Methods of producing peptides derived from chaperonin 60.1

Abstract

Described herein are improved methods for the production of a peptide molecule as described in SEQ ID NO: 1 (DGSVVNKVSELPAGHGLNVNTLSYGDLAAD. In accordance with some embodiments of the invention, the method comprises: forming a peptide by solid phase peptide synthesis (SPPS) on a resin; cleaving and deprotecting the peptides on the resin to form a crude product; purifying the crude product by column chromatography to collect an elution fraction; concentrating the eluted fraction to form a concentrated eluate; and separating the peptide molecules from the concentrated eluate by precipitation and filtration.

Description

Field of the Invention

[0001] The present disclosure, in various aspects and embodiments, relates to improved methods for synthesizing and purifying peptides derived from chaperonin 60.1. Background of the Invention

[0002] Chaperonin polypeptides are a subfamily of heat shock polypeptides, which are well known for their role in polypeptide folding. There are two families of chaperonin polypeptides, the chaperonin 60 (about 60 kDa) family and the chaperonin 10 (about 10 kDa) family. 1 Typically, when the target polypeptide enters the central core of the ring-shaped heptamer, the chaperone helps the polypeptide to fold, and upon subsequent release of energy from ATP, the target polypeptide is released from the central core through conformational changes in the chaperone structure. 2

[0003] Recently, some chaperone polypeptides have been shown to have a role in immunomodulation. Mycobacterium tuberculosis (M. tuberculosis) produces chaperone protein 60.1 (Cpn60.1), which is a polypeptide named based on its amino acid sequence identity with other known chaperone proteins. International Patent Application Publication No. WO2002 / 040037A2 discloses pharmaceutical compositions comprising Cpn60.1 (MtCpn60.1) from Mycobacterium tuberculosis and its encoding nucleic acid molecules. Various therapeutic uses of these molecules are also disclosed, including the treatment and / or prevention of autoimmune diseases, allergic conditions, conditions represented by Th2-type immune responses, and conditions associated with eosinophilia. The present application also discloses a number of specific peptide fragments that can be derived from full-length polypeptides with similar biological activities.

[0004] International Patent Application Publication No. WO2009 / 106819A2 discloses a series of novel peptides that can be derived from MtCpn60.1, including a peptide having the following amino acid sequence (referred to as "peptide 4"): DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1). Peptide 4 exhibits anti-inflammatory activity and has been shown to significantly reduce the recruitment of eosinophils in an animal model of allergic airway inflammation.

[0005] The conventional method currently used to produce the peptide molecule as described in SEQ ID NO: 1 as provided by the Almac group 3,4 The efficiency is low and the purity is low. In addition, isolation by conventional methods requires freeze drying. A method that allows the peptide molecules to precipitate would be preferred.

[0006] Therefore, there exists a need for improved methods of synthesizing and purifying peptide molecules as described in SEQ ID NO:1. SUMMARY OF THE INVENTION

[0007] The present invention provides a method for synthesizing a peptide having SEQ ID NO: 1, the method comprising: (i) attaching an amino acid (AA) to a resin of a solid support via the C-terminus of the AA, and wherein the N-terminus of the amino acid is protected to avoid reaction at the N-terminus, to form a first solid support-bound AA; (ii) deprotecting the N-terminus of the first solid support-bound AA by removing the protecting group; (iii) coupling a second AA to the first solid support-bound AA, wherein the C-terminus of the second amino acid is coupled to the deprotected N-terminus of the first solid support-bound AA to form a second solid support-bound AA, and wherein the second AA comprises a protected N-terminus; and (iv) repeating steps (ii) and (iii). iii) to form the next solid support-bound AA until a solid support-bound AA sequence is formed; (v) cleaving the AA sequence from the solid support to produce a mixture of peptides having a desired sequence ID; (vi) separating the peptide mixture from the solid support by filtration to produce a crude product; (vii) diluting the separated peptide mixture with a solvent to form a precipitate, wherein the precipitate is separated by filtration; (viii) subjecting the separated precipitate to column chromatography to collect an elution fraction containing a purified version of the peptide; (ix) concentrating the elution fraction to form a concentrated eluate containing a purified version of the peptide; and (x) separating the peptide from the concentrated eluate by precipitation followed by filtration.

[0008] A preferred embodiment provides a method for synthesizing the peptide of SEQ ID NO: 1, wherein SEQ ID NO: 1 is DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD.

[0009] Another preferred embodiment provides a method, wherein the C-terminus of AA(A) is attached to the resin-bound amine of aspartic acid from a solid support by treating about 2.5 equivalents of AA alanine(A) with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIEA) in DMF, and further, wherein the N-terminus of AA(A) is protected by using Fmoc as a protecting group.

[0010] Another preferred embodiment provides a method wherein the Fmoc-protected N-terminus of the dipeptide (AD) is deprotected by treating the solid support-bound dipeptide (AD) with a mixture of 10% piperidine and 0.15 MOxyma in DMF three times in succession.

[0011] Yet another preferred embodiment provides a method, wherein A) a solid support-bound dipeptide (AD) comprising a deprotected N-terminus is treated with about 2.5 equivalents of a second AA-alanine (A) in the presence of N,N-diisopropylcarbodiamide (DIC) and ethyl 2-cyano-2-(hydroxyamino)acetate (Oxyma) in DMF, and further, wherein the N-terminus of the second AA (A) is protected by using Fmoc as a protecting group. Another preferred embodiment provides a method, wherein B) the N-terminus of the second AA (A) is deprotected by treating the solid support-bound AA's with a mixture of 10% piperidine and 0.15M Oxyma in DMF three times in a row, and wherein the solid support-bound AA's with a deprotected N-terminus are treated sequentially with the steps in A) and B) until a solid-supported peptide having SEQ ID NO: 1 is formed.

[0012] In another preferred embodiment, a method is provided, wherein the peptide having SEQ ID NO: 1 is obtained by cleaving the solid-supported peptide having SEQ ID NO: 1 from the solid support by treating the solid-supported peptide having SEQ ID NO: 1 with an aqueous solution comprising trifluoroacetic acid (TFA) and triisopropylsilane (TIS), and separating the peptide having SEQ ID NO: 1 from the solid support by passing the mixture through a filter, wherein the peptide having SEQ ID NO: 1 passes through the filter into a filtrate, and wherein the filtered solid material is further washed up to eight times with an aqueous solution comprising TFA and TIS to produce a filtrate comprising the peptide having SEQ ID NO: 1. Another preferred embodiment provides a method, wherein the eluate is diluted by stepwise addition of methyl tert-butyl ether (MTBE), heptane, and MTBE in a volume ratio of 1:0.75:1:1 to produce the peptide having SEQ ID NO: 1 as a precipitate.

[0013] Another embodiment provides a method, wherein the SEQ ID NO: 1 peptide precipitate is further subjected to reverse phase high performance liquid column chromatography using a C4 reverse phase column, wherein the pore size range of the C4 reverse phase column is about And the particle size of the C4 reverse phase column is about 10 μm.

[0014] A preferred method of this embodiment provides a method wherein mobile phase A is about 25 mM to about 50 mM ammonium acetate at a pH of about 7 to about 8.4, and wherein mobile phase B is acetonitrile (ACN).

[0015] Yet another embodiment provides a method, wherein the purification of the peptide precipitate by reverse phase high performance liquid column chromatography using a C4 reverse phase column further comprises loading the C4 reverse phase column to a concentration of about 23 mg of crude product per mL of stationary phase, and wherein the C4 reverse phase column bed has a height of about 20 cm to about 40 cm. Another preferred embodiment provides a method, wherein the mobile phase is collected as an eluent after passing through the C4 reverse phase column, and further wherein the eluent is diluted with 10% tris(hydroxymethyl)aminomethane (Tris) in water at a pH of about 7 to produce a peptide.

[0016] Another aspect of the invention provides a method wherein separating the purified product from the concentrated eluate comprises diluting the concentrated eluate with 0.5× volume acetic acid (AcOH) premixed with ACN to form a reaction mixture. A preferred embodiment of this aspect provides a method wherein the peptide is diluted with MTBE to form a mixture.

[0017] Another preferred embodiment provides a method, wherein separating the peptide molecules from the mixture further comprises aging the reaction mixture at 5°C for 30 minutes to produce a heterogeneous mixture. Yet another preferred embodiment provides a method, wherein the peptides are separated from the heterogeneous mixture by filtering the heterogeneous mixture through a nylon membrane filter, and further wherein the nylon membrane filter is a 10 μm nylon membrane.

[0018] Another preferred embodiment provides a method wherein the separated peptide is further washed with MTBE, the method further comprising humidifying the peptide to remove residual solvent. Yet another preferred embodiment provides a method wherein humidifying the peptide molecules comprises humidifying the peptide molecules with wet N2 until about 90% relative humidity is reached, and then drying with a N2 stream to produce a dry form of the peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various embodiments of the present invention and, together with the description, serve to explain and illustrate the principles of the present disclosure. The drawings are intended only to show the main features of the exemplary embodiments by way of diagrammatic representation.

[0020] Figure 1 is an overlay of chromatograms showing purification of a peptide according to the improved method of the invention described herein (as depicted by the enlarged chromatogram trace farther from the X-axis, "Improved" showing a major peak at about 18.4-18.6 minutes) compared to purification of a peptide according to a conventional method (as depicted by the chromatogram trace closer to the X-axis, and having prominent peaks at 13.2, 17.6, 17.9, 18, 18.2, 18.5, and 19 minutes). Both chromatograms were obtained at 210 nm (+ / - 1 nm).

[0021] Figure 2 yes Figure 1 Figure 1 shows a normalized view of a chromatogram overlay of , where both chromatograms are normalized to the main peak between approximately 18.4-18.6 on the X-axis. Both chromatograms were acquired at 210 nm (+ / - 1 nm).

[0022] Figure 3 is a mass spectrometry (MS) chromatogram of material produced by conventional methods, which includes the peptide peak (main peak) and the labeled impurity peaks. The MS total ion chromatography (TIC) chromatogram is for positive ions. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides a method for synthesizing a peptide having SEQ ID NO: 1, the method comprising: (i) attaching an amino acid (AA) to a resin of a solid support via the C-terminus of the AA, and wherein the N-terminus of the amino acid is protected to avoid reaction at the N-terminus, to form a first solid support-bound AA; (ii) deprotecting the N-terminus of the first solid support-bound AA by removing the protecting group; (iii) coupling a second AA to the first solid support-bound AA, wherein the C-terminus of the second amino acid is coupled to the deprotected N-terminus of the first solid support-bound AA to form a second solid support-bound AA, and wherein the second AA comprises a protected N-terminus; and (iv) repeating steps (ii) and (iii). iii) to form the next solid support-bound AA until a solid support-bound AA sequence is formed; (v) cleaving the AA sequence from the solid support to produce a mixture of peptides having a desired sequence ID; (vi) separating the peptide mixture from the solid support by filtration to produce a crude product; (vii) diluting the separated peptide mixture with a solvent to form a precipitate, wherein the precipitate is separated by filtration; (viii) subjecting the separated precipitate to column chromatography to collect an elution fraction containing a purified version of the peptide; (ix) concentrating the elution fraction to form a concentrated eluate containing a purified version of the peptide; and (x) separating the peptide from the concentrated eluate by precipitation followed by filtration.

[0024] A preferred embodiment provides a method for synthesizing the peptide of SEQ ID NO: 1, wherein SEQ ID NO: 1 is DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD.

[0025] Another preferred embodiment provides a method, wherein the C-terminus of AA(A) is attached to the resin-bound amine of aspartic acid from a solid support by treating about 2.5 equivalents of AA alanine(A) with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIEA) in DMF, and further, wherein the N-terminus of AA(A) is protected by using Fmoc as a protecting group.

[0026] Another preferred embodiment provides a method wherein the Fmoc-protected N-terminus of the dipeptide (AD) is deprotected by treating the solid support-bound dipeptide (AD) with a mixture of 10% piperidine and 0.15 MOxyma in DMF three times in succession.

[0027] Yet another preferred embodiment provides a method, wherein A) a solid support-bound dipeptide (AD) comprising a deprotected N-terminus is treated with about 2.5 equivalents of a second AA-alanine (A) in the presence of N,N-diisopropylcarbodiamide (DIC) and ethyl 2-cyano-2-(hydroxyamino)acetate (Oxyma) in DMF, and further, wherein the N-terminus of the second AA (A) is protected by using Fmoc as a protecting group. Another preferred embodiment provides a method, wherein B) the N-terminus of the second AA (A) is deprotected by treating the solid support-bound AA's with a mixture of 10% piperidine and 0.15M Oxyma in DMF three times in a row, and wherein the solid support-bound AA's with a deprotected N-terminus are treated sequentially with the steps in A) and B) until a solid-supported peptide having SEQ ID NO: 1 is formed.

[0028] In another preferred embodiment, a method is provided, wherein the peptide having SEQ ID NO: 1 is obtained by cleaving the solid-supported peptide having SEQ ID NO: 1 from the solid support by treating the solid-supported peptide having SEQ ID NO: 1 with an aqueous solution comprising trifluoroacetic acid (TFA) and triisopropylsilane (TIS), and separating the peptide having SEQ ID NO: 1 from the solid support by passing the mixture through a filter, wherein the peptide having SEQ ID NO: 1 passes through the filter into a filtrate, and wherein the filtered solid material is further washed up to eight times with an aqueous solution comprising TFA and TIS to produce a filtrate comprising the peptide having SEQ ID NO: 1. Another preferred embodiment provides a method, wherein the eluate is diluted by stepwise addition of methyl tert-butyl ether (MTBE), heptane, and MTBE in a volume ratio of 1:0.75:1:1 to produce the peptide having SEQ ID NO: 1 as a precipitate.

[0029] Another embodiment provides a method, wherein the SEQ ID NO: 1 peptide precipitate is further subjected to reverse phase high performance liquid column chromatography using a C4 reverse phase column, wherein the pore size range of the C4 reverse phase column is about And the particle size of the C4 reverse phase column is about 10 μm.

[0030] A preferred method of this embodiment provides a method wherein mobile phase A is about 25 mM to about 50 mM ammonium acetate at a pH of about 7 to about 8.4, and wherein mobile phase B is acetonitrile (ACN).

[0031] Yet another embodiment provides a method, wherein the purification of the peptide precipitate by reverse phase high performance liquid column chromatography using a C4 reverse phase column further comprises loading the C4 reverse phase column to a concentration of about 23 mg of crude product per mL of stationary phase, and wherein the C4 reverse phase column bed has a height of about 20 cm to about 40 cm. Another preferred embodiment provides a method, wherein the mobile phase is collected as an eluent after passing through the C4 reverse phase column, and further wherein the eluent is diluted with 10% tris(hydroxymethyl)aminomethane (Tris) in water at a pH of about 7 to produce a peptide.

[0032] Another aspect of the invention provides a method wherein separating the purified product from the concentrated eluate comprises diluting the concentrated eluate with 0.5× volume acetic acid (AcOH) premixed with ACN to form a reaction mixture. A preferred embodiment of this aspect provides a method wherein the peptide is diluted with MTBE to form a mixture.

[0033] Another preferred embodiment provides a method, wherein separating the peptide molecules from the mixture further comprises aging the reaction mixture at 5°C for 30 minutes to produce a heterogeneous mixture. Yet another preferred embodiment provides a method, wherein the peptides are separated from the heterogeneous mixture by filtering the heterogeneous mixture through a nylon membrane filter, and further wherein the nylon membrane filter is a 10 μm nylon membrane.

[0034] Another preferred embodiment provides a method wherein the separated peptide is further washed with MTBE, the method further comprising humidifying the peptide to remove residual solvent. Yet another preferred embodiment provides a method wherein humidifying the peptide molecules comprises humidifying the peptide molecules with wet N2 until about 90% relative humidity is reached, and then drying with a N2 stream to produce a dry form of the peptide.

[0035] Described herein is a method for producing a peptide molecule as described in SEQ ID NO: 1 (DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD). The method produces a peptide molecule as described in SEQ ID NO: 1 with improved purity compared to conventional methods for producing a peptide molecule as described in SEQ ID NO: 1. In some embodiments, the method comprises: forming the peptide by solid phase peptide synthesis on a resin; cleaving and deprotecting the peptide on the resin to form a crude product; purifying the crude product by column chromatography to collect elution fractions; concentrating the elution fractions to form a concentrated eluate; and isolating the peptide molecule from the concentrated eluate by precipitation and filtration.

[0036] The peptide according to SEQ ID NO:1 of the present disclosure is produced by solid phase peptide synthesis (SPPS). In SPPS, an amino acid or peptide group is bound to a solid support resin. The peptide is synthesized in the solid phase using a chemical method by which amino acids are added from the C-terminus to the N-terminus. Therefore, the amino acid or peptide group near the C-terminus of the specific fragment is first added to the resin. This occurs by reacting the C-terminal functional group of the amino acid or peptide group with a complementary functional group on the resin support. The N-terminal side of the amino acid or peptide group is masked to prevent unwanted side reactions. The amino acid or peptide group also desirably includes side chain protection. Then the continuous amino acid or peptide group is attached to the carrier-bound peptide material until the target peptide is formed. According to conventional practice, most of these also include side chain protection. At each continuous coupling, the masking group at the N-terminal end of the resin-bound peptide material is removed. Then it is reacted with the C-terminus of the next amino acid whose N-terminus is masked. Therefore, the product of solid phase synthesis is a peptide bound to a resin support. Then, the carrier-bound peptide is usually cut from the carrier and further processed and / or purified.

[0037] Any type of carrier suitable in the practice of solid phase peptide synthesis can be used. In some embodiments, the carrier comprises a resin that can be prepared by a combination of one or more polymers, copolymers or polymers, such as polyamide, polysulfonamide, substituted polyethylene, polyethylene glycol, phenolic resin, polysaccharide or polystyrene. The polymer carrier can also be any solid that is fully insoluble and inert to the solvent used in peptide synthesis. The solid carrier generally comprises a linking portion, to which the peptide grown during synthesis is coupled, and the linking portion can be cut under desirable conditions to release the peptide from the carrier. Suitable solid carriers may have a joint that is photo-cleavable, TFA-cleavable, HF-cleavable, fluoride-cleavable, reducible-cleavable; Pd(O)-cleavable; nucleophilic-cleavable; or free radical-cleavable. The preferred linking portion is cleavable under conditions that the side chain groups of the cleaved peptide are still substantially protected as a whole.

[0038] In some embodiments, a fluorenylmethoxycarbonyl (Fmoc) based solid phase peptide synthesis (SPPS) is performed, wherein the Fmoc group is used to temporarily protect the α-amino group. The Fmoc protecting group can be selectively cleaved from the peptide relative to the side chain protecting group, so that when the Fmoc is cleaved, the side chain protection is left in place. This type of selectivity is important during amino acid coupling to minimize side chain reactions. In addition, the side chain protecting groups can be selectively cleaved relative to the Fmoc to remove them while leaving the Fmoc in place.

[0039] In some embodiments, coupling of Fmoc-AA is performed using about 2.5 equivalents of the amino acid and N,N-diisopropylcarbodiimide (DIC) and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) in dimethylformamide (DMF).

[0040] In some embodiments, coupling Fmoc-AA includes about 28 single couplings. In some embodiments, each coupling uses about 2.5 equivalents of Fmoc-AA. In some embodiments, 12 of the 28 single couplings use a coupling time of about 3 hours. In some embodiments, 12 of the 28 single couplings use about 2.5 equivalents of amino acids and DIC and Oxyma in DMF for 3 hours. In some embodiments, 16 of the 28 single couplings use a coupling time of about 6 hours. In some embodiments, 16 of the 28 single couplings use about 2.5 equivalents of amino acids and DIC and Oxyma in DMF for 6 hours.

[0041] In some embodiments, about 70 equivalents of amino acids are required for coupling (28 single couplings, each coupling using about 2.5 equivalents of amino acids). On the other hand, the conventional method for purifying the peptide molecule as described in SEQ ID NO: 1 requires 27 couplings (18 single couplings 45 minutes and 9 double couplings 45 minutes) using 4 equivalents of amino acids, and thus requires 144 equivalents for coupling. Relative to the conventional method for forming the peptide molecule as described in SEQ ID NO: 1, the method described herein advantageously uses about 50% less raw materials than the conventional method, for example, 144 equivalents of Fmoc-AA in the conventional method compared to 70 equivalents of Fmoc-AA in the improved method.

[0042] After determining that the coupling is complete, the coupling reaction mixture is washed with a solvent, and the coupling cycle is repeated for each subsequent amino acid residue of the peptide material. In order to couple the next amino acid, the N-terminal protecting group (e.g., Fmoc group) is removed from the resin-bound material, usually by treating with a reagent containing 20-50% (based on weight) piperidine in a solvent, such as N-methylpyrrolidone (NMP) or dimethylformamide (DMF). For example, in a conventional method for forming a peptide molecule as described in SEQ ID NO: 1, Fmoc deprotection is performed using 20% ​​piperidine in DMF, and 10 volumes are used for 2 times, each for 20 minutes.

[0043] In some embodiments, about 50% less piperidine is advantageously used for Fmoc deprotection relative to conventional methods. In some embodiments, 10% piperidine in DMF and 0.15M Oxyma are used for Fmoc deprotection. In some embodiments, 6.5 volumes are used for 3 Fmoc deprotections, each for 10 minutes.

[0044] After removing the Fmoc protecting group, washing is performed to remove residual piperidine and Fmoc byproducts (e.g., dibenzofulvene and its piperidine adduct). In some embodiments, washing the resin is performed with a solvent. In some embodiments, the solvent is DMF. In some embodiments, washing with a solvent (e.g., DMF) is performed 9 times using 6.5 volumes, each for 5 minutes. In some embodiments, washing uses 59 volumes of solvent (e.g., DMF) per cycle. In some embodiments, washing uses a total of 1652 volumes of solvent (59 volumes of solvent for 28 cycles).

[0045] The conventional method for forming a peptide molecule as described in SEQ ID NO: 1 requires washing 20 times with 5 volumes, and thus requires 100 volumes per cycle. In total, the conventional method uses 700 volumes of solvent. Advantageously, the improved method described herein requires about 40% less volume of solvent (e.g., DMF) than the conventional method for the washing step.

[0046] In addition, conventional methods for forming peptides using solid phase peptide synthesis generally require a capping step for a period of time (e.g., 10 minutes) with a capping agent. In some embodiments, forming peptides by solid phase peptide synthesis on a resin advantageously does not require a capping step after Fmoc-AA coupling to block the reaction of the unreacted amino acid termini, thereby eliminating a step.

[0047] In some embodiments, after the synthesis is completed, the next step is overall deprotection, i.e., cleaving and deprotecting the peptide on the resin to form a crude product. In some embodiments, cleaving and deprotecting the peptide on the resin comprises treating the resin with a solution comprising trifluoroacetic acid (TFA), H2O, and triisopropylsilane (TIS). In some embodiments, treating the resin with a solution comprises treating with 7 volumes of a solution comprising trifluoroacetic acid (TFA), H2O, and triisopropylsilane (TIS) for 2.5 hours.

[0048] In some embodiments, the resin can then be removed by filtration and rinsed twice with TFA to provide a filtrate with the peptide. In some embodiments, the resin is rinsed twice with 0.5 volumes of TFA. In some embodiments, only 8 volumes of solution are required to cleave the peptide from the resin (cleavage with 7 volumes of cleavage solution and two 0.5 volumes of TFA rinses).

[0049] Conventional methods for forming a peptide molecule as described in SEQ ID NO: 1 typically require about 23 volumes to cleave the peptide (e.g., cleavage with 15.6 volumes of a solution containing ethane-1,2-dithiol (EDT) and rinsing 3 times with 2.5 volumes of TFA). Thus, cleaving the peptide as described herein provides about a 65% volume reduction in cleavage solution and wash solution.

[0050] In addition, in some embodiments, the cleavage solution advantageously does not contain ethane-1,2-dithiol (EDT). Conventional methods for forming peptide molecules as described in SEQ ID NO: 1 typically require EDT in their cleavage solutions, which is problematic because it is an irritating scavenger. Without being bound by any particular theory, it was found that EDT is unnecessary for the cleavage solution in the methods described herein, which eliminates irritating scavengers from the cleavage cocktail.

[0051] After the peptide on the resin is cleaved and deprotected (which provides the peptide in solution), the peptide can be precipitated to form a crude product. In some embodiments, the precipitated crude product comprises progressively adding methyl tert-butyl ether (MTBE) (6 volumes), heptane (8 volumes) and MTBE (8 volumes). In some embodiments, the precipitated crude product comprises progressively adding 6 volumes of MTBE, 8 volumes of heptane and 8 volumes of MTBE. Without being bound by any particular theory, the progressive addition of antisolvents enhances filterability, yet the merging of antisolvents causes gelling of particles and slows down filtration.

[0052] In some embodiments, precipitating the crude product further comprises collecting the precipitate by filtration, washing the precipitate, and then drying to form the crude product. In some embodiments, washing the precipitate with 2 volumes of a solution of MTBE and heptane is performed 4 times.

[0053] In some embodiments, the precipitation of the crude product requires 30 volumes of anti-solvent (6 volumes of MTBE, 8 volumes of heptane, 8 volumes of MTBE are added stepwise, and washed 4 times with 2 volumes of a solution of MTBE and heptane after filtration). Conventional methods for purifying peptide molecules as described in SEQ ID NO: 1 typically require about 59 volumes of anti-solvent (e.g., precipitation with 45.3 volumes of MTBE and washing 3 times with 4.8 volumes of MTBE). Therefore, compared to conventional methods, precipitation as described herein advantageously provides about 50% reduction in anti-solvent volume.

[0054] Conventional methods for forming peptide molecules as described in SEQ ID NO: 1 generally further require grinding of the solid obtained from the precipitation with another anti-solvent (e.g., MTBE). For example, conventional grinding of the solid obtained from the precipitation can be performed with 15 volumes of MTBE for 1.5 hours, separated by filtration, washed 3 times with 5 volumes of MTBE after filtration, and then dried again. Advantageously, grinding of the precipitate is not required in the improved methods for purifying peptide molecules as described in SEQ ID NO: 1 described herein. As a result, in some embodiments, the total volume of the reactive portion during the overall deprotection is 38 volumes (8 volumes during cleavage and 30 volumes during precipitation), which is reduced by about 66% on the reactive portion required in the conventional method (23 volumes for cleavage, 59 volumes for precipitation, and 30 volumes for grinding).

[0055] In some embodiments, the next step after the cleavage and deprotection of the resin-bound peptide is the purification of the crude peptide. In some embodiments, the crude peptide is purified by column chromatography to collect the elution fraction. In some embodiments, the crude product is purified by column chromatography and comprises the use of reversed-phase high performance liquid chromatography and a C4 reverse phase column.

[0056] In some embodiments, the crude peptide is purified by HPLC using a YMC-Pack C4 (butyl) column or a Kromasil C4 column. In some embodiments, the pore size of the C4 reverse phase column is or Preferably, the pore size of the C4 reverse phase column is In some embodiments, the particle size of the C4 reverse phase column is 3 μm, 5 μm or 10 μm. Preferably, the particle size of the C4 reverse phase column is 10 μm.

[0057] In some embodiments, by column chromatography purification of crude product further comprises C4 reverse phase column is loaded to about 20mg to about 35mg crude product per mL stationary phase, or about 23mg to about 30mg crude product per mL stationary phase concentration in the range, or about 25mg crude product per mL stationary phase concentration. In some embodiments, column bed height is between about 10cm to about 30cm, between about 15cm to about 25cm, between about 20cm to about 30cm, or about 25cm. In some embodiments, column bed height is 25cm.

[0058] In contrast, conventional methods for purifying peptide molecules as described in SEQ ID NO: 1 generally only allow loading of up to 3.2 mg of crude product per mL of stationary phase, which is significantly less loading than the improved methods described herein. For example, conventional methods can result in front elution shoulders that severely limit loading. Relative to the purification steps in conventional methods for purifying peptide molecules as described in SEQ ID NO: 1, the higher loading provided in the improved purification steps enhances processability.

[0059] In some embodiments, the crude product is purified by column chromatography comprising at least two purification passes to collect eluted fractions. In some embodiments, mobile phase A is about 25-50 mM ammonium acetate at a pH of about 7-8.4. In some embodiments, mobile phase B is acetonitrile (ACN). In some embodiments, the purification method involves two purification passes through a chromatographic medium, wherein the first chromatographic pass is performed in an ammonium acetate gradient providing a pH of about 7-8.4, and then the second chromatographic pass is performed in an ACN gradient.

[0060] In some embodiments, purification of the crude product by column chromatography includes adding a stabilizer to the eluted fraction, such as 10% by volume tris(hydroxymethyl)aminomethane (Tris) at pH 7. Conventional methods for purifying peptide molecules as described in SEQ ID NO: 1 do not require the addition of stabilizers to the eluted fraction. Instead, purification in conventional methods produces a gelled fraction that needs to be redissolved by pH adjustment after visual inspection. Without being bound by any particular theory, the prevention of gelling provided by the purification step of the improved method is advantageously less invasive than the fragmentation of the gelled fraction required in the conventional method for purifying peptide molecules as described in SEQ ID NO: 1.

[0061] In some embodiments, the crude product is purified by column chromatography further comprising merging elution fractions containing products having a concentration and purity above a desired threshold to form a combined elution fraction. In some embodiments, the crude peptide is purified to>90% (e.g.,>91%,>92%,>93%,>94%,>95%,>96%,>97%,>98% or>99%), and appropriate fractions are combined. In some embodiments, the purity of the peptide can be verified by reverse phase HPLC, and then the purified product can be characterized by liquid chromatography / mass spectrometry (LC / MS) and / or matrix-assisted laser desorption ionization (MALDI) mass spectrometry (i.e., the identity of the peptide can be verified). Conventional methods for synthesizing and purifying peptide molecules as described in SEQ ID NO: 1 cannot produce products with the same high purity as the improved methods described herein, as demonstrated in the following examples.

[0062] In some embodiments, the next step after the crude product is purified by column chromatography to collect the elution fraction is to concentrate the elution fraction (or the combined elution fraction) to form a concentrated eluate. In some embodiments, the concentrated elution fraction or the combined elution fraction includes loading the elution fraction or the combined elution fraction onto a chromatographic column containing a polystyrene divinyl benzene resin or a C4 material, and eluting with an ACN solution containing ammonium acetate to form a concentrated eluate. Performing a concentration step can result in the concentration of the desired peptide in the solvent being increased to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times.

[0063] In some embodiments, the concentration step is followed by a separation step. In some embodiments, the peptide molecules are separated from the concentrated eluate by precipitation and filtration. In some embodiments, the purified product is separated from the concentrated eluate and comprises diluting the concentrated eluate with 0.5× volume acetic acid (AcOH) premixed with ACN to form a reaction mixture. In some embodiments, the peptide molecules are separated from the concentrated eluate and comprise further diluting the reaction mixture with MTBE. In some embodiments, the peptide molecules are separated from the concentrated eluate and comprise aging the reaction mixture for 30 minutes or maintaining at 5°C.

[0064] In some embodiments, separating peptide molecules from the concentrated eluate includes filtering the reaction mixture through a nylon membrane filter to separate the precipitate after aging. In some embodiments, the nylon membrane filter is a 10 μm nylon membrane filter. In some embodiments, separating peptide molecules from the concentrated eluate includes washing the precipitate with MTBE after filtering the reaction mixture through a nylon membrane filter. In some embodiments, the precipitate is dried after washing.

[0065] In some embodiments, the method further comprises humidifying the peptide molecules after precipitation to remove residual solvent. In some embodiments, humidifying the peptide molecules comprises humidifying the peptide molecules with wet N2 until reaching about 90% relative humidity and drying with N2 flow.

[0066] Conventional methods for forming peptide molecules as described in SEQ ID NO: 1 do not use a concentration step. Instead, conventional methods combine and freeze-dry the fractions containing the desired peptide. In addition, conventional methods for forming peptide molecules as described in SEQ ID NO: 1 do not use a humidification step because the peptide is isolated via freeze drying. Synthetic details Peptide Synthesis - General Procedure

[0067] Peptide according to the present invention is synthesized in multiple steps.Synthesis utilizes solid phase peptide synthesis (SPPS), wherein the first amino acid (AA) is covalently bound on a solid support material, and selective protecting group chemistry is used to synthesize stepwise in a single reaction vessel.The first step involves the amino protected amino acid being bound to a solid phase material or resin (most commonly low cross-linked polystyrene beads), forming a covalent bond between the carbonyl group of the amino acid (AA) and the resin.This covalent bond is the bond of an amino group and an ester (an amidoto an ester bond).By treating the solid support-bound AA with a mixture of 10% piperidine and 0.15M Oxyma in DMF, the amino protected by protected groups such as 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Boc) is deprotected.The solid support-bound AA with the amino group of deprotection is now reacted / coupled with the carbonyl group of the next N-protected amino acid.This coupling produces a solid phase with a dipeptide.Repeat this above-mentioned cycle to form a desired peptide chain.After all reactions are completed, the synthesized peptide is cut from the solid support. Example 1

[0068] In this example, a peptide was synthesized having SEQ ID No. 1. As discussed in the general procedure, the SEQ ID No. 1 peptide was synthesized in multiple steps.

[0069] Step 1: The first step involves solid phase peptide synthesis (SPPS) in which the carbonyl / C-terminus of amino acid (AA) 30-alanine (A) whose N-terminus is protected by an Fmoc group is covalently bound to HL-Asp(OtBu)-2CT-resin, which is a solid support to which AA 31D has been attached. This step involves treating HL-Asp(OtBu)-2CT-resin (solid support) and AA (A) with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIEA) at 23°C for about 2 hours to produce Fmoc-AD-2CT-resin. The covalent bonding of the first AA is not monitored directly. The establishment of the correct sequence is checked regularly by running microdissections and analyzing them by HPLC-MS.

[0070] Experimental procedure: Fmoc-L-Ala-OH (2.5 eq.) and TBTU (2.44 eq.) were weighed in appropriate vials. DMF (c=0.25 mol / L for AA) was added to the vial prior to coupling the amino acid. After a clear solution was formed, DIEA (4.90 eq.) was added and the activated AA was stirred for 2-10 min. The pre-activated AA was then transferred to the resin and stirred at RT for 120 min. The resin was washed 4 times with DMF [6.5 ml / g (resin), 5 min each time].

[0071] Step 2: This step involves deprotecting the N-terminal nitrogen by removing the Fmoc protecting group. This deprotection step involves treating the solid support bound dipeptide (AD) with a mixture of 10% piperidine and 0.15M Oxyma in DMF to produce a solid support bound dipeptide (AD) with a deprotected N-terminus. Direct monitoring of this step was not performed. Analysis of microdissection by HPLC-MS did not show any absence of the next amino acid, which would be a direct impurity resulting from incomplete deFmoc (deFmoc) of this step. This analysis was performed during the R&D stage and then omitted after confirming that this step did not produce impurities.

[0072] Experimental procedure: The resin was treated three times with 10% piperidine in DMF containing 0.15 M Oxyma [6.5 ml / g (resin), 10 minutes each time]. The resin was washed 5 times with DMF [6.5 ml / g (resin), 5 minutes each time].

[0073] Step 3: This step involves treating the solid support-bound dipeptide (AD) containing the deprotected N-terminus with 2.5 equivalents of the amino acid alanine AA (A) wherein its N-terminus is protected with an Fmoc group, and N,N-diisopropylcarbodiamide (DIC) and ethyl 2-cyano-2-(hydroxyamino)acetate (Oxyma) in DMF at a temperature ranging from 20 to 24° C. for about 3 hours. The covalent bonding of the AA was not monitored directly. The establishment of the correct sequence was regularly checked by running microdissections and analyzing them by HPLC-MS.

[0074] Experimental Procedure: Fmoc-Ala-OH (2.5 eq.) and OxymaPure (2.5 eq.) were weighed in appropriate vials and dissolved with DMF (c=0.25 mol / L). Prior to coupling the amino acid, DIC (3.75 eq.) was added to the vial and the solution was stirred for 2-15 min]. The activated AA was transferred to the resin and shaken at RT for 180 min. The resin was washed 4 times with DMF [6.5 ml / g (resin), 5 min each time. The example conditions used are provided in Tables 1-7 below. The SPPS sequence is shown in Table 1 below. The example scheme of SPPS is provided in Table 2, and the overall deprotection scheme is provided in Table 3. The 1st and 2nd pass purification conditions are in Tables 4 and 5, respectively. The concentration pass is in Table 6, and Table 7 provides the precipitation, filtration and humidification schemes. Table 1. SPPS sequences Table 2. SPPS protocol Table 3. Overall deprotection Table 4. Conditions for the first purification pass. Table 5. Second pass purification conditions Table 6. Concentration Passes Table 7. Sedimentation, Filtration and Wetting Comparative Example 1. Comparison of Material Purity

[0075] The peptide according to SEQ ID NO: 1 of the present invention was synthesized and purified by conventional methods as described below. Tables 8-12 show the conditions of the comparative (conventional) methods. The comparative SPPS sequences, SPPS schemes, global deprotection, first pass purification and concentration pass schemes are shown in Tables 8-12, respectively. Table 8. Comparative SPPS sequences cycle AA Coupling conditions 0 Wang-resin Swelling 1 Fmoc-L-Asp(OtBu)-OH load 2 Fmoc-L-Ala-L-Ala-OH Single coupling-long 3 Fmoc-L-Leu-OH Single coupling 4 Fmoc-L-Asp(OtBu)-OH Single coupling 5 Fmoc-Gly-OH Single coupling 6 Fmoc-L-Tyr(tBu)-OH Single coupling 7 Fmoc-L-Ser(tBu)-OH Single coupling 8 Fmoc-L-Leu-OH Single coupling 9 Fmoc-L-Asn(Trt)-L-Thr(psiMe,Mepro)-OH Single coupling-long 10 Fmoc-L-Val-OH Single coupling 11 Fmoc-L-Asn(Trt)-OH Double coupling 12 Fmoc-L-Leu-OH Double coupling 13 Fmoc-Gly-OH Single coupling 14 Fmoc-L-His(Trt)-OH Double-coupled-His 15 Fmoc-Gly-OH Single coupling 16 Fmoc-L-Ala*H2O Single coupling 17 Fmoc-L-Pro-OH*H2O Single coupling 18 Fmoc-L-Leu-OH Single coupling 19 Fmoc-L-Glu(OtBu)-OH*H2O Single coupling 20 Fmoc-L-Val-L-Ser(psiMe,Mepro)-OH Single coupling-long 21 Fmoc-L-Lys(Boc)-OH Single coupling 22 Fmoc-L-Asn(Trt)-OH Double coupling 23 Fmoc-L-Val-OH Double coupling 24 Fmoc-L-Val-OH Double coupling 25 Fmoc-L-Val-OH Double coupling 26 Fmoc-L-Ser(tBu)-OH Double coupling 27 Fmoc-Gly-OH Double coupling 28 Boc-L-Asp(OtBu)-OH Single coupling, no Fmoc removal 29 - Final Wash Table 9. Comparative SPPS schemes Table 10. Comparative overall deprotection Table 11. Comparative first pass purification Table 12. Comparative concentration passes Isolation was performed by freeze drying.

[0076] The superposition of the chromatograms comparing the peptide according to Example 1 and the peptide according to Comparative Example 1 is shown in Figure 1 middle. Figure 2 It is normalized to the main peak Figure 1 Normalized display of the chromatograms shown in . Figure 3It is the MS TIC chromatogram of the material (including peptides and impurities) produced by conventional methods.Table 13 compares the retention time (minutes) of impurity peaks and product peaks, the area percentage purity of each peak and the AUC (area under the curve) of each peak. As used herein, the term "impurity" can refer to the impurity related to process and product, including the degradation products that occur, and is measured by area percentage (i.e., impurity percentage or purity), as illustrated in Table 13. The purity of product refers to the area percentage (such as Table 13) of product peaks compared with the total integrated peak area. As such, the purity of the product via the improved method in this embodiment is 96.51%, and the purity of the product via the conventional method is 89.84%. It is noteworthy that in this embodiment, using the improved method, the impurity peak (5.53 area %) at 17.918 minutes in the conventional method does not exist. This front peak is difficult to remove by preparative chromatography. Table 13. Comparative product purity

[0077] Table 14, shown below, provides a chromatogram of material produced by conventional methods ( Figure 3 ) further details of the MS peaks seen in . Table 14. Comparative MS peaks peak quality Notes Main Peak M+0 Target peptide 1 M+127 +Lys 2 M-415 unknown 3 M-57 -Gly 4 Main peak: M+137, M+57 +His, +Gly 5 M+57, M+0 +Gly, isomer 6 M+87 +Ser 7 M+0 Isomers 8 M+0 Isomers 9 M+0,M+71 Isomer, +Ala

[0078] As demonstrated in Example 1 and Comparative Example 1, the material produced by Example 1 advantageously improves the purity of the target peptide, i.e., contains fewer impurities. The method of Example 1 also has multiple advantages over the conventional method of Comparative Example 1, including using less raw material, a smaller volume of solvent, a smaller volume of cleavage mixture, a smaller volume of anti-solvent, eliminating the grinding step, increasing the load during purification to enhance processability, and increasing the concentration step to improve precipitation. Comparative Overview

[0079] The improved method provides unexpectedly high purity and purification (column) loading because there is no front eluting peak that is difficult to purify away. The molecular weight of SEQ ID No. 1 is estimated to be 3112.41 Da, and the accurate mass is 3110.55 Da (molecular formula C 134 H 215 N 37 O 48 The protected FW was estimated to be 4754.81 Da (molecular formula C 254 H 351 N 37 O 52 ). Exemplary differences and advantages of the methods of the present invention relative to conventional methods are summarized in Tables 15-18 below. Table 15. Example SPPS Comparison SPPS Table 16. Example of global deprotection Global deprotection Table 17. Example Purification Steps Pass 1 and 2Purification Steps Pass 1 and 2 Table 18. Example concentration and separation definition

[0080] The terms and abbreviations used in this specification will have the meanings as provided herein. If a particular term is not defined herein, it will have the meaning commonly known to those skilled in the art.

[0081] The term "DMF" refers to the solvent dimethylformamide.

[0082] The term "elution" refers to the eluent or eluate.

[0083] As used herein, the term "Oxyma" means ethyl 2-cyano-2-(hydroxyamino)acetate.

[0084] As used herein, the term "Fmoc" refers to the protecting group 9-fluorenylmethyloxycarbonyl, and the term "Boc" or "t-Boc" is intended to refer to tert-butyloxycarbonyl. Boc and Fmoc groups are used to protect the amino / terminus of amino acids during solid phase peptide synthesis (SPPS).

[0085] The term "SPPS" stands for solid phase peptide synthesis, which is a synthetic method for synthesizing peptides.

[0086] As used herein, the term "AA" means any amino acid.

[0087] The letter "D" stands for aspartic acid.

[0088] The letter "A" represents alanine.

[0089] The letter "G" stands for glycine.

[0090] The letter "S" represents serine.

[0091] The letter "V" stands for valine.

[0092] The letter "N" stands for asparagine.

[0093] The letter "K" stands for lysine.

[0094] The letter "E" stands for glutamine.

[0095] The letter "H" stands for histidine.

[0096] The letter "L" stands for leucine.

[0097] The letter "T" represents threonine.

[0098] The letter "Y" stands for tyrosine.

[0099] The term "TIS" means triisopropylsilane.

[0100] The term "TFA" means trifluoroacetic acid.

[0101] The embodiments of the present disclosure described above are intended to be exemplary only; numerous variations and modifications will be apparent to those skilled in the art. It is intended that all such variations and modifications be within the scope of the present disclosure as defined in any appended claims.

[0102] All references mentioned in this disclosure are incorporated herein by reference in their entirety. The various embodiments of the present disclosure may be characterized by the potential claims listed in the paragraphs after this paragraph (and before the actual claims provided at the end of this application). These potential claims form a part of the written description of this application. Therefore, the subject matter of the following potential claims can be presented as actual claims in subsequent procedures involving this application or any application claiming priority based on this application. The inclusion of such potential claims should not be interpreted as meaning that the actual claims do not cover the subject matter of the potential claims. Therefore, the decision not to present these potential claims in subsequent procedures should not be interpreted as donating the subject matter to the public. References 1 Ranford, JC et al. (2000). "Chaperonins are cell signaling polypeptides:--the unfolding biology of molecular chaperones." Exp. Rev. Mol. Med., 15; 2(8): 1-17. 1 Ranson, N et al. (1998). "Review Article: Chaperones." Biochem. J. 333: 233-242. 1Amblard, M et al. (2006). “Methods and protocols of modern solid phase peptide synthesis.” Molecular Biotechnology, 33(3):239-254. 1 Behrendt, R et al. (2016). “Advances in Fmoc solid-phase peptide synthesis.” Journal of Peptide Science, 22(1):4-27, 1075-2617

Claims

1. A method for synthesizing a peptide having SEQ ID NO: 1, the method comprising: (i) attaching an amino acid (AA) to a resin of a solid support via the C-terminus of the AA, and wherein the N-terminus of the amino acid is protected to avoid reaction at the N-terminus, to form a first solid support-bound AA; (ii) deprotecting the N-terminus of the AA bound to the first solid support by removing the protecting group; (iii) coupling a second AA to the first solid support-bound AA, wherein the C-terminus of the second amino acid is coupled to the deprotected N-terminus of the first solid support-bound AA to form a second solid support-bound AA, and wherein the second AA comprises a protected N-terminus; (iv) repeating steps (ii) and (iii) to form the next solid support-bound AA until a solid support-bound AA sequence is formed; (v) cleaving the AA sequence from the solid support to produce a mixture of peptides having a desired sequence ID; (vi) separating the peptide mixture from the solid support by filtration to produce a crude product; (vii) diluting the separated peptide mixture with a solvent to form a precipitate, wherein the precipitate is isolated by filtration; (viii) subjecting the separated precipitate to column chromatography to collect eluted fractions containing a purified version of the peptide; (ix) concentrating the eluted fractions to form a concentrated eluate comprising a purified version of the peptide; and (x) isolating the peptide from the concentrated eluate by precipitation followed by filtration.

2. The method of claim 1, wherein the peptide SEQ ID NO: 1 is:

3. The method of claim 2, wherein the C-terminus of the AA(A) is attached to the resin-bound amine of aspartic acid from the solid support by treating about 2.5 equivalents of AA alanine(A) with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIEA) in DMF, and further, wherein the N-terminus of the AA(A) is protected by using Fmoc as a protecting group.

4. The method of claim 3, wherein the Fmoc-protected N-terminus of the dipeptide (AD) is deprotected by treating the solid support-bound dipeptide (AD) with a mixture of 10% piperidine and 0.15 M Oxyma in DMF three times in succession.

5. The method of claim 4, wherein the solid support-bound dipeptide (AD) comprising a deprotected N-terminus is treated with about 2.5 equivalents of a second AA-alanine (A) in the presence of N,N-diisopropylcarbodiamide (DIC) and ethyl 2-cyano-2-(hydroxyamino)acetate (Oxyma) in DMF, and further, wherein the N-terminus of the second AA (A) is protected by using Fmoc as a protecting group.

6. The method of claim 5, wherein the N-terminus of the second AA (A) is deprotected by treating the solid support bound AA's with a mixture of 10% piperidine and 0.15 M Oxyma in DMF three times in succession.

7. The method of claim 6, wherein the solid support bound AA's having a deprotected N-terminus are treated sequentially using the steps described in claims 5 and 6 until a solid supported peptide having SEQ ID NO: 1 is formed.

8. The method of claim 7, wherein the peptide having SEQ ID NO: 1 is obtained by: cleaving the solid-supported peptide having SEQ ID NO: 1 from the solid support by treating the solid-supported peptide having SEQ ID NO: 1 with an aqueous solution comprising trifluoroacetic acid (TFA) and triisopropylsilane (TIS), and separating the peptide having SEQ ID NO: 1 from the solid support by passing the mixture through a filter, wherein the peptide having SEQ ID NO: 1 passes through the filter into a filtrate, and wherein the filtered solid material is further washed up to eight times with an aqueous solution comprising TFA and TIS to produce a filtrate comprising the peptide having SEQ ID NO:

1.

9. The method of claim 8, wherein the eluate is diluted by stepwise addition of methyl tert-butyl ether (MTBE), heptane, and MTBE in a volume ratio of 1:0.75:1:1 to produce the peptide having SEQ ID NO: 1 as a precipitate. 10 . The method of claim 9 , wherein the SEQ ID NO: 1 peptide precipitate is further subjected to reverse phase high performance liquid column chromatography using a C4 reverse phase column.

11. The method of claim 10, wherein the pore size of the C4 reverse phase column ranges from about 12. The method of claim 11, wherein the particle size of the C4 reverse phase column is about 10 μm.

13. The method of claim 12, wherein mobile phase A is about 25 mM to about 50 mM ammonium acetate at a pH of about 7 to about 8.

4.

14. The method of claim 13, wherein mobile phase B is acetonitrile (ACN).

15. The method of claim 14, wherein purifying the peptide precipitate by reverse phase high performance liquid column chromatography using a C4 reverse phase column further comprises loading the C4 reverse phase column to a concentration of about 23 mg of crude product per mL of stationary phase.

16. The method of claim 15, wherein the C4 reverse phase column bed has a height of about 20 cm to about 40 cm.

17. The method of claim 16, wherein the mobile phase is collected as an eluate after passing through the C4 reverse phase column, and further wherein the eluate is diluted with 10% tris(hydroxymethyl)aminomethane (Tris) in water at a pH of about 7 to produce the peptide.

18. The method of claim 1, wherein isolating the purified product from the concentrated eluate comprises diluting the concentrated eluate with 0.5 x volume acetic acid (AcOH) premixed with ACN to form a reaction mixture.

19. The method of claim 17, wherein the peptide is diluted with MTBE to form a mixture.

20. The method of claim 19, wherein separating the peptide molecules from the mixture further comprises aging the reaction mixture at 5°C for 30 minutes to produce a heterogeneous mixture.

21. The method of claim 20, wherein the peptide is separated from the heterogeneous mixture by filtering the heterogeneous mixture through a nylon membrane filter.

22. The method of claim 21, wherein the nylon membrane filter is a 10 μm nylon membrane.

23. The method of claim 22, wherein the separated peptide is further washed with MTBE.

24. The method of claim 23, further comprising wetting the peptide to remove residual solvent.

25. The method of claim 24, wherein humidifying the peptide molecules comprises humidifying the peptide molecules with wet N2 until a relative humidity of about 90% is reached, and then drying with a stream of N2 to produce a dry form of the peptide.

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