Field of the invention

The present invention relates to a method for the fermentation of cyclopeptides such as pneumocandins in the presence of increased concentrations of any or all of calcium, copper, iron, magnesium, manganese, molybdenum and zinc ions.

Background of the invention

Cyclopeptides are polypeptides in which the terminal amine and carboxyl groups form an internal peptide bond. Hence, cyclopeptides do not have a terminal amine or carboxyl group, although non-terminal amine and carboxyl groups may be present stemming from individual amino acids such as aspartic acid, glutamic acid, lysine and the like. Several cyclopeptides are known for their advantageous medicinal properties. An excellent example is the class of echinocandins which are potent antifungals. Cyclopeptides can be naturally occurring compounds but may also be obtained by total synthesis or by synthetic or enzymatic modification of naturally occurring or naturally produced precursors; the latter class is referred to as semi synthetic cyclopeptides. Examples of medicinally useful echinocandins are the cyclic hexapeptides anidulafungin, caspofungin, cilofungin and micafungin which are useful in treating fungal infections especially those caused by Aspergillus, Blastomyces, Candida, Coccidioides and Histoplasma. These cyclic hexapeptides are characterized in that they comprise threonine and a proline derivative such as 3-hydroxyproline, 4-hydroxyproline and/or 3-hydroxy-4-methylproline. Anidulafungin, caspofungin and micafungin are all semi synthetic cyclopeptides derivable from naturally occurring echinocandins such as for instance echinocandin B, pneumocandin A ₀ or pneumocandin B ₀ .

Pneumocandin B ₀ (1 , wi t h R-i = C(0)(CH ₂ ) ₈ CH(CH3)CH ₂ CH(CH3)CH ₂ CH ₃ ), first disclosed in US 5,202,309, is a compound that can be obtained fermentatively, for instance in Glarea lozoyensis as described in WO 00/08197. The compound is an important intermediate in the preparation of therapeutically active semi synthetic cyclopeptides such as caspofungin, as described in WO 2010/128096 and references cited therein.

Although nature can provide a substantive part of the complex chemical structure of semi synthetic cyclopeptides, and in many cases having all chiral centers in the required configuration, a major disadvantage nevertheless is that during fermentation often side products are formed that carry through the process and eventually end up as impurities. Only in few cases and after extensive research resulting in unpredictable breakthroughs, can fermentation processes be tuned in such a way as to prevent formation of these impurities. Particularly when these impurities are structurally closely related to the main product, their removal is usually tedious and often requires unprecedented purification approaches as the main products in question are chemically unstable and/or prone to racemization.

I n the case of pneumocandin B ₀ a multitude of structurally related impurities occurring during fermentation has been described. Examples are compounds having an additional methyl function (such as pneumocandin A ₀ , pneumocandin A-i , pneumocandin A ₂ , pneumocandin A ₃ , pneumocandin A4, pneumocandin A ₅ , pneumocandin A ₆ ), compounds lacking one or two hydroxyl groups (such as pneumocandin B-i , pneumocandin B ₂ , pneumocandin B ₅ , pneumocandin B ₆ , pneumocandin E ₀ ), compounds having a 4-hydroxy proline rather than a 3-hydroxy proline moiety (pneumocandin C ₀ ) or compounds having additional hydroxyl groups (such as pneumocandin D ₀ , pneumocandin D ₂ ).

Improvement of the fermentation process is the subject of WO 00/08197, which document addresses increase in the production titer of pneumocandin B ₀ , but also reduction of the formation of unwanted structurally related impurities. Notably favorable results where achieved by supplementing the amino acids arginine, glutamine, hydroxyproline, ornithine, proline or threonine and the trace elements boron, calcium, cobalt, copper, iron, manganese, molybdenum, nickel and zinc. Despite the above improvements, still efforts are required to further optimize the productivity of the process and/or the quality (notably the purity) of the product. For instance, minimizing the pneumocandin C ₀ impurity is the subject of US 2009/0291996 advocating to purify crude pneumocandin B ₀ by chromatography followed by crystallization from a solvent- antisolvent mixture. Given the very high similarity between desired structure and impurity, not only in terms of the many different chemical reactive sites present in both molecules, but also in terms of charge, hydrophilicity and molecular weight, such a separation is laborious, time consuming and usually has a significant negative effect on the recovery yield of the desired product. Hence, there remains a need to further improve the pneumocandin B ₀ fermentation process, either in terms of productivity of the desired product or in terms of reduction of undesired side products or both. Given the limited knowledge available on the mechanism of these highly complex natural processes combined with the endless permutations that could be envisaged in process development studies, such process improvements are difficult to predict and require inventive breakthroughs.

Detailed description of the invention

In the context of the present invention, the term "cyclopeptide" (also known as cyclic peptide or cyclic protein) refers to a polypeptide chain of which the amino and carboxyl termini are linked together with a peptide bond that forms a circular chain. The cyclopeptides of the present invention may be equipped with substituents such as acyl groups connected through an amide bond to an amino group. In addition the cyclopeptides of the present invention comprise natural and/or non-natural amino acids. In the context of the present invention the cyclopeptides comprise an amino acid chosen from the list consisting of 3-hydroxyproline, 3-hydroxy-4-methylproline, 4- hydroxyproline and proline. The number of amino acids linked together in the cyclopeptides of the present invention is from 3 to 20, preferably from 4 to 12, more preferably from 5 to 10, most preferably from 6 to 8. Preferred cyclopeptides in this respect are cyclohexapeptides such as aculeacin, echinocandin B (A30912A), FR901379, L-671329, mulundocandin, pneumocandin (pneumocandin A ₀ , A-i , A ₂ , A ₃ , A4, A5, A ₆ , Bo, Bi , B ₂ , B ₅ , B ₆ , C ₀ , D ₀ , D ₂ , E ₀ ), S31794/F1 and sporiofungin. All such antifungals are structurally characterized by a cyclohexapeptide core, or nucleus, the amino group of one of the amino acids bearing a fatty acid acyl group forming a side chain.

The term "metal ion concentration" refers to the total amount of the metal ion referred to as added prior to or during the fermentation, per amount of initial medium present at the start of the fermentation and can be expressed in g.kg ^"1 , mg.kg ^"1 , mol.kg ^"1 , mmol.kg ^"1 and the like.

The term "nutrient" refers to a chemical that an organism can use to live and grow or to a substance used in an organism's metabolism which must be taken in from its environment. Organic nutrients include carbohydrates, fats, proteins (or their building blocks, amino acids) and vitamins. Inorganic nutrients include water, oxygen and dietary minerals such as metal ions, examples of which are calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, zinc and the like. A nutrient is said to be essential if it must be obtained from an external source, either because the organism cannot synthesize it or produces it in insufficient quantities. The effects of nutrients are dose-dependent and shortages are referred to as deficiencies.

In the first aspect of the present invention there is disclosed a method for the preparation of a cyclopeptide comprising an amino acid chosen from the list consisting of 3-hydroxyproline, 3-hydroxy-4-methylproline, 4-hydroxyproline and proline which method comprises fermenting a culture of Aspergillus sp., Coleophoma sp. or Glarea sp. in the presence of nutrients comprising calcium ions, copper ions, iron ions, magnesium ions, manganese ions, molybdenum ions and zinc ions, characterized in that the metal ion concentration of said calcium ions is at least 0.4 mg.kg ^"1 and/or of said copper ions is at least 0.15 mg.kg ^"1 and/or of said iron ions is at least 3 mg.kg ^"1 and/or of said magnesium ions is at least 70 mg.kg ^"1 and/or of said manganese ions is at least 5 mg.kg ^"1 and/or of said molybdenum ions is at least 0.15 mg.kg ^"1 and/or of said zinc ions is at least 0.7 mg.kg ^"1 .

Suitable microorganisms are Aspergillus sp., Coleophoma sp. and Glares sp. Notably Aspergillus aculeatus (for instance for the production of aculeacin as described in Takeshima et al., J. Biochem. (1989) 105, 606), Aspergillus nidulans and Aspergillus rugulosus (for instance for the production of echinocandin B), Aspergillus sydowii (for instance for the production of mulundocandin as described in Mukhopadhyay et al., J. Antibiotics (1992) 45, 618), Coleophoma empetri F-11899 (for instance for the production of FR901379) or Glarea lozoyensis (for instance for the production of pneumocandins as described in WO 00/08197) are suitable in the context of the present invention. Optimal results in terms of productivity and decrease of side products are achieved when the cyclopeptide contains at least one amino acid chosen from the list consisting of 3-hydroxyproline, 3-hydroxy-4-methylproline, 4-hydroxyproline and proline. A preferred example is pneumocandin B ₀ as produced by Glarea lozoyensis, preferably Glarea lozoyensis CBS 131548.

The predominant conclusion from the prior art such as WO 00/08197 is that fermentation of pneumocandin B ₀ in Glarea lozoyensis benefits from a nutrient medium containing a high residual sugar concentration, trace elements, proline and threonine. The only investigation with respect to metals deals with the trace elements zinc, cobalt and nickel leading to the conclusion that zinc and cobalt reduce the titer of the main product with concomitant increase of the titer of unwanted impurities whereas nickel had no effect on the titer of the main product but increases the formation of almost all impurities. Similar detrimental effects are also reported for copper and zinc by N. Connors et al. {Appl. Microbiol. Biotechnol. (2000) 54, 814-818) and L.A. Petersen et al. {J. Ind. Microbiol. Biotechnol. (2001 ) 26, 216-221 ). Although there is no suggestion as to how to improve cyclopeptide fermentation, metal ion concentrations suggested in WO 00/08197, to be 0.23 mg.kg ^"1 for calcium ions (0.83 mg.kg ^"1 CaCI ₂ .2H ₂ 0), 0.08 mg.kg ^"1 for copper ions (0.21 mg.kg ^"1 CuCI ₂ .2H ₂ 0), 1 .67 mg.kg ^"1 for iron ions (8.3 mg.kg ^"1 FeS0 ₄ .7H ₂ 0), 39.44 mg.kg ^"1 for magnesium ions (0.4 g.kg ^"1 MgS0 ₄ .7H ₂ 0), 2.70 mg.kg ^"1 for manganese ions (8.3 mg.kg ^"1 MnS0 ₄ .H ₂ 0), 0.09 mg.kg ^"1 for molybdenum ions (0.16 mg.kg ^"1 (NH ₄ ) ₆ Mo ₇ 0 ₂₄ .4H ₂ 0) and 0.39 mg.kg ^"1 for zinc ions (1 .7 mg.kg ^"1 ZnS0 ₄ .7H ₂ 0). In the present invention it is surprisingly found that both an increase in biomass growth, productivity and a decrease in unwanted impurities can be obtained when increasing the metal ion concentration of any or all of the metal ions calcium, copper, iron, magnesium, manganese, molybdenum and zinc to a level that is from 1 .5 to 10 times as high as used in the prior art and against what is advocated in the prior art, preferably from 1 .7 to 5 times as high, more preferably from 1 .8 to 2.5 times as high. Thus, preferred metal ion concentrations for these metal ions according to the present invention are: calcium, between 0.4 and 1 mg.kg ^"1 ; copper, between 0.15 and 0.5 mg.kg ^"1 ; iron, between 3 and 10 mg.kg ^"1 ; magnesium, between 70 and 200 mg.kg ^"1 ; manganese, between 5 and 15 mg.kg ^"1 ; molybdenum, between 0.15 and 0.5 mg.kg ^"1 ; zinc, between 0.7 and 2 mg.kg ^"1 . A major advantage of increased biomass growth and productivity is that fermentations can be performed under a carbon limitation regime. Any of the said seven metal ions may be present at the concentration ranges mentioned above, however also two or more or all seven metals may be present at said metal ion concentration ranges.

In one embodiment the cyclopeptide is isolated following its formation during fermentation. Isolation can be carried out according to procedures known to the skilled person such as precipitation, extraction using organic solvents, crystallization, chromatography and combinations thereof.

In another embodiment, for the production of pneumocandin B ₀ a continuous or repeated discontinuous feed of nitrogen in the form of ammonia or ammonium hydroxide during the fermentation is found advantageous as high titers could be obtained while at the same time the consumption of proline could be drastically minimized. Proline is an amino acid that, according to WO 00/08197, is an important nutrient for reduction of the levels of pneumocandin Co, however when following this prior art process also a high consumption of the relatively expensive proline occurs. Yet another advantage is that the present invention not only results in reduction of the amount of proline needed, but also in the circumvention of the need for proline dosing resulting in a less complicated and more robust process design. Preferably said feed of nitrogen in the form of ammonia or ammonium hydroxide during the fermentation is carried out such as to maintain an ammonia concentration in a range of from 0.1 -5 g.L ^"1 . In yet another embodiment, the cyclopeptide obtained according to the present invention may be further converted to other, semi synthetic cyclopeptides. Thus, pneumocandin B ₀ is converted into caspofungin according to known procedures as described in WO 2010/128096 and references cited therein. This may be carried out with pneumocandin B ₀ as directly obtained during fermentation but preferably with pneumocandin B ₀ obtained during fermentation and further purified and/or isolated.

In the second aspect of the present invention there is disclosed a strain of a Glarea sp. that is Glarea lozoyensis CBS 131548, deposited at the Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. The strain of the second aspect is suitable for producing cyclopeptides according to the method of the first aspect and thus exhibits increased productivity and/or decreased side product formation at increased metal ion concentrations, particularly upon production of pneumocandin B ₀ .

Legend to the Figures

Figure 1 depicts the production of pneumocandin B ₀ by Glarea lozoyensis (see Examples for details). X-axis: time in hours after inoculation, Y-axis: production of pneumocandin B ₀ in mg.kg ^"1 .

Figure 2 depicts the ratio of the unwanted impurity pneumocandin C ₀ during the production of pneumocandin B ₀ by Glarea lozoyensis. X-axis: time i n hours after inoculation, Y-axis: ratio (pneumocandin Co / (pneumocandin B ₀ + pneumocandin C ₀ )).

Figure 3 depicts the biomass formation during the production of pneumocandin B ₀ by Glarea lozoyensis. X-axis: time in hours after inoculation, Y-axis: production of biomass in g.kg ^"1 .

Figure 4 depicts the production of pneumocandin B ₀ per gram of biomass by Glarea lozoyensis. X-axis: time in hours after inoculation, Y-axis: production of pneumocandin B ₀ per gram of dry weight biomass in mg.g ^"1 .

All Figures depict results in the four different media outlined below. In all media except medium 1 , the fermentation is carried out under carbon limitation.

Medium 1 (□): Using Expresa yeast extract and metal ion concentrations of

0.23 mg.kg ^"1 for Ca ²⁺ , 0.08 mg.kg ^"1 for Cu ²⁺ , 1 .67 mg.kg ^"1 for Fe ²⁺ , 39.44 mg.kg ^"1 for Mg ²⁺ , 2.70 mg.kg ^"1 for Mn ²⁺ , 0.09 mg.kg ^"1 for Mo ⁶⁺ and 0.39 mg.kg ^"1 for Zn ²⁺ .

Medium 2 (■): As medium 1 with double metal ion concentrations.

Medium 3 ( A ): As medium 1 with double metal ion concentrations and Gistex yeast extract paste LS instead of Expresa yeast extract.

Medium 4 (·): As medium 1 with triple metal ion concentrations and Gistex yeast extract paste LS instead of Expresa yeast extract. EXAMPLES

Equipment used

Laboratory fermentors with a gross volume of 10 L were equipped with a Rushton turbine and a pH and DO probe both of Ingold. Control of fermentations took place with Braun DCCU-2 equipment and a Braun MFCS/WIN system version 3.0 both supplied by B. Braun Biotech International. Shake flasks (2000 mL) equipped with three bottom baffles and a foam plug were used in an Innova 4330 refrigerated incubation shaker.

Stock solutions

Glucose stock solution:

Dextrose (500 g) was dissolved or suspended in approximately 800 mL of de- mineralized water, the volume was adjusted to 1 L and the mixture was sterilized for 20 minutes at 121 °C and cooled down to room temperature before use.

Trace elements stock solution:

The indicated amount of hydrochloric acid was added to approximately 250 mL of de- mineralized water after which all other components were dissolved and the weight was adjusted to 1 kg by addition of water. The mixture was stored at 4°C (shelf life 6 months). Seed fermentation

Expresa 2200S yeast extract medium preparation (Seed medium 1 ):

Gistex yeast extract LS paste medium preparation (Seed medium 2):

Materials of fraction 1 were dissolved or suspended in the order given, in 80% of the final volume of tap water. The pH was brought to 5.7 ± 0.1 with NaOH 4N or H ₂ S0 ₄ 4N and the volume was adjusted to the desired volume. Shake flasks (2000 mL) equipped with three baffles were filled with 500 mL of medium and were sterilized for 20 minutes at 121 °C. After sterilization and cooling down 25 mL of sterile dextrose solution was added to the 2000 mL flasks containing fraction 1 . Seed incubation:

' Glares lozoyensis CBS 131548, deposited at the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. Vials comprise mycelium in glycerol solution

In the next stage, the fermentor was inoculated with the whole content of seed phase 2.

Fermentation

For the fermentation of pneumocandin B ₀ , four different media were used:

Medium 1 :

Based on WO 00/08197, Table 2; concentrations of di-potassium hydrogen phosphate, magnesium sulphate, trace elements stock solution and L-proline were equal. Sodium sulphate was added to increase osmotic pressure. The original recipe contained mono sodium glutamate, which was replaced on N-basis replaced by ammonium sulphate and Difco Yeast extract, which was on N-basis replaced by Expresa yeast extract. Citric acid was added as a chelating agent to prevent precipitations.

Medium 2:

As medium 1 however with double concentrations of di-potassium hydrogen phosphate, magnesium sulphate and trace elements stock solution.

Medium 3:

As medium 1 however with double concentrations of di-potassium hydrogen phosphate, magnesium sulphate and trace elements stock solution and with Gistex yeast extract LS paste instead of Expresa yeast extract.

Medium 4: As medium 1 however with triple concentrations of di-potassium hydrogen phosphate, magnesium sulphate and trace elements stock solution and with Gistex yeast extract LS paste instead of Expresa yeast extract. Also the fermentations using this medium were carried out with a simplified feed scheme in which the sugar, normally dosed during the exponential part of the feed, was now dosed directly in the batch.

Media were prepared in two fractions that are sterilized separately. Fraction 1 is 5/6 of the total volume after sterilization, fraction 2 is 1/6 of the total volume.

Preparation fraction 1 : components were dissolved in tap water, representing 80% of the fraction volume, after which water was added to the appropriate volume and the solution was sterilized for 30 minutes at 121 °C.

Preparation fraction 2: D-fructose was dissolved in tap water, representing 50% of the fraction volume, after which water was added to the appropriate volume and the solution was sterilized for 30 minutes at 121 °C.

Both fractions were added to the fermentor after cooling down and the pH was adjusted to 5.35 ± 0.15 using ammonium hydroxide 12.5%.

Fermentation conditions:

The fermentation was carried out under atmospheric pressure using an amount of seed of 8.3%. The DO probe was calibrated at 1 00% under the fermentation cond itions described below, and before inoculation. During the fermentation the following set points were applied:

Feed preparation:

The feed (D-fructose 200 g.kg ^"1 ) was prepared by dissolving D-fructose in 50% of the desired volume of hot tap water after which water was added to the desired value and the solution was sterilized for 20 minutes at 121 °C.

Feed start:

After start of growth the pH drops to the lower boundary of the control (pH = 5.20) and base was added. When batch sugar was finished the pH value increased . Feed was started when the pH reached a value of 5.35.

Feed profile:

For fermentations 1 -3 using 40 g.kg ^"1 D-fructose as batch dosage the following feed profile was applied:

Time Set point

(Hours after feed start) (g_S.kg ¹ starting weight)

FS - 36 3.0 x e ^(a ° ^{1b x HAha)}

36 EOF 5.15 For fermentation 4 with medium 4 the sugar (70 g.kg ^"1 D-fructose), normally dosed during the exponential part of the profile, was already added to the batch. For this experiment the following feed profile was used:

Time Set point

(Hours after feed start) (g_S.kg ¹ starting weight)

36 EOF 5.15

Applicant's or agent's file reference number28443-WO-PCT | International appliiccaattiion No.

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 136

The indications made below relate to the microorganism referred to in the description

first mentioned on page 5 line 16.

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet

Name of depositary institution

CENTRAAL BUREAU VOOR SCHIMMELCULTURES

Address of depositary institationftncluding postal code andcountry)

Uppsalalaan 8

P.O. Box 85167

NL-3508 AD Utrecht

The Netherlands

Date of deposit 15 December 201 1 Accession Number CBS131548

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet

We inform you that the availability of the microorganism identified above, referred to Rule 13bis PCT,hall be effected only by issue of a sample to an expert nominated by the requester until the publication of the mention of grant of the national patent orwhere applicable, for twenty years from the date of filing if the application has been refused, witlirawn or deemed to be withdrawn.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE(ifthe indications are not for all designated States)

E. SEPARATE FURNISHING OF TNDICATIONSf/eove blank if not applicable)

The indications listed below will be submitted to the International Bureau lnter(specify the general nature of the indications e.g., ''Accession Number of Deposit ")

Form PCT/RO/134 (July 1992)