL 2450 is used. 5. Method according to any one of claims 1 to 3, characterized in that Streptomyces Orientalis, strain NRRL 2451 is used. 6. Process according to any one of claims 1 to 3, characterized in that Streptomyces Orientalis, strain NRRL 2452 is used. 7. Process according to any one of the preceding claims, characterized in that the antibiotic agent is reacted with an acid to form an addition salt of this agent with an acid. 8. Process for the preparation of vancomycin and its <Desc / Clms Page number 24> addition salts with an acid substantially as described above. 9. Vancomycin and its acid addition salts prepared by the process according to any preceding claim. 10. Antibiotic substance of the kind consisting of vancomycin and its addition salts with an acid, said vancomycin being characterized as follows: white amorphous substance soluble in water, insoluble in most organic solvents, stable in organic matter. aqueous solution having a pH of about 2.5 to 9, having a molecular weight of between about 3000 and 3200; containing about 7.17 percent nitrogen, and an emulsion of mineral oil and vancomycin has a maximum absorption spectrum in the infrared region at the following wavelengths, expressed as microns: 3.10- 3.41- 6.05 - 6.30 - 6.72 - 6.84 7.26 - 7.64 - 8.13- 8.50- 8.66- 8.87- 9 , 42 - 9.76 - 10.10 - 10.34 and 11.24. 11. Vancomycin hydrochloride. 12. Vancomycin sulfate.