BACKGROUND OF THE INVENTION [02] Ansamitocins are highly cytotoxic compounds derived from fermentation of microorganisms such as Actinosynnema pretiosum. Ansamitocins have been chemically converted into thiol-containing maytansinoids, whose therapeutic use in the form of cell binding agent-maytansinoid conjugates has been described (US Patents 5,208, 020; 5,416, 064; 6,333, 410; 6,441, 163).

[03] The fermentation process with Actinosynnema spp strains such as Actinosynnema pretiosum produces several ansamitocin species bearing different ester substituents at C-3 (Fig. 1). The various C-3 esters produced include P-3 (iso-butyryl), P-3' (n-butyryl), P-2 (propionyl), P-4 (iso-valeryl), P-4' (n-valeryl). All of these esters can be reductively cleaved to give the C-3 alcohol maytansinol (P-0), which is the precursor for the synthesis of thiol- containing maytansinoids. In addition, minor amounts of undesired ansamitocins which are modified at other sites, such as N-demethyl, 20-O-demethyl, and 19-deschloro are produced.

Upon reductive de-acylation, these ansamitocins do not produce maytansinol.

[04] Processes for the production of ansamitocin from fermentation of Actinosynnema spp have been described (US Patents 4,162, 940; 4,450, 234; 4,228, 239; 4,331, 598; 4,356, 265).

The yield of ansamitocins produced varies, with titers generally ranging from 12 mg/L to 100 mg/L. The ansamitocins are typically recovered and purified by a multistep process involving addition of a filter aid and an organic solvent to the whole fermentation broth, followed by concentrating the organic layer and precipitation with petroleum ether. The precipitate was further purified using silica chromatography and crystallization, followed by further purification by recrystallization or chromatography.

[05] Thus, the process is cumbersome and involves several steps where highly toxic material has to be handled. This renders the scale up of such a process very difficult. In addition, the safety of the human operator has to be ensured throughout the various processing steps.

[06] A recent application (US 2002/0015984 Al) claims certain improvements in the process for the production of ansamitocins. The ansamitocin titer in the fermentation broth are reported to have ranged from 65 to 86 mg/L. The claimed improvements included heat inactivation of the broth at 75 °C, extraction into an aromatic hydrocarbon solvent such as toluene, chromatography through an open silica column, followed by crystallization.

[07] In order to reduce the cost of ansamitocin production, new Actinosynnema spp strains that give significantly higher titers (up to 400 mg/L in fermenters) than those previously described, have been produced. The processes previously described for the production of ansamitocins have several drawbacks, and thus cannot be adapted for the new high producing strains that have been developed. For example, heat inactivation at 75 °C results in some degradation of ansamitocin and a 10 to 20% loss in yield. Extraction of fermentation broth containing high ansamitocin content with aromatic hydrocarbons is inefficient and incomplete, since the ansamitocins are not highly soluble in such solvents. Purification of ansamitocins on open self-packed silica columns has two drawbacks: 1) lot to lot variability in purity and recovery, and 2) significant human exposure resulting in safety concerns.

[08] Thus, a need exists to produce ansamitocins in high yields and also to provide an efficient process for its isolation and purification, while minimizing worker exposure to the highly toxic drug.

SUMMARY OF THE INVENTION [09] One aspect of the invention is a process for the large-scale fermentation of ansamitocin-producing microorganisms, the process being especially applicable to a new, highly productive ansamitocin-producing strain. Another aspect of the invention is a method for isolating the crude ansamitocin, and for preparing purified ansamitocins comprising the following steps: 1) optional inactivation of the culture for example, by exposure to heat at about 50 ° to 55 °C or by addition of 1 % by volume of chloroform, 2) extraction with a non-aromatic organic solvent, 3) concentration of the extract, 4) purification of the concentrate on a silica or alumina column, preferably a pre-packed silica cartridge column, and 5) crystallization of the product.

[10] Other aspects of the invention include the following: A process for preparing purified ansamitocins comprising the steps of : 1) Culturing an ansamitocin-producing microorganism in a liquid culture medium; 2) Treating the culture medium to facilitate solvent extraction of ansamitocins; 3) Extracting ansamitocins from the culture medium with a non-aromatic water immiscible solvent; 4) Concentrating the extracted ansamitocins; and 5) Purifying the ansamitocins by any one of a), b), c) or d): a) absorption chromatography over silica gel or alumina, b) crystallization, c) absorption chromatography over silica gel or alumina followed by crystallization, d) crystallization followed by absorption chromatography over silica gel or alumina.

A process for preparing purified ansamitocins comprising the steps of: 1) Culturing an ansamitocin-producing microorganism in a liquid culture medium; 2) Treating the culture medium to facilitate solvent extraction of ansamitocins; 3) Extracting ansamitocins from the culture medium with a non-aromatic water- immiscible solvent; 4) Concentrating the extracted ansamitocins; and 5) Purifying the ansamitocins by absorption chromatography over silica gel or alumina followed by crystallization.

A process for preparing purified ansamitocins comprising the steps of: 1) Culturing an ansamitocin-producing microorganism in a liquid culture medium; 2) Extracting ansamitocins from the culture medium with a non-aromatic water immiscible solvent; 3) Concentrating the extracted ansamitocins; and 4) Purifying the ansamitocins by any one of a), b), c) or d): a) absorption chromatography over silica gel or alumina, b) crystallization, c) absorption chromatography over silica gel or alumina followed by crystallization, d) crystallization followed by absorption chromatography over silica gel or alumina.

A process for preparing purified ansamitocins comprising the steps of: 1) Culturing an ansamitocin-producing microorganism in a liquid culture medium ; 2) Extracting ansamitocins from the culture medium with a non-aromatic water immiscible solvent; 3) Concentrating the extracted ansamitocins ; and 4) Purifying the ansamitocins by absorption chromatography over silica gel or alumina followed by crystallization.

Ansamitocins prepared by one of the above-described processes.

A cell-binding agent maytansinoid complex prepared by linking at lease one maytansinoid derived from an ansamitocin prepared by any one of the above-described processes, the cell-binding agent preferably being an antibody.

A process for extracting ansamitocins from fermentation broth, said process comprising: 1) Optionally inactivating ansamitocin-producing microorganisms in the fermentation broth; and 2) Extracting the ansamitocins from the fermentation broth with a non- aromatic water-immiscible solvent.

[11] In the above-described methods, it is preferred that the ansamitocin-producing microorganism is Actinosynnema spp. , more preferably Actinosysznenza pretiosuna. The ansamitocin-producing microorganism may alsobeActinosynnema pretiosum ATCC 31565 or strains derived therefrom or Actinosynnema pretiosum PP4-4 (ATCC PTA-3921) or strains derived therefrom.

BRIEF DESCRIPTION OF THE DRAWINGS [12] Figure 1 shows the structure of various ansamitocin C-3 esters that can be made by fermentation, as well as the C-3 alcohol, maytansinol (P-0).

DETAILED DESCRIPTION OF THE INVENTION [13] Methods are provided for culturing a microorganism highly productive for ansamitocins in a liquid culture medium in large fermenters. Methods are also provided for the extraction of the ansamitocin from the culture broth and the microorganism into a non- aromatic water-immiscible organic solvent in which the ansamitocin is highly soluble, and for the purification of the ansamitocin by passage through a silica or an alumina column if needed, preferably these columns are pre-packed cartridges, followed by crystallization of the product. If necessary the microorganism may be inactivated by heat treatment or treatment with chloroform prior to the extraction step.

[14] The purified ansamitocins which could include a mixture of various C-3 esters, such as ansamitocins P-3, P-3', P-4, P-4', P-2 and P-1 (Fig. 1), may be treated with a reducing agent to give the desired C-3 hydroxyl compound, maytansinol (P-0). The purified ansamitocins typically contain only minor amounts of undesirable ansamitocins that have modifications on sites of the molecule other than the C-3 position. Preferably, the ansamitocin-producing strain is Actinosynemma spp. More preferably, the microorganism is Actinosynnema pretiosum. The microorganism can also be Actinosynizenta pretiosum PF4-4 (ATCC PTA-3921) and derivatives thereof and Actinosynnema pretiosum ATCC 31565 and derivatives thereof. The microorganism can be grown by fermentation culture techniques that are known to those skilled in the art, using the specific media described herein or any other media that is described in the art (US Patents 4,162, 940; 4,450, 234; 4228,239 ; 4,331, 598; 4,356, 265).

[15] One embodiment of the method of the invention is the culture of the microorganism in a liquid culture medium. Growth of the bacterial strain PF4-4 is performed under controlled conditions and can employ a wide variety of media and conditions. For example, PF4-4 can be grown under similar conditions and with similar media to those described for ATCC 31565 or ATCC 31281 in issued U. S. Patents 4,137, 230; 4,162, 940; 4, 331, 598 ; 4,356, 265; 4,450, 234; and as described in Hatano et al., Agric. Biol. Chez. 48,1721-1729, 1984. Thus, the strain PF4-4 tolerates a wide variety of carbon sources, which also support fermentative production of ansamitocins. Exemplary growth media are given in Tables 1 and 2. Table 1 shows media that support production of ansamitocins by ansamitocin-producing microorganisms, such as Actinosynnenaa pretiosum PF4-4 and Table 2 shows further media suitable for the propagation and/or growth of Actinosyfzhema pretiosum PF4-4, and other ansamitocin-producing microorganisms.

Table 1. Production Media Composition (entries are % w/v). FM 27-44 FM 112-37 FM 4-4 FM 4-6 FM 4-7 Dextrin 6 6 5 5 5 (Lodex-5) Maltose 4 4 2 2 2 (Difco) Proflo 2. 0 2. 5 2. 75 (Traders) Soybean 1. 5 2. 0 Flour (ADM) Pharmamedia 0. 5 (Traders) CSP 0. 5 0. 5 0. 5 0. 15 0. 15 (Roquette) P. Dry Yeast 0. 25 (Difco) MgS04-7H20 0. 05 (Wako) CaCO3 0. 5 0. 5 0. 6 (Hayashi) (NH4) 2SO4 0. 05 (Wako) KH2P04 005 004 (Wako) 0. 05 0. 06 0. 06 0. 06 (Wako) CaC12-2H20 0. 5 0. 5 (Wako) NaHCO3 0. 2 (Wako) Zeolite 0. 1 FeS04-7H20 0. 0002 0. 0002 0. 0002 0. 0002 (Wako) ZnS04-7H20 0. 0002 CoCl2-6H20 0. 001 0. 0005 0. 0005 (Baker) Nicotinic 0. 0002 Acid MnSO4 H2O 0. 0002 Isobutanol'0. 1 05 05 03 03 (Tedoa) SAG471 0 0. 06 0. 04 0. 04 (Witco) pH 6. 8 6. 8 6. 8 7. 2 7. 35 Sterilization was 121 °C for 20 minutes. Added last.

TABLE 2 Related Media Slant and plate culture, CM41 A¢ar (%, w/v) Yeast extract (Difco) 0.3 Malt extract (Difco) 0.3 Soytone (Difco) 0.5 Glycerol (Difco) 1.0 Bacto Agar (Difco) 2.0 Adjust pH to 6.5 before sterilization; Sterilization: 121 °C, 20 minutes Seed Medium, VM4-1' (%, w/v) Soluble starch (BDH) 2.0 Glucose (Shuling) 1.0 Soybean meal (ADM) 1.0 Corn Steep Liquor (Solulys) 0.5 Soytone (Difco) 0.5 NaCl (Wako) 0.3 CaCO3 (Hayashi) 0.5 pH 6.8 ; Sterilization: 121 °C, 20 minutes [16] Preferred methods for fermentative production of ansamitocins from strain PF4-4 are further described in EXAMPLES 1 and 2 below.

[17] Fermentation: The cultivation may be conducted by culture conditions such as, stationary, shaking, aerobic submerged or any other culture conditions. For good culture growth and high production of ansamitocins in large tank fermenters, aerobic submerged culture is preferred. The ansamitocin production can be further enhanced by feeding of nutrients during the fermentation. For example, when cultivating the organism in FM4-6 medium, additional feeding of glucose for the duration of the fermentation or of glucose for about the first 24 to 72 hours, preferably for about the first 48 h, followed by feeding with glucose and a protein nutrient such as cotton seed flour (for example Proflo or Pharmamedia from Trader's Protein, Memphis, TN) or soybean flour, and an alcohol or an aldehyde to facilitate the formation of the C-3 ester side chain, such as isobutanol, isobutyraldehyde, n- butanol, n-butyraldehyde, n-propanol, n-propionaldehyde, isopropanol, isopropionaldehyde, pentanol, valeraldehyde, isopentanol, isovaleraldehyde until the end of the fermentation can result in doubling of the ansamitocin production. While the culture conditions depend on the media used and the production scale, it is normally preferred to carry out the fermentation in the pH range of about 5 to 9, preferably with a starting pH of about 6.5 to 8.0. More preferably, the pH range is about 7 to 8, even more preferably about 7 to 7.4. The most preferred pH is about 7.2. The temperature can range from about 15° to 35 °C, with a preferred range of about 25° to 30 °C. More preferably, the temperature is about 28 °C. The fermentation is continued until the maximum ansamitocin accumulation has been achieved.

The cultivation time may vary and depends on several factors including the culture method, the composition of the medium, and the temperature. Typically, the fermentation time ranges from 96 to 336 h.

[18] Analysis of ansamitocins: In U. S. Patents 4,331, 598 and 4,450, 234, the parental strain ATCC 31565 is disclosed as producing two classes of ansamitocins that are distinguished by the presence of a methyl or hydroxymethyl group at C-14 (see Fig. 1). For both classes, several different ansamitocins are produced that differ in their respective acyl side chain bound to the C-3 hydroxyl group, and with respect to whether C-14 carries a methyl or hydroxymethyl group (or, in subsequent studies, N-desmethyl). The nomenclature used herein for the permuted compounds is defined above with reference to Fig. 1.

[19] Ansamitocin P-3 is the major product of PF4-4 and the parental strain ATCC 31565, under certain growth conditions. If the bacteria are grown in the presence of valine or isobutyric acid (see U. S. Patent 4,228, 239), isobutyl alcohol or isobutyraldehyde (see U. S.

Patent 4,356, 265) other ansamitocin compounds are present only in minor amounts.

[20] When PF4-4 strain is grown in different fermentation media (designated FM in Table 1), which all contain isobutyl alcohol, ansamitocin P-3 is the predominant ansamitocin produced. In one method to assay the amount of ansamitocins, samples of the fermentation broths are diluted with ethanol or acetonitrile, then shaken strongly and finally centrifuged.

The supernatant is then assayed for ansamitocin P-3 content.

[21] Ansamitocins are preferably fractionated and analyzed by reverse phase high performance liquid chromatography (HPLC), but any suitable technique, such as, for example, MALDI-TOF or thin-layer chromatography may be used. In one method, fermentation broths are extracted with organic solvents, such as ethyl acetate, methylene chloride or chloroform, and the content of P-3 in the organic solvent is determined by HPLC, using reverse phase C18 or C8 columns.

[22] Extraction of Ansamitocins: Ansamitocins can be extracted from the fermentation broth by methods generally employed for the recovery of secondary metabolites. Since the ansamitocins are readily soluble in organic solvents, they can be easily extracted by stirring with non-aromatic water-immiscible solvents such as alkyl acetates wherein the alkyl chain is linear or branched and has 1-5 carbon atoms, dialkylketones and halogenated solvents.

Examples of suitable alkyl acetates include n-butyl acetate, ethyl acetate, and methyl acetate.

An example of a suitable dialkylketone is methyl isobutyl ketone. An example of a suitable halogenated solvent is dichloromethane. Extraction with n-butyl acetate is preferred.

[23] Prior to extraction, the microbes in the fermentation broth may be inactivated, if desired, by exposure to mild heating at about 50° to 55 °C for about 30 minutes to 1 hour, or by addition of 1% (v/v) chloroform (Toru Hasegawa et al. 1983, Iras. J. Syst. Bacteriol.

33: 314-320). The extraction is carried out at a pH near neutral, preferably at a pH of about 6.0 to 7.0, and more preferably about 6.5 to 7.0, and preferably with n-butyl acetate. In order to improve the efficiency of the extraction, the broth may be maintained at a temperature of about 30° and 45 °C during the extraction process. The extraction time depends on several factors including the broth composition, the broth temperature, and the solvent used for extraction and can typically range from 1 hour to 120 hours, depending on the efficiency of the extraction process. The organic solvent can be separated, and concentrated under reduced pressure to give a residue that contains the ansamitocins.

[24] Purification of Ansamitocins: The crude product can be subjected to purification procedures such as adsorption chromatography over silica gel or alumina, followed by recrystallization if needed. Preferably, the chromatography is conducted on a prepacked column, such as a Biotage silica gel cartridge using the Biotage chromatography system. The desired ansamitocins can be eluted from the column using a solvent gradient starting with a mixture of ethyl acetate and hexane and adding increasing amounts of methanol. The fractions containing the desired ansamitocins can be pooled and concentrated. If desired, the ansamitocins can be further purified by crystallization using a solvent such as ethyl acetate to dissolve the product, and then adding a non polar solvent such as heptane or hexane to crystallize out the pure product.

[25] Cell-Binding Asent/Maytansinoid Complexes : The process of the invention can be used to make cell-binding agent/maytasinoid complexes which are useful as tumor-activated pro-drugs. Ansamitocins prepared by the process of the invention can undergo reductive cleavage to maytansinol which can be used as described in U. S. Patent Nos. 5,208, 020, 5,416, 064,6, 333,410 and 6, 441, 163 to produce N-methyl-L-alanine containing maytansinoid derivatives. These derivatives are then conjugated to cell-binding agents, preferably antibodies, via various linkers such as disulfide-containing linkers.

EXAMPLES [26] The invention will now be illustrated by reference to non-limiting examples.

EXAMPLE 1 Production of Ansamitocins : [27] Primary Seed: Seed culture medium VM4-1' (400 ml/flask) comprising of 2% soluble starch, 1% glucose, 1% soybean meal, 0.5% corn steep liquor, (Roquette) 0.5 % Soytone, 0.3% sodium chloride, and 0.5% calcium carbonate was poured into each of eleven 2 L capacity Erlenmeyer flasks. After sterilization, each of the flasks was inoculated with the PF4-4 (ATCC PTA-3921) culture. The flasks were incubated at 28 °C on an orbital shaker at 230 rpm for 48 h.

[28] Secondary Seed: The contents of the primary seed flasks were then pooled. A 300 L fermenter was filled with 100 L of the VM4-1'seed medium. After sterilization, the fermenter was inoculated with 4 L of the pooled primary seed culture. The fermenter was maintained at 28 °C, with agitation at 80 rpm. The dissolved oxygen level was maintained above 30% saturation by aeration and increased agitation if needed. After incubation for 24 h, the secondary seed culture was ready to be transferred into the production vessels.

[29] Production: Two 300 L production fermenters were each filled with 250 L of production medium FM4-6 (see Table 1). After sterilization, the fermenters were each inoculated with 15 L of the secondary seed culture. The fermenters were maintained at 28 °C with agitation at 107 5 rpm and aeration at 0.4 vvm. After day 2, the dissolved oxygen content was maintained above 30% by increasing the agitation rate to a maximum of 170 5 rpm, and the aeration rate to a maximum of 1 vvm. The ansamitocin titer was measured daily by withdrawing a sample of the fermentation broth and diluting into ethanol, followed by quantitation by HPLC analysis. The fermentation was continued until day 10, at which point, the ansamitocin accretion had leveled off. The ansamitocin titer on day 10 in the two fermenters was 251 mg/L and 244 mg/L respectively. The pH of the fermentation broth was adjusted to 6.5 by addition of phosphoric acid. The fermenters were heated up to 55 °C and maintained at this temperature for 1 h to inactivate the microorganism. The fermenters were then cooled down to ambient temperature for extraction with organic solvent.

EXAMPLE 2: Production of Ansamitocins using a fed batch process.

[30] A 1500 L production fermenter was filled with 900 L of production medium FM4-6 (see Table 1). After sterilization, the fermenter was inoculated with 54 L of the secondary seed culture, prepared as described above. The fermenter was maintained at 28 °C with agitation at 107 5 rpm and aeration at 0.4 vvm. From 0 to 48 h, an aqueous solution of 28.5% glucose was fed at a rate of 0.39 mL/L/h. From 48 to 288 h the feed was switched to a stock solution comprising 21. 5% glucose, 7. 1% Proflo and 7. 1% isobutanol, which was fed at the rate of 0.51 mL//L/h. After day 2, the dissolved oxygen content was maintained above 20% by increasing the agitation rate to a maximum of 170 5 rpm, and the aeration rate to a maximum of 1 vvm. The ansamitocin titer was measured daily by withdrawing a sample of the fermentation broth and diluting into ethanol, followed by quantitative analysis by HPLC.

The fermentation was continued until day 13, at which point, the ansamitocin accretion had leveled off. The ansamitocin titer on day 13 in the fermenter was 304 mg/L. The pH of the fermentation broth was adjusted to 6.5 by addition of phosphoric acid.

[31] Heat Inactivation. The fermenter was heated up to 55 °C and maintained at this temperature for 1 h to inactivate the microorganism. The fermenters were then cooled to between 30 and 40 °C for extraction with organic solvent.

EXAMPLE 3. Extraction and Chromatographic Purification of Ansamitocins.

[32] The fermentation broth from Example 2 was mixed with an equal volume of n-butyl acetate. The mixture was maintained between 30 and 40 °C to 45 °C, and stirred gently so that mixing of the two phases was just occurring at the solvent interface. The extraction was continued for up to 5 days, or, until HPLC analysis of the organic layer indicated that >80% of the ansamitocins had been extracted. The organic layer was then separated, and evaporated using a falling film evaporator to a final volume of between 20 and 50 L. The concentrated extract was transferred into a flask containing 2.2 kg of silica gel. The crude ansamitocins were coated onto the silica gel by evaporating the solvent to dryness using a rotary evaporator, operating under reduced pressure. The coated silica was then transferred to a <BR> sample injection module (SIM), obtained from Biotage, Inc. , Charlotesville, VA. The SIM was washed with a mixture of cyclohexane and hexane (2: 1 v/v), and then connected to a Biotage 150M system equipped with a silica cartridge. The desired product was eluted from the column using a mixture of ethyl acetate: hexane: methanol (29.4 : 68. 6: 2.0, v/v/v).

Fractions containing ansamitocins were pooled and the solvent was evaporated under reduced pressure. The product was further dried under high vacuum for 24 h.

EXAMPLE 4. Recrystallization of Ansamitocins.

[33] The dry product from the step above was dissolved in hot ethyl acetate (23 mL/g residue). The mixture was maintained between 60-75 °C, until complete dissolution of the ansamitocins was achieved. Heptane (80 mL/g residue) was added slowly, while maintaining the temperature of the batch between 60-75 °C. After all the heptane had been added, the batch was allowed to cool to room temperature. The crystals were recovered by filtration and then dried under high vacuum to give 221 grams of pure ansamitocins.

Biological Deposits [34] Actinosyiznema pretiosuin strain PF4-4 was deposited on December 11, 2001, with the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108, under the terms of the Budapest Treaty, and accorded Accession No. ATCC PTA-3921. The parent strain ATCC 31565, from which Actiyaosynnema pYetiosum strain PF4-4 was derived, is described in U. S. Patents 4,331, 598 and 4,450, 234, and was deposited with the ATCC on September 11,1979. ATCC 31281 is described in U. S. Patent No. 4,162, 940 and 4, 356,265, and was deposited with the ATCC on March 31,1977.

[35] While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.

[36] All publications referred to herein are expressly incorporated by reference.