Method of crystallizing narasin

Abstract

A method of crystallizing narasin includes heating acetonitrile to a first temperature in a range of 45°C to 55°C, adding narasin having an amorphous structure to the acetonitrile to form a mixture of the acetonitrile and the narasin, stirring, for a first time, the mixture for at least two hours at a second temperature in a range of 45°C to 55°C, and cooling the mixture to a third temperature in a range of 3°C to 10°C and stirring, for a second time, the mixture for at least three hours to obtain crystallized narasin, wherein the crystallized narasin has a polymorph crystalline structure.

Description

[0001] METHOD OF CRYSTALLIZING NARASIN

[0002] RELATED APPLICATION

[0003] This application claims priority under 35 U.S.C. 119 to U.S. Provisional Patent Application No. 63 / 694,185 and U.S. Provisional Patent Application No. 63 / 694,186, both filed September 12, 2024, the disclosures of which are incorporated herein by reference in their entirety.

[0004] FIELD OF DISCLOSURE

[0005] The present disclosure relates to a method of crystallizing narasin.

[0006] BACKGROUND

[0007] FIG. 1 is a conventional structural formula for narasin. Narasin is a polyether antibiotic produced by Streptomyces aureofaciens that is primarily used in animal agriculture. It functions by altering ion transport across cell membranes, which disrupts the energy balance of certain microorganisms, especially gram-positive bacteria and coccidia parasites. Because of this activity, narasin is widely employed as a feed additive to improve feed efficiency, promote weight gain, and control coccidiosis, a parasitic disease that can cause severe intestinal damage in livestock.

[0008] However, conventional methods of crystallizing narasin only produces low-purity narasin and cannot consistently yield a same particular polymorph. The conventional methods also fails to produce narasin that has a stable crystalline structure that can endure solvents and heat. Therefore, there is a need for a narasin crystallization method that produces narasin that has a very stable crystalline polymorph and consistently provides high purity of narasin.

[0009] SUMMARY OF DISCLOSURE

[0010] The method of crystallizing narasin according to the present disclosure may include: preparing acetonitrile; adding amorphous narasin to the acetonitrile; heating a mixture of the acetonitrile and the amorphous narasin to a first predetermined temperature while the mixture is being stirred; and stirring the mixture for a first predetermined time at a second predetermined temperature; stirring the mixture for a second predetermined time at a third predetermined temperature to obtain crystallized narasin having a polymorph crystalline structure. In addition, in the method, the crystallized narasin may be characterized by one or more of: peaks in an XRPD diffractogram at 20 values of [4.3, 8.6, 9.2, 9.5, 12.7, 5.1, 17.5 °] ± 0.1°, but absent or substantially absent of a peak at 5 to 8, 10 to 11, 14 °20; peaks in an XRPD diffractogram at 20 values of [15.2, 16.9, 19.3, 15.3, 17.0 °] + 0.1°, but absent or substantially absent of a peak at 5 to 8, 10 to 11, 14 °20 in an XRPD diffractogram as substantially illustrated in FIG. 14.

[0011] In addition, in the method, the crystallized narasin may exhibit an XRPD diffractogram as substantially illustrated in FIG. 14 and a TGA / DSC thermograph as substantially illustrated in FIG. 15.

[0012] In addition, in the method, the first predetermined temperature may be 45 °C to 55 °C.

[0013] In addition, in the method, a ratio of an amount of the narasin having an amorphous structure to the acetonitrile may be 1:3.5 to 1 :4.5.

[0014] In addition, in the method, the second predetermined temperature may be 45 °C to 55°C.

[0015] In addition, in the method, the first predetermined temperature and the second predetermined temperature may be same.

[0016] In addition, in the method, the third predetermined temperature may be 2°C to 10°C.

[0017] In addition, in the method, the stirring for the second time may be performed for about 17 hours.

[0018] In addition, in the method, the crystallized narasin may have a lipid content of 0.05% or less.

[0019] In addition, in the method, the polymorph crystalline structure may have an anhydrate form, and the crystallized narasin may be water-free and solvent-free.

[0020] In addition, in the method, the crystallized narasin may have a melting point in a range of 188°C to 193°C and a melting enthalpy of 57 J / g to 59 J / g.

[0021] In addition, in the method, the crystallized narasin may be vapor-resistant, and the crystallized narasin may be capable of maintaining the polymorph crystalline structure when the narasin is exposed to solvents.

[0022] In addition, in the method, the crystallized narasin may be capable of maintaining the polymorph crystalline structure up to 9 days after water is introduced to the crystallized narasin.

[0023] In addition, in the method, the stirring of the mixture at the third predetermined temperature may be performed for at least 5 hours. In addition, the method may further include: after the stirring for the second time, filtering the mixture to obtain the crystallized narasin having the polymorph crystalline structure; and drying the crystallized narasin in a vacuum at a fourth predetermined temperature in a range of 30°C to 50°C for at least 15 to 19 hours.

[0024] In addition, in the method, the crystallized narasin may have a purity of 97 % or above.

[0025] In addition, in the method, the crystallized narasin may have a solubility in a range of 0.0015 to 0.0020 mg / mL.

[0026] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

[0027] BRIEF DESCRIPTION OF DRAWINGS

[0028] FIG. 1 illustrates a conventional structural formula for narasin.

[0029] FIG. 2 illustrates a flowchart for a conventional method of isolating narasin.

[0030] FIG. 3 illustrates a flowchart for another conventional method of isolating narasin.

[0031] FIG. 4 illustrates a flowchart for a method of isolating narasin according to one embodiment of the present disclosure.

[0032] FIG. 5 illustrates an example of a lab-scale IL glass jacketed reactor according to the one embodiment of the present disclosure.

[0033] FIG. 6 illustrates an example of an industry-scale 20L glass jacketed reactor according to the one embodiment of the present disclosure.

[0034] FIG. 7 illustrates an example of an industry-scale 50L stainless steel (or metal) jacketed reactor according to the one embodiment of the present disclosure.

[0035] FIG. 8 illustrates a flowchart of the specific steps S 110, S130 included within the step SI 00 according to FIG. 4.

[0036] FIG. 9 illustrates a flowchart of the specific steps S210, S230, S250, S270 included within the step S200 according to FIG. 4.

[0037] FIG. 10 illustrates a flowchart for specific steps S610, S630, S650, S670 within the step S600 according to FIG. 4.

[0038] FIG. 11 illustrates the form and color changes of the filtrate (i.e., the slurry solution) in the steps of the isolation method according to the one embodiment of the present disclosure. FIG. 12 illustrates a result of the high-performance liquid chromatography (HPLC) on the crystallized narasin according to another embodiment of the present disclosure.

[0039] FIG. 13 illustrates an XRPD result of the narasin isolated according to the isolation method of the one embodiment of the present disclosure.

[0040] FIG. 14 illustrates an XRPD result of the narasin crystallized according to the crystallization method of the another embodiment of the present disclosure.

[0041] FIG. 15 illustrates thermogravimetric and Fourier-transform infrared spectroscopy (TG-FTIR) and differential scanning calorimetry (DSC) results of the crystallized narasin according to the one embodiment of the present disclosure.

[0042] FIG. 16 illustrates a microscope view of the amorphous narasin according to the one embodiment of the present disclosure and a microscope view of the crystallized narasin according to the another embodiment of the present disclosure.

[0043] FIG. 17 shows a dynamic vapor sorption (DVS) test result of the crystallized narasin crystallized narasin according to the another embodiment of the present disclosure.

[0044] FIG. 18 illustrates different examples of crude narasin broth with different concentrations and potencies according to the one embodiment of the present disclosure.

[0045] FIG. 19 illustrates an example of the crystalline polymorph product that results from the crystallization method according to the another embodiment of the present disclosure.

[0046] FIG. 20 illustrates examples of the steps of the crystallization method being performed according to the another embodiment of the present disclosure.

[0047] FIG. 21 illustrates an overview of the isolation method according to the one embodiment of the present disclosure and the crystallization method according to the another embodiment of the present disclosure.

[0048] FIG. 22 illustrates a flowchart for a method of crystallizing narasin according to another embodiment of the present disclosure.

[0049] DETAILED DESCRIPTION

[0050] Method of Isolating Narasin

[0051] Hereinafter a method of isolating narasin will be described. FIG. 4 illustrates a flowchart for a method of isolating narasin according to one embodiment of the present disclosure. According to the one embodiment of the present disclosure, the method of isolating narasin may include: a step SI 00 of adding acetone to a crude narasin broth to perform acetone filtration to obtain acetone soluble from the crude narasin broth; a step S200 of adjusting a pH of a filtrate from the acetone filtration to 4.5 or less, the filtrate including the acetone soluble; a step S300 of adding a first heptane and / or hexanes to the filtrate to remove residual lipids; a step S400 of adding polysorbate 80 to the filtrate; a step S500 of stirring the filtrate for 15 to 19 hours; and a step S600 of washing the filtrate with second heptane and / or hexanes and water may be performed.

[0052] Hereinafter, “hexanes” may mean a mixture of all six -carbon alkane isomers.

[0053] Hereinafter, the step SI 00 of adding acetone to a crude narasin broth will be explained in detail.

[0054] The crude narasin broth may be prepared from fermentation of Streptomyces aureofaciens . Any fermentation method known to those skilled in the art may be used to produce the crude narasin broth, and the present disclosure is not limited to any particular fermentation method. For example, a submerged aerobic fermentation of Streptomyces aureofaciens in a nutrient-rich culture medium may be used to produce the crude narasin broth. See FIG. 18 which illustrates different types of crude narasin broth, each having a different concentration and potency.

[0055] The crude narasin broth from the fermentation may be used as a starting material of the isolation method of the present disclosure. The crude narasin broth may be added to a reactor to start the isolation method of the present disclosure. Reactors that may be used in the isolation of narasin include batch reactors, stainless steel jacketed reactors, metal jacketed reactors, glass jacketed reactors, continuous stirred-tank reactors (CSTRs), plug-flow reactors (PFRs), packed-bed reactors, fluidized-bed reactors, tubular reactors, and fermenters / bioreactors. For the purposes of the present disclosure, the most preferred forms of the reactors include stainless steel jacketed reactors, metal jacketed reactors, and glass jacketed reactors. The reactors can be industry-scale or lab-scale. For example, the reactors can be IL lab-scale reactors. In another example, the reactors can be 20L or 50L industryscale reactors. FIG. 5 illustrates an example of a lab-scale IL glass jacketed reactor. FIG. 6 illustrates an example of an industry-scale 20L glass jacketed reactor. FIG. 7 illustrates an example of an industry-scale 50L stainless steel (or metal) jacketed reactor. A same reactor can be used throughout the entire isolation method without any cleaning of the reactors between the steps of the isolation method, which will be described more in detail below.

[0056] The crude narasin broth added into the reactor may have a narasin potency in a range of 50 mg / g to 150 mg / g, which means 50-150 mg of narasin per gram of the crude narasin broth. More preferably, the potency of the crude narasin broth may be in a range of 80 mg / g to 120 mg / g. Most preferably, the potency of the crude narasin broth may be in a range of 95 mg / g to 105 mg / g. For example, the potency of the narasin in the crude narasin broth may be about 100 mg / g.

[0057] The concentration of the narasin in the crude narasin broth may be in the range of 0.05 g / mL to 0.15 g / mL. More preferably, the concentration of the narasin in the crude narasin broth may be in a range of 0.08 g / mL to 0.12 g / mL. Most preferably, the concentration of the narasin in the crude narasin broth may be in a range of 0.095 g / mL to 0. 105 g / mL. For example, the concentration of the narasin in the crude narasin broth is about 0.1 g / mL.

[0058] In an alternative to the crude narasin broth having the above-mentioned potencies and concentrations, a crude narasin broth that is more concentrated, also referred to as EV450 herein, may be used as a starting material. EV450 may be prepared by adding amyl alcohol to the crude narasin broth mentioned above to make sure all organic and inorganic materials are dissolved in the crude narasin broth and evaporating water in the crude narasin broth to reduce the volume of the crude narasin broth and increase the concentration.

[0059] When the crude narasin broth that is more concentrated (e.g., EV450) is used as a starting material, a potency of the EV450 may be in a range of 300 mg / g to 500 mg / g. More preferably, the potency of the EV450 may be in a range of 380 mg / g to 450 mg / g. More preferably, the potency of the EV450 may be in a range of 395 mg / g to 405 mg / g. For example, the potency of the EV450 may be 436 mg / g (i.e., 436 mg of narasin per gram of EV450 material). In another example, the potency of the EV450 may be 400 mg / g (i.e., 400 mg of narasin per gram of EV450 material).

[0060] The concentration of the more concentrated crude narasin broth (e.g., EV450) may be in a range of 0.3 g / mL to 0.5 g / mL. More preferably, the concentration of the EV450 may be in a range of 0.35 g / mL to 0.45 g / mL. Most preferably, the concentration of the EV450 may be in a range of 0.39 g / mL to 0.41 g / mL. For example, the concentration of the EV450 may be 0.4 g / mL. For example, the concentration of the EV450 may be 0.436 g / mL.

[0061] Regardless of the form of the crude narasin broth, the crude narasin may have a concentration in a range of 0.05 g / mL to 0.5 g / mL.

[0062] An amount or a weight of the more concentrated crude narasin broth (e.g., EV450) as the starting material may be in a range of 4 kg to 8 kg. More preferably, the amount of the crude narasin broth may be in a range of 5 kg to 7 kg. Most preferably, the amount of crude narasin broth may be in a range of 6.1 kg to 6.6 kg. For example, the amount of crude narasin broth may be 6.25 kg. This amount of the EV450 is suitable for use in a 50 L Jacketed Reactor (metal, stainless steel, or glass). However, the present disclosure is not limited to the above-mentioned ranges, and other ranges which are not mentioned herein can be used by those skilled in the art in light of the present disclosure. For example, the amount of the crude narasin broth may be the ones that are suitable for 20L, 30L, 40L reactors or IL, 2L, 3L reactors and so forth. For example, the amount of the crude narasin broth may be 0.1 , 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 200, 300, 500, 1000 times the above-mentioned ranges of the amount of the crude narasin broth as a starting material.

[0063] Hereinafter, “amount” mas mean volume, mass, and / or weight.

[0064] When the more concentrated crude narasin broth (e.g., EV450) is used, a volume of the acetone may be 3 times to 6 times the amount of the narasin (i.e., the narasin as active substance, as opposed to the whole broth). More preferably, the volume of the acetone may be 4 times to 5.5 times the amount of the narasin. Most preferably, the volume of the acetone may be 4.6 times to 5.0 times the amount of the narasin. For example, the volume of the acetone may be 4.8 times the amount of the narasin in the crude narasin broth as the starting material.

[0065] In an alternative, a volume of the acetone added to the crude narasin broth may be 10 L to 14 L. More preferably, the volume of the acetone may be 11 L to 13 L. Most preferably, the volume of the acetone may be 11.5 L to 13.5 L. For example, the volume of the acetone added to the crude narasin broth may be 12 L.

[0066] For example, when 12 L or 12 kg of EV450, the concentrated crude narasin broth, is used, the amount of narasin as an active substance included in the crude narasin broth is about 4.8 kg (with the concentration of the crude narasin broth being 0.4 g / mL). Therefore, when the volume of the acetone is 4.8 times the amount of narasin as the active substance, the amount of acetone will be approximately 23 kg or 23 L.

[0067] More specifically, the step SI 00 of adding the acetone to the crude narasin broth to perform acetone filtration may include: a step SI 10 of, after the acetone is added to the crude narasin broth, stirring and heating a mixture of the acetone and the crude narasin broth to a temperate of 50°C - 60°C; and a step S130 of filtering the mixture to retain the filtrate including the acetone soluble. FIG. 8 illustrates a flow chart of the specific steps SI 10, SI 30 included within the step S100.

[0068] In the step SI 10 of stirring and heating a mixture of the acetone, the mixture may be heated to a temperature in a range of 50°C - 60°C. More preferably, the mixture may be heated to a temperature in a range of 53°C - 57°C. Most preferably, the mixture may be heated to a temperature in a range of 54°C - 56°C. For example, the mixture may be heated to about 55 °C. The stirring may be performed while the mixture is being heated to the above- mentioned temperature ranges.

[0069] In the step SI 10 of stirring and heating, once the temperature of the mixture reaches the above-mentioned ranges, the stirring of the mixture of the acetone and the crude narasin broth may continue for additional 0.5 hours to 1.5 hours. More preferably, the stirring of the mixture of the acetone and the crude narasin broth may continue for additional 0.8 hours to 1.2 hours. Most preferably, the stirring of the mixture of the acetone and the crude narasin broth may continue for additional 0.9 hours to 1.1 hours. For example, the stirring of the mixture of the acetone and the crude narasin broth may continue for additional 1 hour.

[0070] The stirring of the mixture of the acetone and the crude narasin may be performed at a predetermined stirring rate. For example, the stirring rate of the mixture may be in a range of 150 RPM (revolution per minute) to 500 RPM. More preferably, the stirring rate of the crude narasin may be in a range of 200 RPM to 250 RPM. Most preferably, the stirring rate of the crude narasin may be in a range of 210 RPM to 230 RPM. For example, the stirring rate of the crude narasin may be 220 RPM.

[0071] Stirrers that may be used in the stirring step SI 10 include magnetic stirrers, head stirrers, marine propellers, Rushton turbines, pitched-blade turbines, anchor impellers, helical ribbon impellers, static mixers, four-blade PTFE (20L reactor), stainless steel propeller (50L reactor), and airlift or bubble-column systems. For purposes of the present disclosure, head stirrers are the most preferred form of the stirrers. For example, the head stirrers include four- blade PTFE (20L reactor) and stainless steel propeller (50L reactor). For example, for the 50L Jacketed Metal Reactor, the stainless steel propeller (50L reactor) may be used to stir the mixture. See FIG. 7 shows an example of the stainless steel propeller for the 50L metal reactor.

[0072] In the step SI 30, the mixture from the step SI 10 may be filtered to obtain the filtrate including the acetone soluble from the crude narasin broth. The acetone filtration method may include one or more filtration methods selected from centrifuge filtration, gravity filtration, vacuum filtration, and pressure filtration.

[0073] Here, the filtrate including the acetone soluble may be retained to be used in next steps, and resulting residual solids may be discarded. At this stage, the filtrate including the acetone soluble has a clear dark brown color. See FIG. 21 which illustrates the clear dark brown color in Step 1 which may be substantially equivalent to Step 100. Hereinafter, “dark brown” may mean a color that has a strong absorption in the blue / violet (-400-500 nm) region and an additional absorption in the green (-500-570 nm) region, and leaves mainly red-orange-yellow (-570-700 nm) wavelengths reflected.

[0074] FIG. 9 illustrates a flowchart of the specific steps S210, S230, S250, S270 included within the step S200. In the step S200 of adjusting pH of the filtrate to 4.5 or less, the filtrate may be transferred to the same reactor used in the step SI 00 or a different reactor. The step of S200 of adjusting the pH of the filtrate may include: a step S210 of cooling the filtrate to a predetermined temperature; a step S230 of adding a first o-phosphoric acid to the filtrate; a step S250 of adding water to the filtrate; a step S270 of adding a second o-phosphoric acid.

[0075] In the step S210, the filtrate may be cooled to a temperature in a range of 25°C - 35°C. More preferably, the filtrate may be cooled to a temperature in a range of 28°C - 32°C. Most preferably, the filtrate may be cooled to a temperature in a range of 29°C - 31°C. For example, the filtrate may be cooled to a temperature of 30°C. While the filtrate is being cooled to the above-mentioned temperature ranges, the filtrate may also be stirred in the reactor. For example, the filtrate may be stirred for about 0.4 to 0.6 hours. For example, the filtrate may be stirred for 0.5 hours.

[0076] The same reactor may be used throughout the entire isolation method of the one embodiment of the present disclosure. More particularly, the same reactor may be used in the steps S100, S200, S300, S400, S500, including the specific steps included in each of the steps S100, S200, S300, S400, S500. This is made possible by the use of heptane or hexanes (instead of activated carbon), which washes and removes residual lipids or oily substances carried over from the fermentation process.

[0077] In the step S230, a first predetermined amount of 85% o-phosphoric acid may be added to the filtrate. The amount of 85% o-phosphoric acid added to the filtrate may be in a range of 300 mL to 500 mL. More preferably, the amount of 85% o-phosphoric acid added to the filtrate may be in a range of 350 mL to 400 mL. Most preferably, the amount of 85% o- phosphoric acid added to the filtrate may be in a range of 373 mL to 377 mL.

[0078] In an alternative, the amount of 85% o-phosphoric acid added to the filtrate may be 0.1 times to 0.2 times the amount of narasin (as an active substance) in the crude narasin broth as the starting material. More preferably, the amount of 85% o-phosphoric acid added to the filtrate may be 0.13 times to 0.17 times the amount of narasin (as an active substance) in the crude narasin broth. Most preferably, the amount of 85% o-phosphoric acid added to the filtrate may be 0.14 times to 0.16 times the amount of narasin (as an active substance) in the crude narasin broth. For example, the amount of 85% o-phosphoric acid added to the filtrate may be 0.15 times the amount of narasin in the crude narasin broth as the starting material.

[0079] In the step S250, a predetermined amount of water, such as Milli-Q water, may be added to the filtrate. The amount of water added to the filtrate may be in a range of 11.0 L to 13.0 L. More preferably, the amount of water added to the filtrate may be in a range of 11.5 L to 12.5 L. Most preferably, the amount of water added to the filtrate may be in a range of 11.9 L to 12.1 L.

[0080] In an alternative, the amount or volume of water, such as Milli-Q water, added to the filtrate may be 3 times to 6 times the amount of the narasin included in the crude narasin broth as the starting material (i.e., the narasin as active substance, as opposed to the whole broth). More preferably, the amount or volume of water added to the filtrate may be 4 times to 5.5 times the amount of the narasin. Most preferably, the amount or volume of water added to the filtrate may be 4.6 times to 5.0 times the amount of the narasin. For example, the amount or volume of water added to the filtrate may be 4.8 times the amount of the narasin in the crude narasin broth as the starting material. The amount of water added to the filtrate may be the same as the amount of acetone added to the crude narasin broth in the step S 100.

[0081] In this step S250, the water may be added slowly at a predetermined rate in a range of 1 mL / min to 3 mL / min. More preferably, the water may be added at a predetermined rate in a range of 1.5 mL / min to 2.5 mL / min. Most preferably, the water may be added at a predetermined rate in a range of 1.9 mL / min to 2.1 mL / min. For example, the water may be added at a predetermined rate of 2.0 mL / min.

[0082] In the step S270, in addition to the first predetermined amount of the 85% o- phosphoric acid, a second predetermined amount of 85% o-phosphoric acid may be added to the filtrate. More specifically, the second predetermined amount of 85% o-phosphoric acid added to the filtrate may be in a range of 200 mL to 300 mL. More preferably, the amount of 85% o-phosphoric acid added to the filtrate may be in a range of 230 mL to 260 mL. Most preferably, the amount of 85% o-phosphoric acid added to the filtrate may be in a range of 245 mL to 255 mL. For example, the amount of 85% o-phosphoric acid added to the filtrate may be 250 mL.

[0083] In an alternative, the second predetermined amount of 85% o-phosphoric acid added to the filtrate may be 0.05 times to 0.15 times the amount of narasin (as an active substance) in the crude narasin broth as the starting material. More preferably, the second predetermined amount of 85% o-phosphoric acid added to the filtrate may be 0.08 times to 0.12 times the amount of narasin (as an active substance) in the crude narasin broth. Most preferably, the second predetermined amount of 85% o-phosphoric acid added to the filtrate may be 0.11 times to 0. 12 times the amount of narasin (as an active substance) in the crude narasin broth. For example, the second predetermined amount of 85% o-phosphoric acid added to the filtrate may be about 0.1 times the amount of narasin in the crude narasin broth as the starting material.

[0084] In the step S200, the pH of the filtrate may be adjusted to 4.5 or lower by adding the 85% o-phosphoric acid to the filtrate. More preferably, the pH of the filtrate may be adjusted to 1 .5 to 3.5. Most preferably, the pH of the filtrate may be adjusted to 2 to 3. A pH probe may be used to check the pH of the filtrate after the step S200 is completed.

[0085] After the step S200, the temperature of the filtrate may be lowered to 15°C to 25 °C by using the jacketed reactor. More preferably, the temperature of the filtrate may be lowered to 18 °C to 22°C. Most preferably, the temperature of the filtrate may be lowered to 19°C to 21 °C. For example, the temperature of the filtrate may be lowered to about 20°C.

[0086] After the filtrate is cooled to the above-mentioned temperatures, the step S300 of adding an alkane to the filtrate may be performed. The alkane may be heptane and / or hexanes. The amount of the heptane and / or hexanes may be in a range of 4.0 L to 6.0 L. More preferably, the amount of the heptane and / or hexanes may be in a range of 4.5 L to 5.5 L. Most preferably, the amount of the heptane and / or hexanes may be in a range of 4.9 L to 5.1 L. For example, the amount of the heptane and / or hexanes may be 5.0 L.

[0087] In an alternative, the amount of hexanes or heptane added to the filtrate may be 1.5 to 2.5 times the amount of narasin (i.e., the active substance) included in the crude narasin broth as the starting material. More preferably, the amount of heptane and / or hexanes added to the filtrate may be 1.8 to 2.2 times the amount of narasin as the active substance. Most preferably, the amount of heptane and / or hexanes added to the filtrate may be 1.9 to 2.1 times the amount narasin. For example, the amount of heptane and / or hexanes added to the filtrate may be 2.0 times the amount of narasin as the active substance.

[0088] In the step S300, the temperature of the filtrate may be maintained at the range of 15°C to 25°C. More preferably, the temperature of the filtrate may be maintained at 18°C to 22°C. Most preferably, the temperature of the filtrate may be maintained at 19°C to 21 °C. For example, the temperature of the filtrate may be maintained at 20°C.

[0089] In the step S400 of adding Polysorbate 80 (PS80), a predetermined amount of PS80 solution may be added to the filtrate that is added with the heptane or the hexanes in the step S300. The PS80 solution may have a concentration in a range of 2% to 10%. More preferably, the PS80 solution may have a concentration in a range of 4% to 8%. Most preferably, the PS80 solution may have a concentration in a range of 6.5% to 7.0%. For example, the PS80 solution may be a 6.8% PS80 solution. For example, when the concentration of the PS80 solution is 3%, the 150 mL of the PS80 solution may contain 4.5 grams of PS80 in the solution.

[0090] The amount of PS 80 solution used in the step S400 may be in a range of 8 L to 12 L. More preferably, the amount of PS80 solution may be in a range of 9 L to 11 L. Most preferably, the amount of PS80 solution may be in a range of 9.3 L to 9.7L. For example, the amount of PS80 solution may be 9.5 L. In another specific example, the PS80 may be prepared by adding 650 g of PS80 to 9.5 L of water (i.e., to have a 6.5% concentration of PS80) in a separate beaker, and the mixture of the PS80 and water may be added to the filtrate.

[0091] In an alternative, the amount of PS80 solution added to the filtrate in the step S400 may be 2.5 times to 5.5 times the amount of narasin as an active substance included in the crude narasin broth as the starting material. More preferably, the amount of PS80 solution added to the filtrate in the step S400 may be 3 times to 4.5 times the amount of narasin as an active substance included in the crude narasin broth as the starting material. Most preferably, the amount of PS80 solution added to the filtrate in the step S400 may be 3.6 times to 4.0 times the amount of narasin as an active substance included in the crude narasin broth as the starting material. For example, the amount of PS 80 solution added to the filtrate in the step S400 may be 3.8 times the amount of narasin as an active substance included in the crude narasin broth as the starting material.

[0092] When and while the PS 80 solution is being added to the second solution in the step S400, the temperature of the filtrate may be maintained at a temperature of 15°C to 25°C. More preferably, the temperature of the filtrate may be maintained at 18°C to 22°C. Most preferably, the temperature of the filtrate may be maintained at 19°C to 21 °C. For example, the temperature of the filtrate may be maintained at 20°C.

[0093] After the water is added in the step S250, the filtrate may have a slurry form. The slurry form, although slightly different in contents and colors, may be maintained even after each of the steps S300 and S400. See FIGS. 11 and 21 illustrating the slurry forms of the filtrate after the steps S250, S300, or S400. Hereinafter, “slurry” may mean a mixture of solids and liquid.

[0094] After the PS 80 solution is added to the filtrate, the filtrate added with the PS 80 solution may have a narasin concentration in a range of 30 g / L to 80 g / L. More preferably, the filtrate added with the PS 80 solution may have a narasin concentration in a range of 40 g / L to 60 g / L. Most preferably, the filtrate added with the PS80 solution may have a narasin concentration in a range of 54 g / L to 56 g / L.

[0095] After the step S400, a step S500 of stirring the filtrate for 15 hours to 19 hours may be performed. More preferably, the filtrate may be stirred for 16 hours to 18 hours. Most preferably, the filtrate may be stirred for 16.5 hours to 17.5 hours. For example, the filtrate may be stirred for 17 hours.

[0096] While the filtrate added with the PS 80 solution is being stirred, the temperature of the filtrate may be maintained at a temperature range of 15°C to 25°C. More preferably, the temperature of the filtrate may be maintained at 18 °C to 22°C. Most preferably, the temperature of the filtrate may be maintained at 19°C to 21°C. For example, the temperature of the filtrate may be maintained at 20°C.

[0097] After the step S500 of stirring the filtrate, the filtrate in the reactor may be drained into a container (e.g., a plastic container). The size of the plastic container may be about 4-6 gallons, but the present disclosure is not limited thereto, and containers of other various sizes may be used to accommodate various amounts of the filtrate.

[0098] Hereinafter, the filtrate after step S500 will be referred to as a solution having a slurry form (e.g., a slurry solution) for the ease of description.

[0099] Then, a step S600 of washing the slurry solution (i.e., the filtrate after the step S600) with heptane and / or hexanes and water may be performed. The slurry solution may also have a wetcake form. FIG. 10 illustrates a flowchart for specific steps S610, S630, S650, S670 within the step S600. The step S600 of washing the slurry solution may include: a step S610 of filtering solids from the slurry solution by filtration, a step S630 of washing the solids from the slurry solution with heptane and / or hexanes for a first time, a step S650 of washing the solids with water, and a step S670 of washing the solids with heptane and / or hexanes for a second time.

[0100] The step S610 may be performed to filter the slurry solution drained into the container to obtain the solids in the slurry solution. The filtration method may include one or more filtration methods selected from centrifuge filtration, gravity filtration, vacuum filtration, and pressure filtration.

[0101] When the vacuum filtration method is used in the second filtration, a filter may be wet with heptane and / or hexanes. Then, the vacuum may be turned on for the vacuum filtration. Then, the slurry solution (for example 2.5-3.0 gallons of the slurry solution) drained into the container may be poured on the filter. Once the filtration is finished, the residual solids may be transferred to a second container. This process may be repeated using a fresh filter paper each time until the entire residual solids (e.g., the slurry) of the slurry solution is filtered.

[0102] Then, in the step S630 of washing the solids with heptane and / or hexanes, the solids filtered from the slurry solution may be transferred to a clean filter paper wet with heptane and / or hexanes in a filtration system, such as 20L filtration system. Then, heptane and / or hexanes may be poured over the solids in the 20 L filtration system until all the solids are covered with heptane and / or hexanes.

[0103] Here, the amount of heptane and / or hexanes may be 1 .5 times to 2.5 times the amount of narasin (i.e., the active substance) in the crude narasin broth as the starting material. More preferably, the amount of heptane / hexanes may be 1.8 times to 2.2 times the amount of narasin (i.e., the active substance) in the crude narasin broth as the starting material. Most preferably, the amount of heptane / hexanes may be 1.9 times to 2.1 times the amount of narasin (i.e., the active substance) in the crude narasin broth as the starting material. For example, the amount of heptane / hexanes may be 2 times the amount of narasin (i.e., the active substance) in the crude narasin broth as the starting material.

[0104] Then, the solids and the heptane and / or hexanes may be mixed with a glass stir bar to make sure all solids and / or the narasin crystals included in the solids are wet with the heptane and / or hexanes covering the solids. Then, the mixture of the solids and the heptane and / or hexanes stirred with the glass stir bar may be filtered in the 20L filtration system to obtain / retain the residual solids. The step S630 may be repeated one more time to obtain the residual solids.

[0105] In the step S650, the step S630 may be repeated, except water, such as Milli-Q water, is used to wet a clean filter paper and wash the solids, instead of the heptane and / or hexanes. The step S650 may be performed two times in total. Here, the amount of water may be 1.5 times to 2.5 times the amount of narasin (i.e., the active substance) in the crude narasin broth as the starting material. More preferably, the amount of water may be 1 .8 times to 2.2 times the amount of narasin (i.e., the active substance) in the crude narasin broth as the starting material. Most preferably, the amount of water may be 1.9 times to 2.1 times the amount of narasin (i.e., the active substance) in the crude narasin broth as the starting material. For example, the amount of water may be 2 times the amount of narasin (i.e., the active substance) in the crude narasin broth as the starting material.

[0106] In the step S670, the residual solids filtered and washed in the step S650 may be washed with heptane and / or hexanes for a second time using the same process of step S630. Then, the residual solids obtained in the step S670 may sit in the 20L filtration system for a predetermined time. Specifically, the solids may sit for 15 hours to 19 hours. More preferably, the solids may sit for 16 hours to 18 hours. Most preferably, the solids may sit for 16.5 hours to 17.5 hours. For example, the residual solids may sit for 17 hours.

[0107] Then, the residual solids after the step S670 may result in a wetcake form having a light beige color. See FIG. 11 illustrating the light beige color wetcake of the residual solids after the step S670. Hereinafter, “light beige” may mean a color that absorbs light at around 400-500 nm and reflects light above 500 nm.

[0108] Then, the residual solids obtained after the step S670 may be transferred to a vacuum oven and may be dried at a predetermined temperature in a range of 35°C to 45°C. More preferably, the predetermined temperature is in a range of 38°C to 42°C. Most preferably, the predetermined temperature is in a range of 39°C to 41 °C. For example, the predetermined temperature at which the residual solids are dried is 40°C.

[0109] Also, the drying step in the vacuum oven may be performed for 20 hours to 28 hours. More preferably, the drying step in the vacuum oven may be performed for 22 hours to 26 hours. Most preferably, the drying step in the vacuum oven may be performed for 23 hours to 25 hours. For example, the drying step in the vacuum oven may be performed for 24 hours.

[0110] Then, the vacuum-drying step results in a final product of the isolated narasin. The isolated narasin is an amorphous narasin (i.e., narasin having an amorphous structure). “Amorphous” may mean a solid material that lacks a long-range, ordered crystal structure. Amorphous narasin may include narasin having an amorphous chemical structure or a mixture of narasin having an amorphous chemical structure and narasin having a crystalline structure.

[0111] The method of isolating narasin according to the present disclosure is reduced or streamlined compared to conventional methods, such as those that are shown in FIGS. 2 and 3. As such, the manufacturing cost of narasin is reduced. Also, the method of isolating narasin according to the present disclosure does not require use of sodium bicarbonate. Also, the method of isolating narasin according to the present disclosure does not require concentration / dis dilation of methanol (MeOH), thereby reducing the manufacturing operation cost of narasin. Also, the method of isolating narasin according to the present disclosure uses a low-cost heptane as a solvent to remove significantly residual lipids from fermentation process. Also, the method of isolating narasin according to the present disclosure does not require use of activated carbon to remove coloration (e.g., orange-color or dark orange color) from narasin broth. Activated carbon is conventionally used to isolated narasin as a kilo-scale laboratory process. Removing activated carbon from manufacturing will reduce manufacturing cost, because use of activated carbon requires extensive cleaning of reactor and parts to remove all activated carbon prior to use of the reactor for the next step. Hereinafter, “orange” may mean a color that reflects / transmits light around 590-620 nm (the orange region of the visible spectrum) and absorbs mainly blue (-450-495 nm) and some violet (-400-450 nm). Hereinafter, “dark orange” may mean a color that strongly absorb light in the blue region, roughly around 460-480 nm.

[0112] Method of Crystallizing Narasin

[0113] FIG. 22 illustrates a flowchart for a method of crystallizing narasin according to another embodiment of the present disclosure. The method of crystallizing narasin includes: a step S1000 of preparing acetonitrile, a step S2000 of adding amorphous narasin to the acetonitrile, a step S3000 of heating a mixture of the acetonitrile and the amorphous narasin to a first predetermined temperature, a step S4000 of stirring the mixture for a first predetermined time at a second predetermined temperature; and a step S5000 of stirring the mixture for a second predetermined time at a third predetermined temperature.

[0114] In the step SI 000, acetonitrile may be added into a jacketed reactor (e.g., a metal or stainless steel jacketed reactor). The jacketed reactor may be a 50L reactor, for example, but the present disclosure is not limited thereto, and other volume reactors may be used.

[0115] Reactors that may be used include batch reactors, stainless steel jacketed reactors, metal jacketed reactors, glass jacketed reactors, continuous stirred-tank reactors (CSTRs), plug-flow reactors (PFRs), packed-bed reactors, fluidized-bed reactors, tubular reactors, and fermenters / bioreactors. For the purposes of the present disclosure, the most preferred forms of the reactors include stainless steel jacketed reactors, metal jacketed reactors, and glass jacketed reactors. The reactors can be industry-scale or lab-scale. For example, the reactors can be IL lab-scale reactors. In another example, the reactors can be 20L or 50L industryscale reactors.

[0116] The amount of acetonitrile added to the reactor may be 3.5 times to 4.5 times the amount of narasin (i.e., the active substance) that may be added to the acetonitrile. More preferably, the amount of acetonitrile added to the reactor may be 3.8 times to 4.2 times the amount of narasin (i.e., the active substance). Most preferably, the amount of acetonitrile added to the reactor may be 4.0 times to 4.1 times the amount of narasin (i.e., the active substance).

[0117] The minimum amount of the acetonitrile is 3.5 times the amount of the narasin (i.e., the active substance). Below that, the amorphous narasin does not crystallize. The maximum amount of the acetonitrile is 4.5 times the amount of the narasin (i.e., the active substance). Above that, acetonitrile will dissolve crystal narasin and lower the yield of crystallized narasin and lose product in the mother liquor.

[0118] The amount of narasin included in the final product of the isolation method above (i.e., the amorphous narasin) may be calculated based on the limit of detection (LOD) data and the weight information of the final product. The LOD may be collected using Computrac.

[0119] Prior to adding the amorphous narasin to acetonitrile, the acetonitrile may be preheated. The temperature at which the acetonitrile is pre-heated may be in a range of 45°C to 55°C. More preferably, the temperature may be 47°C to 53°C. Most preferably, the temperature may be 49°C to 51°C. For example, the temperature may be 50°C.

[0120] In the step S2000 of adding amorphous narasin to the acetonitrile, the amorphous narasin may be added to the acetonitrile in the reactor while the acetonitrile is being stirred or agitated. Here, the amorphous narasin may be a narasin having an amorphous chemical structure or a mixture of narasin having the amorphous chemical structure and narasin having a crystal structure.

[0121] The amorphous narasin may be final products of the isolation method of the present disclosure described above. In an alternative, the amorphous narasin may be any isolated narasin from conventional isolation methods, such as those found in FIGS. 2 and 3.

[0122] Stirrers that may be used in the stirring of the step S2000 include magnetic stirrers, head stirrers, marine propellers, Rushton turbines, pitched-blade turbines, anchor impellers, helical ribbon impellers, static mixers, four-blade PTFE (20L reactor), stainless steel propeller (50L reactor), and airlift or bubble-column systems. For purposes of the present disclosure, head stirrers are the most preferred form of the stirrers. For example, the head stirrers include four-blade PTFE (20L reactor) and stainless steel propeller (50L reactor), For example, for the 50L Jacketed Metal Reactor, the stainless steel propeller (50L reactor) may be used to stir the mixture.

[0123] Then, in the step S3000, the mixture of the acetonitrile and the amorphous narasin may be heated to a first predetermined temperature while the mixture is being stirred. The first predetermined temperature may be in a range of 45°C to 55°C. More preferably, the first temperature may be 47°C to 53°C. Most preferably, the first temperature may be 49°C to 51 °C. For example, the first temperature may be 50°C.

[0124] The stirring in the step S3000 may be performed at a rate in a range of 150 RPM (revolution per minute) to 500 RPM. More preferably, the stirring rate of the crude narasin may be in a range of 200 RPM to 280 RPM. Most preferably, the stirring rate of the crude narasin may be in a range of 230 RPM to 250 RPM. For example, the stirring rate of the crude narasin may be 240 RPM.

[0125] In the step S4000, the mixture of the acetonitrile and the starting material (i.e., the amorphous narasin) may be stirred for a first predetermined time at a second predetermined temperature. The stirring of the mixture for the first predetermined time at the second predetermined temperature ensures conversion of the narasin having the amorphous structure to the narasin having a polymorph structure having a crystalline form. Preferably, the mixture may be stirred for at least two hours at the second temperature. More preferably, the mixture may be stirred for 2 hours to 8 hours. Most preferably, the mixture may be stirred for 2 hours to 4 hours. For example, the mixture may be stirred for about 3 hours.

[0126] Stirrers that may be used in the stirring of step S4000 may include magnetic stirrers, head stirrers, marine propellers, Rushton turbines, pitched-blade turbines, anchor impellers, helical ribbon impellers, static mixers, four-blade PTFE (20L reactor), stainless steel propeller (50L reactor), and airlift or bubble-column systems. For purposes of the present disclosure, head stirrers are the most preferred form of the stirrers. For example, the head stirrers include four-blade PTFE (20L reactor) and stainless steel propeller (50L reactor), For example, for the 50L Jacketed Metal Reactor, the stainless steel propeller (50L reactor) may be used to stir the mixture.

[0127] Also, the second temperature may be in a range of 45°C to 55°C. More preferably, the second predetermined temperature may be in a range of 47°C to 53°C. Most preferably, the second predetermined temperature may be in a range of 49°C to 51 °C. For example, the second predetermined temperature may be about 50°C. In an alternative, the first predetermined temperature and the second predetermined temperature may be the same.

[0128] In the step S5000, the mixture of the acetonitrile and the starting material may be cooled to a third predetermined temperature and may be stirred again for a second predetermined time. Preferably, the third predetermined temperature may be in a range of 2°C to 10°C. More preferably, the third predetermined temperature may be in a range of 3°C to 7°C. Most preferably, the third predetermined temperature may be in a range of 4°C to 6°C. For example, the third predetermined temperature may be about 5 °C. The third predetermined temperature may be maintained while the mixture is being stirred for the second predetermined time.

[0129] The second predetermined time may be at least 3 hours. More preferably, the second predetermined time may be at least 7 hours. Most preferably, the second predetermined time may be in a range of 16 hours to 18 hours. For example, the second predetermined time may be about 17 hours.

[0130] Then, after the step S5000, the reactor may be emptied and drained into a 4-6 gallon container (e.g., 5 gallon container). Then, the mixture may be filtered to obtain the crystallized narasin having the polymorph crystalline structure. The filtration of the mixture may be performed by one or more filtration methods selected from centrifuge, gravity filtration, vacuum filtration. For example, a 20L vacuum filtration system may be used, in which the mixture is filtered under vacuum and with a filter paper wet with acetonitrile.

[0131] During the filtration of the mixture, a filter cake or a residue may be collected and the filtrate liquid that has passed through the filter may be discarded. The filter cake or the residue is in a wetcake form after the collection. The filter cake or the residue includes the crystallized narasin. Hereinafter, “wetcake” may mean a solid after filtration but still contains residual solvent.

[0132] Then, the crystallized narasin in the wet cake form may be dried in a vacuum at a fourth predetermined temperature for a third predetermined time. Preferably, the fourth predetermined temperature may be in a range of 30°C to 50°C. More preferably, the fourth predetermined temperature may be in a range of 35°C to 45°C. Most preferably, the fourth predetermined temperature may be in a range of 39°C to 41°C. For example, the fourth temperature may be about 40°C.

[0133] Also, the third predetermined time may be in a range of 15 hours to 19 hours. More preferably, the third predetermined time may be in a range of 16 hours to 18 hours. Most preferably, the third predetermined time may be in a range of 16.5 hours to 17.5 hours. For example, the third predetermined time may be about 17 hours.

[0134] A chemical structure of the final product is shown in Formula I below.

[0135] Chemical Formula: C43H72OH Exact Mass: 764.51 Molecular Weight: 765.04

[0136] Formula I The final product of the crystallization method of the present disclosure is a particular polymorph of the narasin shown in Formula I. Polymorphism is the ability of a substance to exist in more than one crystalline form, where the molecules or atoms are arranged differently in the crystal lattice, even though the chemical composition is the same. All polymorphs have the same chemical formula. What differs is the packing of molecules / ions and the symmetry of the crystal structure. This difference in arrangement can lead to very different physical properties, such as melting point, solubility, stability, density, color, and hardness.

[0137] The particular polymorph (see FIG. 14) that results from the crystallization method of the present disclosure has very high stability in heat and liquid, compared to conventional polymorphs. Also, the crystallization method of the present disclosure consistently yields the polymorph of the present disclosure that is very stable, as seen the purity result in Tables 1 and 2 and the high-performance liquid chromatography result in FIG. 12. There, the purity of the polymorph that results from the crystallization method of the present disclosure is higher than 97%. As such, the method of crystallizing narasin according to the present disclosure consistently yields the crystallized final product having very high purity. Every time the method of crystallizing narasin according to the present disclosure is repeated, the same polymorph (i.e., the polymorph according to FIG. 14) in high purity results (97% purity) has always consistently resulted.

[0138] Also, the Total Lipids (i.e., impurities carried over from the fermentation) of the crystallized final product is 0.05 % area or less, because the isolation method of the present disclosure (i.e., using heptane and / or hexanes) significantly removes residual lipids carried over from the fermentation process, such that the lipid content is very close to 0 %. Also, the method of crystallizing narasin according to the present disclosure consistently results in a particular polymorph of narasin (i.e., the crystallized narasin having high stability).

[0139] Hereinafter, examples of the present invention will be described in detail so that the present disclosure can be easily carried out by those skilled in the art. However, the present disclosure may be embodied in various different forms and is not limited to the examples described herein.

[0140] Example 1 : Narasin Isolation Method Using EV450

[0141] 6.25 Kg of EV450 was added to 50 L Metal Jacketed Reactor. 12.0 L (4.8 Vol) Acetone was added to the EV450. The solution of EV450 and acetone was heated to 55 C while stirring. The solution was stirred for an additional hour at 55 °C. The solution was cooled to 30 C while being stirred. 375 mL (0.15 Vol) o-phosphoric acid was added to the solution. 12.0 L (4.8 Vol) Milli-Q water was added slowly at a rate of 2mL / min. 250 mL (0.10 Vol) o-phosphoric acid was added to the solution. pH was checked using a probe that the pH is between 2-3. Then, the solution was cooled to 20 °C. Then, 5.0 L (2.0 Vol) Hexane was added. Then, 9.5 L (3.8 Vol) 6.8 % Polysorbate 80 Solution (9.5 L Water + 650 g Polysorbate 80 dissolved in separate beaker and then poured in) was added. The solution was stirred for 17 hours at 20 °C. Then, the reactor was drained into 5 Gallon plastic containers.

[0142] Then, the slurry in the solution was filtered on 20 L vacuum filtration system. Specifically, a filter paper was wet using hexane from squirt bottle. The vacuum was turned on. 2.5-3.0 Gallons of the solution containing the slurry were poured on the filter. Once filtration is finished the residual solids were transferred into a secondary container. This filtration process was repeated until the entire slurry is filtered.

[0143] Then, the slurry or the solids were transferred onto a clean filter paper wet with hexane in 20 L filtration system. Hexanes were poured over the solids until all are covered. Then, the mixture of the solids and the hexane was mixed with glass stir bar to make sure all the solids are wet with hexanes. Then, the solids were again filtered under vacuum. Then, this washing with hexanes was repeated one more time.

[0144] The washing process was repeated 2 more times, except with Milli-Q water instead of the hexanes.

[0145] Then, the washing process was repeated 1 more times with hexanes. These washing processes are crucial for eliminating the dark coloration of the slurry formed. For example, the orange color of the slurry may be removed by these washing processes. The wetcake that results from these washing processes has a light beige color, as seen in FIG. 11.

[0146] Then, the solids in the wetcake form sat on the filter for 17 hours.

[0147] Then, the solids in the wetcake form were transferred to drying trays. Then, the solids were dried in Vacuum Oven at 40 °C under vacuum for approximately 24 hours.

[0148] Example 2: Narasin Crystallization Method Using Amorphous Narasin Isolated from Example 1

[0149] LOD of amorphous narasin was collected using Computrac. The weight and LOD information were used to determine the amount of Narasin in the amorphous narasin isolated in Example 1. 5.0 Vol of Acetonitrile was added to clean 50 L reactor (Approximately 40 L Acetonitrile). While someone is agitating acetonitrile add the amorphous Narasin. Then, the lid was closed and the agitator was turned on. The solution of acetonitrile and the amorphous narasin was heated to 50 C while being stirred. Then, the solution was stirred at 50 C for 5 hours. Then, the solution was stirred for 17 hours at 5 C. Then, the reactor was emptied into 5 Gallon Container. Then, a 20 L vacuum filtration system was used to filter slurry under vacuum with a filter paper wet with Acetonitrile. Then, the solids (e.g., the wetcake) having a light beige color onto drying trays. Then, the solids were dried at 40 C under vacuum using vacuum oven for approximately 24 hours.

[0150] Comparative Example 1 : Conventional Method of Isolating Narasin

[0151] FIG. 2 shows a flow chart for the method of Comparative Example 1 . Whole broth (132 liters) of narasin is filtered with 3% filter aid. Then, the filtered broth (97 liters) is extracted twice with ethyl acetate (97 liters). The mycelial solids from the filtration are extracted twice with methanol (60 liters). Then, the ethyl acetate extract from the filtered broth is concentrated to 500 ml and then added to 10 liters of hexanes. This forms a first supernatant. The aqueous methanol extract (120 liters) from the mycelial solids is concentrated to 10 liters of water, adjusted to pH 7.5, and then extracted twice with ethyl acetate (10 liters). This ethyl acetate extract is concentrated to 500 ml and added to hexanes (8 liters). This forms a second supernatant. Precipitates from both hexanes additions are discarded, and the first and second supernatants are concentrated to a residue, yielding active oil of 20.6 g and 6.9 g, respectively, which represents isolated narasin.

[0152] Comparative Example 2: Conventional Method of Isolating Narasin

[0153] FIG. 3 shows a flow chart for the method of Comparative Example 2. Whole broth (60 liters) of narasin has its pH adjusted to 3.0, is stirred for 1 hour, and then filtered with 3% filter aid. The filtered broth is discarded. The mycelial solids are extracted twice with 5% KHC03 in methanol (30 liters). The aqueous methanol extract (60 liters) is concentrated to 7 liters of water, adjusted to pH 7.5, and then extracted twice with ethyl acetate (7 liters). The ethyl acetate extract is concentrated to an oil. This oil is dissolved in acetone(1.5 liters), added with water (1.5 liters), and adjusted to pH 3.0. The supernatant is discarded, and the remaining crude narasin is dissolved in 1.5 liters of acetone, followed by the addition of 400 ml of water, resulting in isolated narasin crystals (74 g).

[0154] Comparative Example 3: Conventional Method of Crystallizing Narasin

[0155] The factors present in the crude narasin preparations from either procedure of Comparative Example 1 or Comparative Example 2 are separated by silica-gel chromatography. The isolated narasin is dissolved in benzene and applied to a silica gel (Grace-Davison, Grade 62) column, which is packed in benzene. An initial wash with benzene removed inactive oils. Narasin methyl ester and narasin B were eluted in separate fractions using benzene - ethyl acetate (9:1). Narasin was then eluted with benzene - ethyl acetate (4:1). The elution of these factors was monitored by TLC using a vanillin-H2SO4 spray. Fractions containing only (1) narasin methyl ester and (2) narasin B, respectively, were combined, concentrated to an oily residue, dissolved in acetone, and water was added to induce crystallization. The crystals were recovered by filtration and dried in vacuo to yield narasin methyl ester and narasin B free acid. Fractions containing (3) narasin were concentrated to a residue, dissolved in acetone, and an equal volume of water was added. The pH of this mixture was adjusted to 3 with dilute HC1 to allow crystallization. The narasin crystals are collected and recrystallized from acetone - water (2: 1).

[0156] Table 1 below summarizes the yield (%), assay (%), total lipids, what solvent used in crystallization procedures, polymorph results, advantages, and disadvantages of the crystallization method of Example 2 and other crystallization methods.

[0157] Table 1

[0158] Experimental Example 1. Analysis of Residual Lipids

[0159] Residual lipid contents of the products of Example 2 have been compared with residual lipid contents of products of Comparative Example 3 (Narasin Reference standard (RS0302) using high-performance liquid chromatography (HPLC). As shown in FIG. 12 and Table 2 below, it was confirmed that the final product of Example 2 results in an extremely low lipid content and high purity. For example, the % area of the narasin in the final product of Example 2 is 99.76 %, whereas the % area of the narasin in the final product of Comparative Example 3 is 96.86 %. Also, the % area of the total lipids in the final product of Example 2 is 0.05 %, whereas the % area of the total lipids in the final product of Comparative Example 3 is 2.28 %. This test results confirm that the use of heptane and / or hexanes in the isolation method, namely the method of Example 1 , results in significantly removing and washing the lipid contents carried over from the fermentation.

[0160] TABLE 2

[0161] Experimental Example 2: Powder X-ray Diffraction (XRPD) Result of Example 2

[0162] FIG. 13 is an XRPD result of the narasin isolated according to the isolation method of Example 1 , which is the starting material for the crystallization method of Example 2. FIG. 6 suggests a mixture of amorphous material with possible small amount of crystalline particles.

[0163] On the other hand, FIG. 14 is an XRPD result of the narasin crystallized according to the crystallization method of Example 2. The pattern, as shown in FIG. 7, which shows sharp peaks, corresponds to a crystallized polymorph of the narasin. See also Table 3 below, which shows the peak list of the XRPD result of the narasin that is crystallized according to the crystallization method of the present disclosure. Table 3 also provides peak information for the polymorph crystallized structure according to FIG. 14.

[0164] Table 3

[0165] No. 2-theta D (ang.) Height FWHM Int. 1 (cps Int. Asym.

[0166] (deg) (cps) (deg) deg) W(deg) factor

[0167] 1 4.271 (7) 20.67(3) 11643(311 ) 0.283(10) 4709(75) 0.404(17) 2.0(2)

[0168] 2 8.595(4) 10.280(5) 24009(447) 0.414(4) 11102(115) 0.462(13) 2.81 (14)

[0169] 3 9.24(3) 9.56(3) 9368(279) 0.394(18) 4098(1086) 0.44(13) 2.0(4)

[0170] 4 9.553(7) 9.250(6) 22220(430) 0.35(4) 8706(1147) 0.39(6) 1.8(4)

[0171] 5 12.706(8) 6.961 (4) 23350(441 ) 0.483(7) 12704(192) 0.544(18) 4.1 (4)

[0172] 6 14.62(3) 6.053(13) 3614(174) 0.8(3) 3050(124) 0.84(7) 0.41 (7)

[0173] 7 15.222(18) 5.816(7) 5755(219) 0.509(18) 3120(121 ) 0.54(4) 0.41 (7)

[0174] 8 16.921 (18) 5.236(6) 6927(240) 0.40(3) 2953(590) 0.43(10) 2.9(7)

[0175] 9 17.459(12) 5.075(4) 11652(312) 0.69(6) 8595(533) 0.74(7) 3.6(5)

[0176] 10 19.308(5) 4.5933(13) 5391 (212) 0.576(17) 3394(114) 0.63(5) 4.1 (8)

[0177] 11 20.80(4) 4.268(8) 2481 (144) 0.43(4) 1141 (120) 0.46(7) 0.9(3)

[0178] 12 21.573(17) 4.116(3) 1786(122) 0.38(6) 736(101 ) 0.41 (8) 1.9(11 ) 13 22.87(9) 3.886(15) 834(83) 0.52(11) 465(93) 0.56(17) 0.46(12)

[0179] 14 23.72(7) 3.748(11 ) 883(86) 0.29(7) 271 (76) 0.31 (12) 1.2(4)

[0180] 15 25.57(3) 3.481 (4) 1297(104) 0.53(3) 731 (48) 0.56(8) 0.84(19)

[0181] 16 26.48(6) 3.364(7) 564(69) 0.47(7) 279(31 ) 0.50(12) 0.7(4)

[0182] 17 27.46(5) 3.245(6) 795(81) 0.58(5) 487(40) 0.61 (11) 1.2(4)

[0183] 18 43.52(8) 2.078(4) 338(53) 0.74(9) 353(41 ) 1.0(3) 0.7(3)

[0184] 19 53.70(16) 1.706(5) 64(23) 2.2(5) 169(40) 2.7(16) 1.0(10)

[0185] 20 76.45(3) 1.2449(5) 364(55) 0.20(4) 101 (11 ) 0.28(7) 1.1 (11 )

[0186] 21 80.63(3) 1.1906(3) 360(55) 0.14(2) 64(9) 0.18(5) 0.8(7)

[0187] 22 4.283(6) 20.61 (3) 11923(315) 0.261 (10) 4889(73) 0.410(17) 2.6(3)

[0188] 23 8.606(4) 10.266(5) 25997(465) 0.382(4) 11726(132) 0.451 (13) 3.29(17)

[0189] 24 9.32(7) 9.48(7) 16988(376) 0.42(7) 8446(3120) 0.50(19) 1.4(2)

[0190] 25 9.598(11 ) 9.207(11 ) 19704(405) 0.23(7) 5328(3024) 0.27(16) 1.8(6)

[0191] 26 12.671 (7) 6.980(4) 32170(518) 0.354(8) 15528(178) 0.483(13) 2.25(19)

[0192] 27 14.684(15) 6.028(6) 4268(189) 0.375(15) 1702(87) 0.40(4) 2.3(5)

[0193] 28 15.282(14) 5.793(5) 8091 (260) 0.489(17) 4215(245) 0.52(5) 1.5(2)

[0194] 29 15.814(17) 5.599(6) 3015(159) 0.38(7) 1213(249) 0.40(10) 4(3)

[0195] 30 16.956(12) 5.225(4) 7800(255) 0.360(10) 3046(92) 0.39(2) 2.9(5)

[0196] 31 17.468(8) 5.073(2) 13346(333) 0.699(8) 9929(115) 0.74(3) 3.6(2)

[0197] 32 19.310(12) 4.593(3) 6445(232) 0.608(11 ) 4170(77) 0.65(4) 3.7(4)

[0198] 33 19.993(18) 4.437(4) 802(82) 0.24(7) 297(52) 0.37(10) 1 (2)

[0199] 34 20.79(3) 4.270(7) 3129(161) 0.42(4) 1404(137) 0.45(7) 0.8(3)

[0200] 35 21.585(15) 4.114(3) 2239(137) 0.39(5) 921 (117) 0.41 (8) 2.0(10)

[0201] 36 22.86(8) 3.888(14) 1080(95) 0.56(10) 642(116) 0.59(16) 0.41 (9)

[0202] 37 23.74(7) 3.745(11 ) 994(91) 0.25(6) 261 (81 ) 0.26(11 ) 1.6(6)

[0203] 38 25.56(3) 3.482(4) 1540(113) 0.50(3) 817(59) 0.53(8) 0.8(2)

[0204] 39 26.49(5) 3.363(6) 705(77) 0.30(5) 227(28) 0.32(7) 1.2(7)

[0205] 40 27.45(5) 3.247(6) 858(85) 0.54(5) 494(40) 0.58(10) 1.3(5)

[0206] 41 33.88(2) 2.6435(18) 565(69) 0.20(7) 118(45) 0.21 (10) 2(4)

[0207] 42 43.60(5) 2.074(2) 544(67) 0.60(7) 511 (47) 0.9(2) 1.1 (3)

[0208] 43 53.70(10) 1.705(3) 99(29) 1.8(3) 185(39) 1.9(9) 0.9(6)

[0209] Experimental Example 3: TG-FTIR Thermogram of Final Product of Example 2 FIG. 15 shows the thermogravimetric and Fourier-transform infrared spectroscopy (TG-FTIR) and differential scanning calorimetry (DSC) results of the crystallized narasin according to the present disclosure. A TG-FTIR measurement was conducted on the crystallized narasin of the present disclosure at a heating rate of 10°C / min up to 300°C. The TG-FTIR result reveals a 0.5% to 0.6% mass loss (e.g., 0.56% mass loss) from 26 to 200°C that corresponds to water. Based on this result, the narasin crystallized according to the crystallization method of Example 2 is a new polymorph having the polymorph crystalline structure having an anhydrate form and is water-free and solvent-free. Also, as the decomposition does not occur until about 200°C, the polymorph (i.e., the final product) that results from Example 2 is thermally and chemically very stable.

[0210] In the DSC result, the crystallized narasin has a melting peak observed at 200.68°C, with an onset at 187.5°C. And, a melting enthalpy is between 57 J / g to 60 J / g, such as 59.34 J / g. The sharp and high peak observed shows a crystalline polymorph structure of the final product of Example 2.

[0211] Experimental Example 4: Microscope Views of Final Product of Example 2

[0212] FIG. 16 shows comparison between the microscope view of the product of Example 1 (Left) and the microscope view of the product of Example 2 (Right). The microscope view of the product of Example 1 (Left) shows an amorphous structure, whereas the microscope view of the product of Example 2 (Right) shows a crystalline form.

[0213] Experimental Example 5: Dynamic Vapor Sorption Result of Example 2

[0214] Specifically, FIG. 17 shows a dynamic vapor sorption (DVS) test result of the crystallized narasin of Example 2. The change of the relative sample weight (a red curve) and the relative humidity (a blue curve) are shown with a function of time. The relative sample weight is calculated from the mass change of the sample during the DVS measurement. The behavior of crystallized narasin was investigated in the presence of variable water vapor pressure at 25 °C using DVS. The measurement started at 50% relative humidity and the sample lost 0.8% of water by decreasing the relative humidity to 0%. When increasing the relative humidity to 95% and upon storage at 95%, the sample adsorbed 1.3% of water. The second DVS cycle is almost identical to the first one. At the end of the measurement, the relative humidity was 50%, and the sample mass was the same as at the start of the experiment. No hysteresis (e.g., a mass change) was observed in course of the DVS measurement, suggesting that no hydrate formation was obtained. The result shows that no form conversion of the crystallized narasin to other forms was observed during the DVS test or after 24 hours of equilibration in water in course of the solubility test. However, by slurring the crystallized narasin at a water activity of 0.9 for 10 days, conversion into other form was observed. Thus, the crystallized narasin is also vapor- resistant, and the crystallized narasin is capable of maintaining the polymorph crystalline structure when the narasin is exposed to solvents. For example, the crystallized narasin is capable of maintaining the polymorph crystalline structure up to 9 days after water is introduced to the crystallized narasin. Therefore, the crystallized narasin produced according to the methods of the present disclosure has a very low solubility and good shelf-life and is a crystalline form that is very stable.

[0215] The foregoing description of exemplary embodiments is provided to illustrate certain principles and practical applications. It is not intended to be exhaustive or to limit the scope to the specific embodiments disclosed. Those skilled in the art will recognize that modifications, variations, and substitutions may be made to the described methods, conditions, and chemical components without departing from the overall technical concept.

[0216] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and other parameters used in the specification and claims are to be understood as being modified in all instances by the term “about.” Ranges may be expressed herein as from “about A” to “about B,” or from “approximately A” to B, and are intended to encompass all values between A and B inclusive. The disclosure of specific values and ranges is not intended to exclude other values and ranges that may be suitable.

[0217] The operations of the present invention may be performed in different orders, in parallel, or simultaneously, depending on the embodiment and implementation.

[0218] The terminology used herein is only for the purpose of describing particular embodiments and is not intended to limit the present disclosure. Singular expressions include plural expressions unless otherwise specified in the context thereof. Throughout the present specification, it is to be understood that when any part is referred to as “comprising,” “including,” “containing,” and “having” any component, it does not exclude other components, but may further comprise other components, unless otherwise specified.

[0219] The subject matter described herein is not limited to the particular materials, solvents, reagents, or conditions disclosed, as these may vary depending on the desired application or the judgment of the skilled artisan. All such modifications and equivalents are intended to be included within the scope defined by the appended claims.

Claims

CLAIMS1. A method of crystallizing narasin, the method comprising: preparing acetonitrile; adding amorphous narasin to the acetonitrile; heating a mixture of the acetonitrile and the amorphous narasin to a first predetermined temperature while the mixture is being stirred; and stirring the mixture for a first predetermined time at a second predetermined temperature; stirring the mixture for a second predetermined time at a third predetermined temperature to obtain crystallized narasin having a polymorph crystalline structure.

2. The method of claim 1, wherein the crystallized narasin is characterized by one or more of: peaks in an XRPD diffractogram at 20 values of [4.3, 8.6, 9.2, 9.5, 12.7, 5.1, 17.5 °] ± 0.1°, but absent or substantially absent of a peak at 5 to 8, 10 to 11, 14 °20; peaks in an XRPD diffractogram at 20 values of 115.2, 16.9, 19.3, 15.3, 17.0 ° | ± 0.1°, but absent or substantially absent of a peak at 5 to 8, 10 to 11, 14 °20 in an XRPD diffractogram as substantially illustrated in FIG. 14.

3. The method of claim 1, wherein the crystallized narasin exhibits an XRPD diffractogram as substantially illustrated in FIG. 14 and a TGA / DSC thermograph as substantially illustrated in FIG. 15.

4. The method of claim 1, wherein the first predetermined temperature is 45°C to 55°C.

5. The method of claim 1, wherein a ratio of an amount of the narasin having an amorphous structure to the acetonitrile is 1:3.5 to 1 :4.5.

6. The method of claim 1, wherein the second predetermined temperature is 45°C to 55°C.

7. The method of claim 1, wherein the first predetermined temperature and the second predetermined temperature are same.

8. The method of claim 1, wherein the third predetermined temperature is 2°C to 10°C.

9. The method of claim 1, wherein the stirring for the second time is performed for about 17 hours.

10. The method of claim 1, wherein the crystallized narasin has a lipid content of 0.05% or less.11 . The method of claim 1 , wherein the polymorph crystalline structure has an anhydrate form, wherein the crystallized narasin is water-free and solvent-free.

12. The method of claim 1 , wherein the crystallized narasin has a melting point in a range of 188°C to 193°C and a melting enthalpy of 57 J / g to 59 J / g.

13. The method of claim 1, wherein the crystallized narasin is vapor-resistant, and the crystallized narasin is capable of maintaining the polymorph crystalline structure when the narasin is exposed to solvents.

14. The method of claim 13, wherein the crystallized narasin is capable of maintaining the polymorph crystalline structure up to 9 days after water is introduced to the crystallized narasin.

15. The method of claim 1, wherein the stirring of the mixture at the third predetermined temperature is performed for at least 5 hours.

16. The method of claim 1, further comprising: after the stirring for the second time, filtering the mixture to obtain the crystallized narasin having the polymorph crystalline structure; and drying the crystallized narasin in a vacuum at a fourth predetermined temperature in a range of 30°C to 50°C for at least 15 to 19 hours.

17. The method of claim 13, wherein the crystallized narasin has a purity of 97% or above.

18. The method of claim 11, wherein the crystallized narasin has a solubility in a range of 0.0015 to 0.0020 mg / mL.

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