Method for purifying methanobactin and methanobactin purified thereby

Abstract

The present invention relates to a method for method for purifying methanobactin, preferably, methanobactin SB2 (mb-SB2). More specifically, the present invention relates to a method for culturing and purifying methanobactin SB2 (mb-SB2) with high purity and high yield.

Description

METHOD FOR PURIFYING METHANOBACTIN AND METHANOBACTIN PURIFIED THEREBY

[0001] CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 675,355 filed on July 25, 2024 with the United States Patent and Trademark Office, the disclosures of which are herein incorporated by reference in their entirety.

[0003] This application was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare　(RS-2025-02213496, Republic of Korea).

[0004] The present invention relates to a method for purifying methanobactin, preferably, methanobactin SB2 (mb-SB2). More specifically, the present invention relates to a method for culturing and purifying methanobactin SB2 (mb-SB2) with high purity and high yield.

[0005] Methylocystis sp. strain SB2 is a type of bacteria known as a methanotroph and for its ability to use methane as carbon and energy source. Many methanotrophs have been found to synthesize a protein known as methanobactin (MB). Methanobactin SB2 (mb-SB2), as a kind of MB, is produced by fermentation with Methylocystis sp. strain SB2 under methane and oxygen gases.

[0006] The structure and utility of mb-SB2 are well described in US patent No. 11000568 (incorporated herein by reference), and the cultivation method for mb-SB2 is disclosed in papers such as Methods in Enzymology. 2011; 495: 259-269 (incorporated herein by reference). However, the disclosed method had limitations in efficiently producing mb-SB2 and did not meet the production quantities required for non-clinical and clinical trials necessary for drug development. Therefore, a new method was needed to increase production yield, particularly to efficiently purify mb-SB2 in the downstream process.

[0007]

[0008] The present inventors, for the first time, suggest a novel preparation method by efficiently methanobactin, preferably, methanobactin SB2(hereinafter, also described as mb-SB2) in the downstream process.

[0009] It is, therefore, an object of the present invention to provide a　method　for　purification of methanobactin from a sample comprising the combined use of ion-exchange　chromatography　and　reverse phase chromatography. The　method　comprises the steps of subjecting the sample (if necessary concentrated) to ion-exchange　chromatography　and subjecting the eluate to　reverse phase chromatography.

[0010] In order to achieve the object, the present invention provides a method for purifying methanobactin, preferably, methanobactin SB2 (mb-SB2). More specifically, the present invention relates to a method for culturing and purifying methanobactin, preferably, methanobactin SB2 (mb-SB2) with high purity comprising the combined use of ion-exchange chromatography and reverse phase chromatography.

[0011] The present invention achieves increased production yield, particularly by efficiently purifying methanobactin, preferably, mb-SB2 in the downstream process.

[0012] Figure 1 presents Chromatogram explaining a, b WHand VR.

[0013] Figures 2 and 3 are chromatograms for Run-1 (Fig. 2) and Run-2 (Fig. 3) confirming repeatability of existing method using Diaion HP20.

[0014] Figure 4 is a chromatogram for anion exchange chromatography (AEX) using DEAE-FF in order to choose optimized conditions for AEX Run on column.

[0015] Figures 5 and 6 are chromatograms for anion exchange chromatography (AEX) using DEAE- where in 3+5 or 5+5 column volumes (CVs) of washes are given with low pH buffer and low pH with EDTA buffer.

[0016] Figure 7, 8 and 9 are a chromatogram for anion exchange chromatography (AEX) using DEAE for adjustment of column volumes (CVs) of wash buffers

[0017] Figure 10 is a chromatogram for anion exchange chromatography (AEX) using DEAE to include pH 7 buffer in wash buffers

[0018] Figure 11, 12 and 13 are a chromatogram for anion exchange chromatography (AEX) using DEAE with multiple number of cycles to check the effect on binding

[0019] Figure 14 is a chromatogram for anion exchange chromatography (AEX) using DEAE for checking the maximum loading capacity

[0020] Figure 15 is a chromatogram for anion exchange chromatography (AEX) using DEAE with a column inner diameter of 26 mm

[0021] Figure 16 is a chromatogram for anion exchange chromatography (AEX) using DEAE with a column inner diameter of 50 mm

[0022] Figure 17 and 18 are a chromatogram for anion exchange chromatography (AEX) using DEAE with a column inner diameter of 140 mm

[0023] Figure 19 is a chromatogram for HP20 Diaion resin with a column inner diameter of 50 mm to compare with DEAE Sepharose FF resin

[0024] Figure 20 is a for DEAE Sepharose FF resin with a column inner diameter of 50 mm to compare with HP 20 Diaion resin

[0025] Figure 21, 22 and 23 are a chromatogram with HP 20 Diaion resin chromatography after anion exchange chromatography using DEAE

[0026] Figure 24 is a chromatogram for chromatography with HP 20 resin and Sodium acetate pH 7.0 of washing buffer

[0027] Figure 25 is a chromatogram for chromatography with HP 20 resin and Sodium acetate pH 4.0 of washing buffer

[0028] Figure 26 and 27 are a chromatogram for C18 reverse phase column chromatography with methanol of equilibration buffer

[0029] Figure 28 is a chromatogram for C18 reverse phase column chromatography with highly purified water of equilibration buffer

[0030] Figure 29 is a chromatogram for C18 reverse phase column chromatography with Tris buffer of equilibration buffer

[0031] Figure 30 and 31 are a chromatogram for C18 reverse phase column chromatography with Ammonium Acetate buffer of equilibration buffer

[0032] Figure 32 is a chromatogram for C18 reverse phase column chromatography with 10 (v / v)% methanol of elution solvent

[0033] Figure 33 is a chromatogram for C18 reverse phase column chromatography with 20 (v / v)% methanol of elution solvent

[0034] Figure 34 is a chromatogram for C18 reverse phase column chromatography with 5 (v / v)% and 10% methanol of elution solvents

[0035] Figure 35 is a chromatogram for C18 reverse phase column chromatography with 10 (v / v)% methanol of elution solvent

[0036] Figure 36 is a chromatogram for C18 reverse phase column chromatography with 10 (v / v)% methanol of equilibration buffer, loading solvent and elution buffer.

[0037] Hereinafter, the present invention will be described in detail.

[0038]

[0039] METHANOBACTIN

[0040] Methanobactins are low molecular mass copper-binding molecules produced by many methanotrophic bacteria and have been demonstrated to mediate copper acquisition from the environment (Semrau et al., 2010. FEMS Microbiol. Rev 34:496-531). it has been demonstrated that methanobactins hold considerable potential for treatment of a variety of copper-related diseases and conditions (Lichtmannegger et al., 2016. J Clin Invest. 126(7):2721-35), and, due to their excellent copper binding affinities (Choi et al., 2006. Biochemistry 45: 1442-1453) and tolerance in vivo, are promising new agents for a massive and fast depletion of excess copper levels in patients in need thereof. Due to their beneficial properties, methanobactins are considered to be particularly useful for acute de-coppering therapy in Wilson Disease patients.

[0041]

[0042] The term “methanobactin” as used herein includes naturally occurring methanobactins and functional variants, fragments and derivatives thereof which retain the capability of complexing copper (i.e., Cu(I) and Cu(II)), and preferably bind Cu(I) with a binding affinity that is comparable or even higher than that of the naturally occurring methanobactins.

[0043]

[0044] The examples of the methanobactin are known in Krentzet al.(Biochemistry (2010) DOI: 10.1021 / bi1014375), El Ghazouaniet al.(PNAS, May 29, 2012, vol. 109, no. 22, pp. 8400-8404) and DiSpirito et al. (Microbiology and Molecular Biology Reviews, June 2016, Vol. 80, No. 2), the disclosures of which are herein incorporated by reference in their entirety.

[0045]

[0046] In some specific embodiment, the methanobactin may comprise or consist of the following general formula (I):

[0047] R1―(X)2-5―R2(I)

[0048] wherein

[0049] R1and R2are each a 5 or 6-membered heterocycle comprising N and associated with an enethiolate;

[0050] and each X is independently selected from any amino acid.

[0051]

[0052] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its recognized definition such as an amino acid selected from the group consisting of: alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (GIn or Q); glutamic acid (GIu or E); glycine (GIy or G); histidine (His or H); isoleucine (Ile or I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); pro line (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (VaI or V), although modified, synthetic, or rare amino acids may be used as desired. Generally, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, IIe, Leu, Met, Gly, Phe, Pro, VaI); a negatively charged side chain (e.g., Asp, GIu); a positively charged sidechain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, GIn, Ser, Thr, Trp, and Tyr). The term encompasses naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0053]

[0054] It is further contemplated that the methanobactin may be derived from bacteria, including methanotroph and non-methanotroph bacteria, such as Methylocystis spec., Methylosinus spec., Methylomicrobium spec. and Methylococcus spec. For instance, the methanobactin may be selected from (a) a Methylosinus trichosporium OB3b methanobactin (mb-OB3b) (b) a Methylocystis strain SB2 methanobactin (mb-SB2), (c) a Methylococcus capsulatus Bath methanobactin (mb-Bath) (d) a Methylomicrobium album BG8 methanobactin (mb-BG8), (e) a Methylocystis strain M methanobactin, (f) a Methylocystis hirsuta CSC1 methanobactin and (g) a Methylocystis rosea methanobactin (mb-rosea), (h) a Methylosinus sp. strain LW3 methanobactin (mb-LW3), (i) a Methylosinus sp. strain LW4 methanobactin (mb-LW4), (j) a Methylocystis sp. strain LW5 (mb-LW5), (k) a Methylosinus sp. strain PW1 methanobactin (mb-PW1), (I) a Methylocystis parvus OBBP methanobactin (mb-OBBP), (m) a Cupriavidus basiliensis B-8 methanobactin (mb-B-8), (n) a Pseudomonas extremaustralis 14-3 methanobactin (mb-14-3), (o) a Azospirillum sp. stain B510 methanobactin (mb-B510), (p) a Tistrella mobilis KA081020-065 (mb-mobilis) methanobactin and (q) a Comamonas composti DSM 21721 methanobactin (mb-21721).

[0055]

[0056] Preferably, the methanobactin may be methanobactin SB2 (mb-SB2).

[0057]

[0058] One of the non-limiting examples of said mb-SB2 may be of the formula (I):

[0059] (I)

[0060] .

[0061]

[0062] For the purpose of the invention the methanobactin as defined above also includes the pharmaceutically acceptable salt(s) thereof. The phrase "pharmaceutically acceptable salt(s)", as used herein, means those salts of methanobactins that are safe and effective for treatment. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, choline etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0063]

[0064] PURIFICATION METHOD FOR METHAOBACTIN

[0065] In an aspect, the present invention relates to a　method　for purifying methanobactin, preferably, methaobactin SB2 (mb-SB2), in particular for purifying methanobactin from a crude　preparation in the culture medium with a high degree of purity and high production yield.

[0066]

[0067] In a specific aspect, the method may comprise comprising the following steps:

[0068] a) subjecting a sample comprising the methanobactin to an anion exchange chromatography to produce a first eluate;

[0069] b) lyophilizing or nano-filtering the first eluate obtained in the step a) in order to concentrate the first eluate

[0070] c) subjecting the first eluate from step b) to a reverse phase chromatography to produce a second eluate;

[0071] d) lyophilizing the second eluate obtained in the step c).

[0072]

[0073] In one aspect, the purification method according to the present invention is characterized in that it is performed under basic conditions. More specifically, the basic conditions mean maintaining pH 6.0-9.0, preferably pH 6.5-7.5.

[0074]

[0075] The inventors surprisingly confirmed that while mb-SB2, a methanobactin, is rapidly degraded under acidic conditions such as a pH 1.0-pH 6.0 buffer solution, when purified while maintaining basic conditions, the degradation of methanobactin is significantly reduced, thereby maintaining its stability.

[0076]

[0077] In order to implement such basic conditions, the manufacturing method according to the present invention specifically selects first purifying a sample containing methanobactin using anion exchange chromatography, and then purifying methanobactin using C18 reversed phase chromatography.

[0078]

[0079] The step a) is subjecting a sample comprising the methanobactin to an anion exchange chromatography to produce a first eluate. Preferably, the methanobactin may be methanobactin SB2 (mb-SB2).

[0080]

[0081] The sample may be a culture medium from Methanotrophs. In some embodiemtns, the sample may be constituted by freshly collecting cell culture supernatant medium perfused through a bioreactor. It is preferably clarified by filtration. The crude solution can then be concentrated, if necessary, and subjected to filtration with TFF filter or hollow fiber filter.

[0082] After the above preliminary steps, the sample is then subjected to anion-exchange　chromatography.

[0083] The anion chromatography may be preferably carried out with　resin such as SP Sepharose FF, Ni+ NTA, Q Sepharose resins, DEAE Sepharose FF or PPG-600M.

[0084]

[0085] In some embodiments, the anion exchange chromatography may be performed by a process comprising the steps of: (i) loading a sample comprising methanobactin onto an anion exchange column, wherein said methanobactin are bound to said anion exchange column; (ii) washing said anion exchange column in a) containing said bound methanobactin with one or more wash buffers; (iii) eluting said methanobactin from the column in (ii) with an elution buffer; and (iv) collecting said mb-SB2.

[0086]

[0087] In some embodiments, the anion exchange chromatography process comprises a buffer system. In some embodiments, the buffer system comprised one or more elution buffers. In some embodiments, the buffer system comprises one or more wash buffers.

[0088]

[0089] In some embodiments, the pH of the sample is from pH 6.0 to pH 9.0, preferably, 6.5 to pH 7.5.

[0090]

[0091] In some embodiments, one or more wash buffer may be used in the anion exchange chromatography. The wash buffer comprises optionally at least one chelating agent.

[0092]

[0093] In some embodiments, the one or more elution buffers comprise a NaCl concentration of 0.1 M to 0.7 M, 0.2 M to 0.6 mM, or 0.5 M.

[0094]

[0095] In some embodiments, methanobactin is eluted in the anion exchange chromatography step using one elution buffer. In some embodiments, methanobactin is eluted in the anion exchange chromatography step using a gradient elution method comprising more than one elution buffer. In some embodiments, the elution buffer comprises optionally at least one chelating agent.

[0096]

[0097] In some embodiments, the pH of the wash buffer for the anion exchange chromatography step is from pH 6.0 to pH 9.0, preferably, 6.5 to pH 7.5.

[0098]

[0099] In some embodiments, the pH of the elution buffer for the anion exchange chromatography step is from pH 6.0 to pH 9.0, preferably, 6.5 to pH 7.5.

[0100]

[0101] In some embodiments, the one or more buffers (including wash and / or elution buffers) optionally comprise one or more chelating agents. In some embodiments, the elution buffer includes at least one chelating agent. The chelating agent can be a divalent cation chelating agent. In some embodiments, the at least one chelating agent is a divalent cation chelating agent. In some embodiments, the divalent cation chelating agent is selected from the group consisting of EDTA, EGTA, CDTA, and citrate. In some embodiments, the divalent cation chelating agent is selected from the group consisting of NTA, DTPA, EDDS, EDTA, EGTA, CDTA, and citrate. In some embodiments, the chelating agent is EDTA.

[0102]

[0103] Any of the buffers (buffer systems) described herein can be selected from the group consisting of glycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TrisHCl (Tris(hydroxymethyl)-aminomethane), histidine, imidazole, acetate citrate, citrate, acetate, MES, phosphate, TrisHCl, Bis-Tris, Histidine, Imidazol, ArgininHCl, LysinHCl, and 2-(N-morpholino)ethanesulfonic acid, as single buffers or as a combination of two or more buffers. In some embodiments, the buffer comprises TrisHCl (Tris(hydroxymethyl)-aminomethane).

[0104]

[0105] In some specific embodiments, the wash buffer may comprise (i) Tris buffer and EDTA at pH 4.0 or (ii) Tris buffer at pH 7.0.

[0106]

[0107] In some specific embodiments, the elution buffer may comprise (i) Tris buffer or (ii) Tris and NaCl buffer at pH 7.0

[0108]

[0109] In some embodiments, the column volume (CV) of the elution of the present method is 5-30 CV.

[0110]

[0111] In some embodiments, the flow rate of one or more elution steps of the present method is about 10 cm / h to about 200 cm / h.

[0112]

[0113] In some embodiments, the one or more buffers further comprise one or more nonionic detergents. In some embodiments, the nonionic detergent is selected from the group consisting of Triton X-100,　Tween　80, and　Tween　20.

[0114]

[0115] In some further embodiments, the anion exchange chromatography process may further comprise the steps of pre-equilibration, equilibration and regeneration.

[0116]

[0117] Chromatography equilibration is　the process of preparing a chromatographic column to ensure a stable and consistent environment before sample injection.　This involves establishing a stable baseline by allowing the mobile phase to flow through the column until interactions between the stationary and mobile phases are consistent throughout.　Proper equilibration is crucial for reliable and reproducible chromatographic results.　In some embodiment, the equilibration may be performed with Tris buffer at pH 7.0.

[0118]

[0119] Chromatography regeneration is　the process of cleaning and restoring the performance of a chromatography column, after it has been used and its efficiency has decreased due to contamination or fouling.　This process typically involves removing accumulated sample components and mobile phase impurities that can hinder separation performance. In some embodiment, the regeneration may be performed with NaCl solution or NaOH solution.

[0120]

[0121] The step b) is a step of lyophilizing or nano-filtering the first eluate obtained in the step a) in order to concentrate the first eluate. After the lyophilization or the nano-filtration, the concentrated first eluate may be reconstituted with water (preferably, purified water), methanol,etc. The reconstituted first eluate may be subjected to step c).

[0122] Lyophilization is carried out using techniques common in the art and should be optimized for the composition being developed (Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res. 13:243-9 (1996)).

[0123] A lyophilization cycle is, in one aspect, composed of three steps: freezing, primary drying, and secondary drying (A. P. Mackenzie, Phil Trans R Soc London, Ser B, Biol 278:167 (1977)). In the freezing step, the solution is cooled to initiate ice formation. Furthermore, this step induces the crystallization of the bulking agent. The ice sublimes in the primary drying stage, which is conducted by reducing chamber pressure below the vapor pressure of the ice, using a vacuum and introducing heat to promote sublimation. Finally, adsorbed or bound water is removed at the secondary drying stage under reduced chamber pressure and at an elevated shelf temperature. The process produces a material known as a lyophilized cake. Thereafter the cake can be reconstituted with either sterile water or suitable diluent for injection.

[0124] The lyophilization cycle not only determines the final physical state of excipients but also affects other parameters such as reconstitution time, appearance, stability and final moisture content. The composition structure in the frozen state proceeds through several transitions (e.g., glass transitions, wettings, and crystallizations) that occur at specific temperatures and the structure may be used to understand and optimize the lyophilization process. The glass transition temperature (Tg and / or Tg′) can provide information about the physical state of a solute and can be determined by differential scanning calorimetry (DSC). Tg and Tg′ are an important parameter that must be taken into account when designing the lyophilization cycle. For example, Tg′ is important for primary drying. Furthermore, in the dried state, the glass transition temperature provides information on the storage temperature of the final product.

[0125] In a specific exemplified embodiment, the lyophilization step may be conducted by using tray type lyophilization.

[0126] As used herein, the term "tray-type lyophilization" refers to a freeze-drying process in which a liquid or semi-solid composition is dispensed into open trays and subjected to a series of drying steps under reduced pressure. The process typically includes (i) a freezing step in which the composition is solidified under controlled temperature conditions; (ii) a primary drying step in which frozen solvent, typically water, is removed via sublimation under vacuum; and (iii) a secondary drying step in which residual bound moisture is removed. Tray-type lyophilization enables uniform heat transfer across the surface of the product and allows for efficient processing of bulk materials. Due to its scalability and ability to preserve the structural and functional integrity of thermosensitive substances, tray-type lyophilization is widely used in the preparation of pharmaceutical, biotechnological, and diagnostic products.

[0127] In the present invention, the tray-type lyophilization may be performed using a programmed setting ranging from -40°C to 30°C over 2,000-3,500 minutes under a pressure range of 200 to 30 microbar.

[0128]

[0129] Nanofiltration (NF) is a membrane-based separation technology that operates under pressure to selectively remove small molecules and multivalent ions from a solution. In purification processes involving chromatography, nanofiltration is often employed downstream to concentrate the target compound, remove residual solvents or buffer components, and eliminate low molecular weight impurities. NF membranes typically have a molecular weight cutoff between 100 and 1000 Da, allowing for the retention of larger bioactive molecules while permitting smaller contaminants to pass through. This step enhances product purity and facilitates solvent exchange or buffer adjustment, thereby improving the efficiency and yield of the overall purification process.

[0130] In the present invention, said nanofiltration is performed for concentration and diafiltration of the sample to bring the sample conditions for reverse column chromatography. The nanofiltration is performed by cleaning the filter using water for injection (WFI) followed by equilibration of filter using WFI. The load is concentrated to the desired volume at a TMP of 6-7 bar. The filter is then washed with AEX Elution Buffer B, and the entire load is recovered. Nano Filtration unit of 100-150Da was used with membrane area of 1.8" X 12". AEX Desired fractions is used as Nanofiltration input.

[0131]

[0132] In the step c), the first eluate from step b) (lyophilized or nano-filtered then reconstituted) is subjected to a reverse phase chromatography to produce a second eluate. The reverse phase chromatography step is preferably performed on silica based column, more preferably, Silica　C18 column　(C18 column chromatography) and is effective in removing higher amounts of salts and maintaining purity.

[0133]

[0134] In some embodiments, the reverse phase chromatography may be performed by a process comprising the steps of: (i) loading the first eluate comprising mb-SB2 onto a reverse column; (ii) eluting said mb-SB2 from the column in (i) with an elution buffer; and (iii) collecting said mb-SB2.

[0135] In some embodiments, the reverse column is C18 column.

[0136]

[0137] In some embodiments, the buffer (equilibration buffer, loading buffer, post load washing buffer and / or washing buffer) of the reverse phase　chromatography　comprises an organic solvent. In some embodiments, the buffer of the reverse phase　chromatography　comprises an alcohol, preferably methanol, more preferably, 10-50(v / v)% methanol, preferably, 10-30(v / v)% methanol.

[0138]

[0139] In some further embodiments, the process may further comprise the steps of equilibration, and regeneration.

[0140]

[0141] In some embodiments, the regeneration step may be used 90 (w / w)% methanol as regeneration buffer.

[0142]

[0143] In some embodiments, the pump flow of the reverse phase　chromatography　system is about 50 mL / min to about 10 L / min, 65 mL / min to about 8 L / min, 75 mL / min to about 6 L / min, about 85 mL / min to about 1 L / min, or about 100 mL / min to about 800 mL / min.

[0144]

[0145] The resulting second eluate is then lyophilized in step c).

[0146] Unless otherwise stated, the lyophilization of the step c) may be applied as is to the lyophilization in step b) mentioned above.

[0147]

[0148] ****

[0149] It must be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0150] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0151] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0152] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes, however, also the concrete number, e.g., about 20 includes 20.

[0153] The term “less than” or “greater than” includes the concrete number. For example, less than 20 means less than or equal to. Similarly, more than or greater than means more than or equal to, or greater than or equal to, respectively.

[0154] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.

[0155] When used herein “consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0156] In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

[0157] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0158] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0159]

[0160] Hereinafter, the present invention will be described in more detail through Examples.

[0161] These Examples are intended to describe the present invention in more detail, and the scope of the present invention is not limited to these Examples.

[0162]

[0163] 1.DEFINITIONS

[0164]

[0165] Column Chromatography

[0166]

[0167] Column Volume (CV, ml): Total volume of material, both solid and liquid, in the column, i.e., the volume of the support particle plus the void volume. It gives the volume of resin in column.

[0168]

[0169] CV = πr2h

[0170]

[0171] where, r=radius of the column (cm), h=height to which resin is packed in the column(cm)

[0172]

[0173] Compression factor (CF): The permeability of the packed bed depends on the particle size of the resin which can be compressed to an extent as per the resin’s recommendation. That compression limit is called as Compression factor.

[0174]

[0175] Compression factor = Bed height measured after settling by gravity (cm) / Packed bed height (cm)

[0176]

[0177] Slurry Concentration (%): Slurry consists of the solid particles suspended in a liquid in some proportion. This proportion of solid in the medium is called slurry concentration.

[0178]

[0179] Settled bed Volume (SV): Minimum volume of solvent necessary to wet the defined quantity of sorbent within the column.

[0180]

[0181] SV = (100 * Column Volume) / (100 - % Compression)

[0182]

[0183] Bed height(h): Height of the slurry packed into the column described in cm.

[0184]

[0185] Cross sectional area of column (A): A = πr2where r is the radius of the column in cm2

[0186]

[0187] Volumetric flow rate(ml / min): Is the volume of fluid which passes per unit time through the column, given in ml / min or L / Hr.

[0188]

[0189] Linear flow rate (cm / hr): Volumetric flow rate(ml / min) per unit cross section area (cm2) of a given column.

[0190]

[0191] Linear flow rate(cm / h) = Volumetric flow rate (ml / min) * 60 / Cross sectional area of column(cm2)

[0192]

[0193] Slurry Volume (ml): Is the ratio of settled bed volume and slurry concentration.

[0194]

[0195] Slurry Volumn = Settled bed volume (ml) / Slurry concentration

[0196]

[0197] HETP (Height equivalent to a theoretical plate): is calculated by the formula.

[0198] HETP = L / N

[0199] Where:

[0200] L - Column bed height (in cm)

[0201] N - Number of Theoretical Plates calculated by the following equation:

[0202] N = 5.54 (VR / WH)2

[0203] Where:

[0204] VR = retention volume, which is the volume from the start of tracer injection until the peak on the chromatogram, as shown in Figure 01

[0205] WH = peak width at half height expressed in ml, as shown in Figure 01

[0206]

[0207] Peak asymmetry: is calculated with the following equation:

[0208] AF = b / a

[0209] Where:

[0210] a-is the peak width (left half) at 10% of peak height.

[0211] b-is the peak width (right half) at 10% of peak height.

[0212]

[0213] Figure 1 presents Chromatogram explaining a, b WH and VR..

[0214]

[0215] 2.Upstream Process

[0216] In order to produce mb-SB2, Methylocystis sp. strain SB2 is inoculated into the Nitrate Mineral Salt (NMS) medium (Whittenbury et al., 1970) along with copper and then feed with methane and oxygen (or air) gases as a source of carbon and oxygen at 30oC. The more specific cultivation method is descried in US patent No. 8,629,239 (incorporated herein by reference).

[0217]

[0218] 2.1.Culture of mb-SB2

[0219]

[0220] - Add NMS media with corresponding stock solutions into the bioreactor

[0221] - Sterilize the medium and reactor

[0222] - Once medium cooled, turn on the gases (methane and oxygen or air)

[0223] - Inoculate Methylocystic sp. Strain SB2 into bioreactor

[0224] - Turn on the agitator and set the temperature to 30oC

[0225] - Monitor OD600and D.O. (dissolved oxygen) value and adjust gassing ratio

[0226]

[0227] 2.2.Cell Clarification (Harvesting)

[0228] - Once the target concentration of mb-SB2 dissolved in the medium is reached, proceed to filter with TFF filter or hollow fiber filter

[0229] - Obtain clarified harvest

[0230]

[0231] mb-SB2 secreted from these culture medium was used in all subsequent experiments

[0232]

[0233] 3.Checking the Repeatability of existing methods

[0234]

[0235] 3.1.Specification of Diaion HP20:

[0236]

[0237]

[0238] 3.2.Run-1

[0239]

[0240] Experimental Design:

[0241] Column Packing Details:

[0242] Column Used: XK16 / 40

[0243] Resin Used: HP20 Diaion

[0244] Bed height: 20 cm

[0245] Column Volume: 40ml

[0246]

[0247] Operational Details:

[0248] Sample Details:

[0249] Volume: 1342ml

[0250] pH: 7.0

[0251] Conductivity: 5.93mS / cm

[0252] Load preparation: No load preparation was done

[0253]

[0254]

[0255] Chromatogram: Figure 2

[0256]

[0257] Results:

[0258] As per the UPLC analysis, a peak is observed in Post load wash. Elution didn’t show any peak. The peak in post load wash is confirmed to be copper bound impurity.

[0259]

[0260] 3.3.Run-2

[0261]

[0262] To check the repeatability of mb-SB2 Sample on HP20 Resin by executing the step as per the process given by sending unit.

[0263]

[0264] Experimental Design:

[0265] Column Packing Details:

[0266] Column Used: XK16 / 40

[0267] Resin Used: HP20 Diaion

[0268] Bed height: 20 cm

[0269] Column Volume: 40ml

[0270]

[0271] Operational Details:

[0272] Initial Sample Details:

[0273] Volume: 50ml

[0274] pH: 7.09

[0275] Conductivity: 8.76mS / cm

[0276] Load preparation: No load preparation was done

[0277]

[0278]

[0279] Chromatogram: Figure 3

[0280]

[0281] Results: As per the UPLC analysis, a peak is observed in Post load wash. Elution didn’t show any peak. The peak in post load wash is confirmed to be copper bound impurity.

[0282]

[0283] 3.4.Conclusions:

[0284] In above two trials, the repeatability was not observed. Hence, it was decided that the purification process would be developed further for capturing of mb-SB2 product

[0285]

[0286] 4.Process Development

[0287]

[0288] 4.1.Evaluation of Resin for Capture of mb-SB2:

[0289] Multiple resins screening studies were performed to capture and elute the product, and better resin was finalized based on overall performance in terms of impurity removal. Clarified cell culture fluid is directly loaded onto the resin in batch mode.

[0290]

[0291] 4.1.1.Experimental Design:

[0292] Different resins were selected for screening, based upon their interaction with the product of interest. Five different resin such as SP Sepharose FF, Ni+ NTA, Q Sepharose resins, DEAE Sepharose FF and PPG-600M were chosen for screening.

[0293]

[0294]

[0295] 4.1.2.Experimental Details:

[0296]

[0297]

[0298] 4.1.3.Results:

[0299] Based on the HPLC analysis of FT, PLW, wash and Elution, it was observed that the product was bound to SP Sepharose, Ni-NTA, DEAE FF- and Q-Sepharose FF resins, and in case of PPG resin, the product was not bound to resin itself.

[0300] In SP-Sepharose and Ni NTA, the product of interest was not seen during elution. In DEAE- & Q- Sepharose FF experiment, the desired product was completely bound to resin and eluted out with salt concentration at pH 7.0.

[0301]

[0302] 4.1.4.Conclusion:

[0303] As per the results of above 5 trials, the product binding and elution was seen only in DEAE Sepharose FF and Q Sepharose FF trials and observed the better purity in DEAE resin trial. Hence the DEAE Sepharose FF has been chosen for further purification process trials. But the peak eluted was copper bound impurity only.

[0304]

[0305] 4.2.Optimization of AEX (Anion Exchange chromatography) for capture of mb-SB2

[0306]

[0307] 4.2.1.AEX Run on Column

[0308] Based upon the above screening trials on multiple resin, the better purity with removal of other impurities were observed in the DEAE-FF resin. Hence, the below experiments were performed to confirm results on column trials. The copper bound impurity when incubated with 20 mM EDTA did not yield free mb-SB2 in batch mode. The below mentioned trials were done with low pH buffer and low pH buffer with EDTA to see if leads to conversion of copper bound mb-SB2 to free mb-SB2.

[0309]

[0310] 4.2.1.1.Experimental Design

[0311] Based on the batch mode trials, the DEAE condition was chosen, and higher scale column chromatography is performed to check the purity of desired product after capture on DEAE-FF. The condition used for the run is as given below.

[0312]

[0313] Column Details:

[0314] Column Used: XK26 / 40

[0315] Resin Used: DEAE Sepharose

[0316] Bed height (cm): 20

[0317] Column Volume (ml): 106

[0318]

[0319] Run Details:

[0320]

[0321] 4.2.1.2.Chromatogram: Figure 4

[0322]

[0323] 4.2.1.3.Results

[0324] The DEAE-FF showed the conversion of copper bound mb-SB2 to free mb-SB2 i.e., the desired product in Elution with more than 90% of purity, along with the separation of product from other impurities (seen in separate fractions).

[0325]

[0326] 4.2.2.Optimization of No. of AEX Washes

[0327] As per previous experiment, it has been observed that DEAE-FF gives good conversion of copper bound impurity to free mb-SB2. Hence, this experiment is designed to optimize the number of washes of low pH and low pH EDTA buffer.

[0328]

[0329] 4.2.2.1.Experimental Design

[0330] The design of experiments is as given below where in 3+5 or 5+5 CVs of washes are given with low pH buffer and low pH with EDTA buffer, respectively.

[0331]

[0332] 4.2.2.2.Experimental Details:

[0333]

[0334] 4.2.2.3.Chromatogram: Figure 5, Figure 6

[0335]

[0336] From the results of both the experiments, it has been observed that similar profile of separation obtained for the product. mb-SB2 and confirms the repeatability of cycle conditions with purity of > 85%.

[0337]

[0338] 4.2.2.4.Conclusion:

[0339] There was not much difference observed in both the cycles. Based on the results in Experiment 5.1 and 5.2, it was concluded that only 25mM Tris-Cl pH4.0+20mM EDTA wash can be given instead of giving two low pH washes such as 25mM Tris-Cl pH4.0 followed by 25mM Tris-Cl pH4.0+20mM EDTA.

[0340]

[0341] 4.2.3.Inclusion of Wash2

[0342] As per the previous conditions, the resin does not achieve back the pH 7.0 during elution, and hence to do so Wash2 was included in the run with pH 7 buffer.

[0343]

[0344] 4.2.3.1.Experimental Design:

[0345]

[0346] 4.2.3.2.Chromatogram: Figure 10

[0347]

[0348] Inclusion of Wash2 did not have any significant impact on the purity or yield of the product. Similar result was obtained from the run as observed in the previous run along with similar profile.

[0349]

[0350] 4.2.3.3.Conclusion

[0351] Since there is no impact observed in the quality of product, it was concluded to include wash 2 so that the pH can be brought back to elution pH conditions.

[0352]

[0353] 4.2.4.Optimization of loading conductivity on AEX

[0354] It has been seen that on DEAE-FF resin, at conductivity of 2.9mS / cm, the desired product binds to the column completely without any losses in flow through and washes. As the broth conductivity ranges between 8-10mS / cm, the binding at different conductivity levels by dilution was scouted in below set of experiments to avoid loss of product in flow through.

[0355]

[0356] 4.2.4.1.Experimental Design:

[0357] The multiple number of cycles was performed at multiple conductivities to check the effect on binding on column

[0358]

[0359]

[0360]

[0361] 4.2.4.2.Chromatogram: Figure 11, 12, 13

[0362] From above chromatograms, it has been observed that in none of the experiments the desired product was lost in Flowthrough and washes which shows the complete binding to the column at max. 6.2mS / cm conductivity

[0363]

[0364] 4.2.4.3.Conclusion:

[0365] Based on above trials, product is binding completely up to 6.2mS / cm conductivity.

[0366]

[0367] 4.2.5.Optimization of Loading capacity on AEX:

[0368] As per previous experiments, after dilution with water during load preparation, the fermentation broth was loaded to maximum capacity to check the impact on purity profile.

[0369]

[0370] 4.2.5.1.Experimental Design

[0371] The 160L of harvest broth was diluted to 2.9mS / cm conductivity and loaded on BPG140 / 500 column with 2.3 L of resin to understand the maximum loading capacity by following the below condition of cycle.

[0372]

[0373]

[0374] 4.2.5.2.Chromatogram: Figure 14

[0375] In this experiment, only 78.8% purity observed in fraction 3 of eluate. As per other experiments, the desired purity should be >85%. Hence maximum loading can cause the improper separation of desired product in elution.

[0376]

[0377] 4.2.5.3.Conclusion:

[0378] For further experiments, 1 mL resin for 25 mL of harvest is recommended to be used for purification of mb-SB2 using AEX resin. Based on the results of above experiment, it is recommended that the DBC studies need to be evaluated to achieve higher DBC along with higher purity.

[0379]

[0380] 4.2.6.Finalized DEAE-FF Cycles at higher scale

[0381] As per previous selected conditions the DEAE-FF resin has been chosen for capture of mb-SB2 from fermentation broth. To check the repeatability and consistency of process the following cycles have been considered.

[0382]

[0383] 4.2.6.1.Experimental Design:

[0384] The multiple number of cycles was performed as per finalized condition with cycles ID are as given below.

[0385]

[0386]

[0387] 4.2.6.2.Chromatogram: Figure 15, 16, 17, 18

[0388] All experiment showed the proper separation of mb-SB2 in elution with >85% purity and recovery. Hence it is confirmed to use the above conditions for capture of mb-SB2.

[0389]

[0390] 4.2.6.3.Conclusion

[0391] As per above experiments, the profiles and purity were observed similar even at higher scale.

[0392]

[0393] 4.2.7.Description of finalized AEX Step

[0394] The final process based on above experiment is described in below sections.

[0395]

[0396]

[0397] 4.2.8.Comparison between AEX verses HP20

[0398] Here in below set of experiments, both AEX and HP20 runs were performed simultaneously to check the comparison of mb-SB2 purity.

[0399]

[0400] 4.2.8.1.Experimental Design

[0401]

[0402] 4.2.8.2.Operational Details:

[0403]

[0404] Run Details:

[0405]

[0406]

[0407] 4.2.8.3.Chromatogram: Figure 19, 20

[0408]

[0409] 4.2.8.4.Results:

[0410] In comparison of both the cycles of mb-SB2 capture, DEAE gives the higher purified mb-SB2 in elution fraction consisting of 88.5%. Due to observation of the desired mb-SB2 in post load wash of HP20, the number of CV’s were reduced to 0.2CV of PLW to avoid the loss in washes. But as observed the elution F1 do also consist of only 66.21% purity which fails the desired requirement of mb-SB2 purity at capture step.

[0411]

[0412] 4.2.8.5.Conclusion:

[0413] Higher purity was observed in AEX elute than HP20 Eluate.

[0414]

[0415] 4.3.Polishing or Final Purification of mb-SB2:

[0416] The AEX eluate contains higher amount of salts. To remove salts and to maintain purity, the following trials were taken.

[0417]

[0418] 4.3.1.Polishing on HP20

[0419] To achieve the purity greater than 90% of mb-SB2, the polishing purification has been evaluated using resin HP20

[0420]

[0421] 4.3.1.1.Optimization of Equilibration Condition on HP20:

[0422] To achieve the desired product quality of > 90% purity, the DEAE pure eluate fractions were loaded on HP20 on different pH to check the impact on separation of mb-SB2. Here in below experiments, three cycles were performed where one was control cycle of HP20 details as per client suggestion and remaining two were at equilibration at pH 7.0 and 7.5 in Tris buffer.

[0423]

[0424] 4.3.1.1.1.Experimental Design:

[0425] The three experiments details are as given below

[0426]

[0427] 4.3.1.1.2.Operational Details:

[0428]

[0429]

[0430] 4.3.1.1.3.Chromatogram: Figure 21, 22, 23,

[0431]

[0432]

[0433] 4.3.1.1.4.Conclusion:

[0434] Among all three cycles, in control cycle itself there was no separation found. In remaining cycles < 75% Purity was found which was not as per desired purity. Hence further optimization needs to be done.

[0435]

[0436] 4.3.1.2.Optimization of Wash condition on HP20:

[0437] Based on previous experiment the Equilibration was scouted and finalized at 25mM Tris buffer pH 7.0 where as to improvise the purity the washes were optimized with different salts along with different molarities

[0438]

[0439] 4.3.1.2.1.Experimental Design:

[0440] The three experiments details are as given below.

[0441]

[0442]

[0443] 4.3.1.2.2.Operational Details:

[0444]

[0445] 4.3.1.2.3.Chromatogram: Figure 24, 25

[0446]

[0447]

[0448] 4.3.1.2.4.Conclusions

[0449] As per rationale the washes with different pH were tried to increase the purity, whereas during experiments there were no such increase in purity profile was observed. Hence it was concluded that no impact of different pH washes on separation of mb-SB2.

[0450] Overall, there were not much separation was observed with HP20 resin with multiple scouting of equilibration buffer as well as washes. Hence it was decided to optimize the polishing on different resin.

[0451]

[0452] 4.3.2.Polishing on C4 Column:

[0453] To achieve the Purity greater than 90% of mb-SB2, the polishing purification has been evaluated using C4 Column.

[0454] To achieve the desired product quality of > 90% purity, the DEAE pure eluate fractions were loaded on C4 column and run was performed as per the in house protocol to check the separation profile of mb-SB2.

[0455]

[0456] 4.3.2.1.Experimental Design:

[0457] The three experiments details are as given below.

[0458]

[0459] 4.3.2.2.Operational Details:

[0460]

[0461]

[0462] 4.3.2.3.Results:

[0463] No separation was found during C4 column run of mb-SB2. Hence cannot be used for further polishing of mb-SB2

[0464]

[0465]

[0466] 4.3.3.Polishing on C18 Column:

[0467]

[0468] 4.3.3.1.Scouting of equilibration buffer

[0469] The AEX elute is further processed on C18 run to achieve more purity of the product.

[0470]

[0471] 4.3.3.1.1.Experimental Design:

[0472] Different buffers were selected for screening. Methanol, Tris, Ammonium acetate and WFI were used as equilibration to perform the experiment.

[0473]

[0474] 4.3.3.1.2.Experimental Details:

[0475] To screen for different equilibration buffer, 4 different Equilibration buffer was used to perform the experiment.

[0476]

[0477] S. No.01

[0478]

[0479] S. No.02

[0480]

[0481] S. No.03

[0482]

[0483] S. No.04

[0484]

[0485] S. No.05 & S. No.06

[0486]

[0487] 4.3.3.1.3.Operational Details:

[0488] The process flow along with buffer details are mentioned in the above table. The experiment was carried out using the buffers mentioned in the above table in the respective experiment.

[0489]

[0490] 4.3.3.1.4.Results and Discussion:

[0491] The results obtained from the trial runs are given in Table below

[0492]

[0493] 4.3.3.1.5.Chromatograms: Figure 26, 27, 28, 29, 30, 31

[0494]

[0495] 4.3.3.1.6.Results:

[0496] From the above experiments it can be observed that Methanol is giving highest purity in comparison to others and hence methanol was decided to be used as the Equilibration buffer.

[0497]

[0498] 4.3.3.1.7.Conclusions:

[0499] Methanol as Equilibration buffer was taken into consideration compared to other buffers since maximum purity was observed in methanol buffer.

[0500]

[0501] 4.3.3.2.Evaluation of Methanol concentration in Equilibration buffer:

[0502]

[0503] 4.3.3.2.1.Experimental Design:

[0504] Methanol at different concentration was tried as Equilibration to achieve good purity.

[0505]

[0506] 4.3.3.2.2.Experimental Details:

[0507] Different methanol concentration was used as EQB buffer for C18 Run. The experimental details are listed below

[0508]

[0509] S. No.1

[0510]

[0511] S. No.2

[0512]

[0513] S. No.3

[0514]

[0515] S. No.4

[0516]

[0517] 4.3.3.2.3.Operational Details:

[0518] EQB buffer was passed for 2CV’s followed by loading and then PLW was given using Equilibration Buffer. Further process details of respective experiment is given in the above tables.

[0519]

[0520] 4.3.3.2.4.Results:

[0521]

[0522] 4.3.3.2.5.Chromatograms: Figure 32, 33, 34, 35

[0523]

[0524] 4.3.3.2.6.Conclusions:

[0525] As seen in the result section, 5% methanol is also giving good results in terms of purity, but the yield is comparatively very less. And hence 10% Methanol has been considered as Equilibration Buffer and post load wash since product is eluting in post load wash with high purity and yield. 10% Methanol was concluded as Equilibration buffer and post load wash since good purity along with recovery was obtained compared to 5% methanol where only 22% Recovery was obtained.

[0526]

[0527] 4.3.3.3.Description of finalized process on C18 Column:

[0528] The final process based on above experiment is described in below sections.

[0529]

Claims

1.A method for purifying a methanobactin comprising the following steps:a) subjecting a sample comprising the methanobactin to an anion exchange chromatography to produce a first eluate;b) lyophilizing or nano-filtering the first eluate obtained in the step a) in order to concentrate the first eluatec) subjecting the first eluate obtained from step b) to a reverse phase chromatography to produce a second eluate;d) lyophilizing the second eluate obtained in the step c).2.The method of claim 1, wherein the methanobactin comprises the following general formula (I):R1―(X)2-5―R2 (I)whereinR1and R2are each a 5 to 6-membered heterocycle comprising N and associated with an enethiolate; and each X is independently selected from any amino acid.3.The method of claim 2, wherein the methanobactin is methanobactin SB2 (mb-SB2).4.The method of claim 1, wherein the sample is a culture medium from Methanotrophs.5.The method of claim 1, wherein the anion exchange chromatography is selected from a group consisting of SP-Sepharose column chromatography, Ni+NTA column chromatography, Q-Sepharose column chromatography, DEAE column chromatography or PPG-600M column chromatography.6.The method of claim 5, wherein the anion exchange chromatography is DEAE column chromatography.7.The method of claim 6, wherein the anion exchange chromatography is performed by a process comprising the steps of:(i) loading a sample comprising methanobactin onto an anion exchange column, wherein said methanobactin are bound to said anion exchange column;(ii) washing said anion exchange column in a) containing said bound methanobactin with one or more wash buffers;(iii) eluting said methanobactin from the column in (ii) with an elution buffer; and (iv) collecting said methanobactin.8.The method of claim 7, wherein the pH of the wash buffer is from pH 6.0 to pH 9.0.9.The method of claim 7, wherein the elution buffer comprises optionally at least one chelating agent.10.The method of claim 7, wherein the pH of the elution buffer is from pH 6.0 to pH 9.0.11.The method of claim 1, wherein the step a) and step c) are conducted under pH 6.0- pH 9.0.12.The method of claim 1, wherein the lyophilization is tray-type lyophilization, and the tray-type lyophilization is performed using a programmed setting ranging from -40°C to 30°C over 2,000-3,500 minutes under a pressure range of 200 to 30 microbar.13.The method of claim 1, wherein the reverse phase chromatography is C18 column chromatography.14.The method of claim 1, wherein the reverse phase chromatography is performed by a process comprising the steps of: (i) loading the first eluate comprising methanobactin onto a reverse column; (ii) eluting said methanobactin from the column in (i) with an elution buffer; and (iii) collecting said methanobactin.15.The method of claim 1, wherein the reverse column is C18 column.16.The method of claim 1, wherein the reverse phase　chromatography　uses 10-50 (v / v)% methanol as a buffer.17.The method of claim 1, wherein the buffer is equilibration buffer, loading buffer, post load washing buffer or washing buffer.18.Methanobactin purified by a method according to any one of claims 1 to 17.

Images