Methods for Producing Peptides Derived from Chaperonin 60.1

Abstract

Described herein is an improved method for making the peptide molecule shown in SEQ ID NO:1 (DGSVWNKVSELPAGHGLNVNTLSYGDLAAD). According to some embodiments of the present disclosure, the method includes forming a peptide on a resin by solid phase peptide synthesis (SPPS), cleaving and deprotecting the peptide on the resin to form a crude product, purifying the crude product by column chromatography to collect an eluate fraction, concentrating the eluate fraction to form a concentrated eluate, and isolating the peptide molecule from the concentrated eluate by precipitation and filtration.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present disclosure, in various aspects and embodiments, relates to improved methods for the synthesis and purification of peptides derived from chaperonin 60.1. [Background technology]

[0002] 2. Background of the Invention Chaperonin polypeptides are a subgroup of heat shock polypeptides well known for their role in polypeptide folding. There are two families of chaperonin polypeptides: the chaperonin 60 (approximately 60 kDa) and the chaperonin 10 (approximately 10 kDa) families. 1 Typically, chaperonins assist the folding of a target polypeptide when it enters the central core of its ring-shaped heptamer, and then upon the release of energy from ATP, the target polypeptide is released from the central core by a conformational change in the chaperonin structure. 2 .

[0003] More recently, several chaperonin polypeptides have been shown to have a role in immune regulation. Mycobacterium tuberculosis (M. tuberculosis) produces a polypeptide, chaperonin 60.1 (Cpn60.1), named based on its amino acid sequence identity to other known chaperonins. International Patent Application, Publication No. WO2002 / 040037A2 (Patent Document 1) disclosed pharmaceutical compositions containing Cpn60.1 (MtCpn60.1) from Mycobacterium tuberculosis and its encoding nucleic acid molecules. Various therapeutic uses of these molecules have also been disclosed, including the treatment and / or prevention of autoimmune disorders, allergic conditions, conditions typified by Th2-type immune responses, and conditions associated with eosinophils. The application also disclosed a number of specific peptide fragments obtainable from the full-length polypeptide having similar biological activity.

[0004] International Patent Application, Publication No. WO2009 / 106819A2 (Patent Document 2) discloses the amino acid sequence: We have disclosed a novel set of peptides obtainable from MtCpn60.1, including a peptide having the following structure: TIFF2025506650000001.tif5128 (referred to as "Peptide 4") Peptide 4 has been shown to exert anti-inflammatory activity and significantly reduce eosinophil recruitment in an animal model of allergic airway inflammation.

[0005] Conventional methods provided by the Almac Group that are currently used to generate the peptide molecule depicted in SEQ ID NO:1. 3,4 However, isolation using conventional methods is inefficient and results in low purity. Moreover, isolation using conventional methods requires lyophilization. A method that allows precipitation of the peptide molecules would be preferable.

[0006] Thus, there is a need for improved methods for synthesizing and purifying the peptide molecule depicted in SEQ ID NO:1. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2002 / 040037A2 [Patent Document 2] WO2009 / 106819A2 Summary of the Invention

[0008] The present invention relates to a method of synthesizing a peptide as set forth in SEQ ID NO:1, comprising the steps of: (i) attaching an amino acid (AA) to a solid support resin through the C-terminus of the AA to form a first solid support-bound AA, wherein the N-terminus of the amino acid is protected to avoid reaction at the N-terminus; (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 to form a second 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, wherein the second AA comprises a protected N-terminus; (iv) coupling a second AA to the first solid support-bound AA to form a second 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, wherein the second AA comprises a protected N-terminus; (v) cleaving the AA sequence from the solid support to obtain a mixture of peptides with desired sequence IDs; (vi) separating the peptide mixture from the solid support by filtration to obtain a crude product; (vii) diluting the separated peptide mixture with a solvent to form a precipitate, which is isolated by filtration; (viii) subjecting the isolated precipitate to column chromatography to collect an eluate fraction comprising a purified version of the peptide; (ix) concentrating the eluate fraction 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.

[0009] In a preferred embodiment, SEQ ID NO:1 is A method for synthesizing a peptide of SEQ ID NO:1, which is TIFF2025506650000002.tif3128, is provided.

[0010] Another preferred embodiment provides a method, wherein the C-terminus of AA alanine (A) is coupled to the resin-bound amine of aspartic acid of a solid support by treating about 2.5 equivalents of AA (A) with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIEA) in DMF, and further, the N-terminus of AA (A) is protected by using Fmoc as a protecting group.

[0011] A further preferred embodiment provides a method, wherein the Fmoc-protected N-terminus of the dipeptide (AD) is deprotected by three successive treatments of the solid support-bound dipeptide (AD) with a mixture of 10% piperidine and 0.15 M Oxyma in DMF.

[0012] Yet another preferred embodiment provides a method in which A) a solid support-bound dipeptide (AD) containing 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 the N-terminus of the second AA (A) is further protected by using Fmoc as a protecting group. A further preferred embodiment provides a method in which B) the N-terminus of the second AA (A) is deprotected by three successive treatments of the solid support-bound AA with a mixture of 10% piperidine and 0.15M Oxyma in DMF, and the solid support-bound AA with the deprotected N-terminus is treated successively with steps A) and B) until a solid supported peptide having the sequence SEQ ID NO:1 is formed.

[0013] In a further preferred embodiment, a method is provided in which 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 isolating the peptide having SEQ ID NO:1 from the solid support by passing the mixture through a filter, where the peptide having SEQ ID NO:1 passes through the filter into a filtrate, and where the filtered solid is further washed up to eight times with an aqueous solution comprising TFA and TIS to obtain a filtrate comprising the peptide having SEQ ID NO:1. In a further preferred embodiment, a method is provided in which 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 obtain the peptide having SEQ ID NO:1 as a precipitate.

[0014] Another embodiment provides a method, wherein the SEQ ID NO:1 peptide precipitate is further subjected to reversed-phase high performance liquid column chromatography using a C4 reversed-phase column, the pore size of the C4 reversed-phase column being in the range of about 100-120 Å and the particle size of the C4 reversed-phase column being about 10 μm.

[0015] 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 mobile phase B is acetonitrile (ACN).

[0016] Yet another embodiment provides a method, wherein purifying the peptide precipitate by reversed-phase high performance liquid column chromatography using a C4 reversed-phase column further comprises loading the C4 reversed-phase column to a concentration of about 23 mg crude product per mL of stationary phase, the C4 reversed-phase column bed having a height of about 20 cm to about 40 cm. Another preferred embodiment provides a method, wherein the mobile phase is collected as an eluate after passing through the C4 reversed-phase column, and the eluate is further diluted with 10% tris(hydroxymethyl)aminomethane (Tris) in water at a pH of about 7 to obtain the peptide.

[0017] Another aspect of the invention provides a method, wherein isolating the purified product from the concentrated eluate comprises diluting the concentrated eluate with 0.5x volume of 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.

[0018] Another preferred embodiment provides a method, wherein isolating the peptide molecules from the mixture further comprises aging the reaction mixture at 5° C. for 30 minutes to obtain a heterogeneous mixture. Yet another preferred embodiment provides a method, wherein the peptides are isolated 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.

[0019] A further preferred embodiment provides a method, wherein the isolated peptide is further washed with MTBE, further comprising the step of humidifying the peptide to remove residual solvent. Yet another preferred embodiment provides a method, wherein the step of humidifying the peptide molecule comprises humidifying the peptide molecule with wet N2 until it achieves a relative humidity of about 90%, followed by drying with a stream of N2 to obtain the peptide in a dry form. [Brief description of the drawings]

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various aspects of the invention and, together with the detailed description, serve to explain and explain the principles of the present disclosure. The drawings are intended only to diagrammatically illustrate major features of exemplary aspects. [Figure 1] Chromatographic overlay showing purification of a peptide according to the improved method of the invention described herein (shown in the zoomed-in chromatogram trace "Improved" further from the x-axis, showing the major peaks at approximately 18.4-18.6 minutes) compared to purification of a peptide according to a conventional method (shown in the chromatogram trace closer to the x-axis, with significant peaks at 13.2, 17.6, 17.9, 18, 18.2, 18.5, and 19 minutes). Both chromatograms were acquired at 210 nm (+ / - 1 nm). [Diagram 2] Normalized view of the chromatogram overlay from Figure 1 where both chromatograms were normalized about the main peak between approximately 18.4 and 18.6 on the x-axis. Both chromatograms were acquired at 210 nm (+ / - 1 nm). [Diagram 3] Mass spectrum (MS) chromatogram of conventionally produced material containing peptide (main peak) and labeled impurity peaks. The MS total ion chromatogram (TIC) chromatogram is for positive ions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description of the Invention The present invention relates to a method of synthesizing a peptide as set forth in SEQ ID NO:1, comprising the steps of: (i) attaching an amino acid (AA) to a solid support resin through the C-terminus of the AA to form a first solid support-bound AA, wherein the N-terminus of the amino acid is protected to avoid reaction at the N-terminus; (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 to form a second 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, wherein the second AA comprises a protected N-terminus; (iv) coupling a second AA to the first solid support-bound AA to form a second 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, wherein the second AA comprises a protected N-terminus; (v) cleaving the AA sequence from the solid support to obtain a mixture of peptides with desired sequence IDs; (vi) separating the peptide mixture from the solid support by filtration to obtain a crude product; (vii) diluting the separated peptide mixture with a solvent to form a precipitate, which is isolated by filtration; (viii) subjecting the isolated precipitate to column chromatography to collect an eluate fraction comprising a purified version of the peptide; (ix) concentrating the eluate fraction 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.

[0022] In a preferred embodiment, SEQ ID NO:1 is TIFF2025506650000003.tif3128 provides a method for synthesizing a peptide of SEQ ID NO:1.

[0023] Another preferred embodiment provides a method, wherein the C-terminus of AA alanine (A) is coupled to the resin-bound amine of aspartic acid of a solid support by treating about 2.5 equivalents of AA (A) with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIEA) in DMF, and further, the N-terminus of AA (A) is protected by using Fmoc as a protecting group.

[0024] A further preferred embodiment provides a method, wherein the Fmoc-protected N-terminus of the dipeptide (AD) is deprotected by three successive treatments of the solid support-bound dipeptide (AD) with a mixture of 10% piperidine and 0.15 M Oxyma in DMF.

[0025] Yet another preferred embodiment provides a method in which A) a solid support-bound dipeptide (AD) containing 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 the N-terminus of the second AA (A) is further protected by using Fmoc as a protecting group. A further preferred embodiment provides a method in which B) the N-terminus of the second AA (A) is deprotected by three successive treatments of the solid support-bound AA with a mixture of 10% piperidine and 0.15M Oxyma in DMF, and the solid support-bound AA with the deprotected N-terminus is treated successively with steps A) and B) until a solid supported peptide having the sequence SEQ ID NO:1 is formed.

[0026] In a further preferred embodiment, a method is provided in which 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 isolating the peptide having SEQ ID NO:1 from the solid support by passing the mixture through a filter, where the peptide having SEQ ID NO:1 passes through the filter into a filtrate, and where the filtered solid is further washed up to eight times with an aqueous solution comprising TFA and TIS to obtain a filtrate comprising the peptide having SEQ ID NO:1. In a further preferred embodiment, a method is provided in which 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 obtain the peptide having SEQ ID NO:1 as a precipitate.

[0027] Another embodiment provides a method, wherein the SEQ ID NO:1 peptide precipitate is further subjected to reversed-phase high performance liquid column chromatography using a C4 reversed-phase column, the pore size of the C4 reversed-phase column being in the range of about 100-120 Å and the particle size of the C4 reversed-phase column being about 10 μm.

[0028] 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 mobile phase B is acetonitrile (ACN).

[0029] Yet another embodiment provides a method, wherein purifying the peptide precipitate by reversed-phase high performance liquid column chromatography using a C4 reversed-phase column further comprises loading the C4 reversed-phase column to a concentration of about 23 mg crude product per mL of stationary phase, the C4 reversed-phase column bed having a height of about 20 cm to about 40 cm. Another preferred embodiment provides a method, wherein the mobile phase is collected as an eluate after passing through the C4 reversed-phase column, and the eluate is further diluted with 10% tris(hydroxymethyl)aminomethane (Tris) in water at a pH of about 7 to obtain the peptide.

[0030] Another aspect of the invention provides a method, wherein isolating the purified product from the concentrated eluate comprises diluting the concentrated eluate with 0.5x volume of 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.

[0031] Another preferred embodiment provides a method, wherein isolating the peptide molecules from the mixture further comprises aging the reaction mixture at 5° C. for 30 minutes to obtain a heterogeneous mixture. Yet another preferred embodiment provides a method, wherein the peptides are isolated 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.

[0032] A further preferred embodiment provides a method, wherein the isolated peptide is further washed with MTBE, further comprising the step of humidifying the peptide to remove residual solvent. Yet another preferred embodiment provides a method, wherein the step of humidifying the peptide molecule comprises humidifying the peptide molecule with wet N2 until it achieves a relative humidity of about 90%, followed by drying with a stream of N2 to obtain the peptide in a dry form.

[0033] Described herein is a method for making a peptide molecule as shown in TIFF2025506650000004.tif5128. The method produces a peptide molecule as shown in SEQ ID NO:1 with improved purity compared to conventional methods for making the peptide molecule as shown in SEQ ID NO:1. In some embodiments, the method includes forming a peptide on a resin by solid phase peptide synthesis, cleaving and deprotecting the peptide on the resin to form a crude product, purifying the crude product by column chromatography to collect an eluate fraction, concentrating the eluate fraction to form a concentrated eluate, and isolating the peptide molecule from the concentrated eluate by precipitation and filtration.

[0034] 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 attached to a solid support resin. Peptides are synthesized in the solid phase using a chemistry that adds amino acids from their C-terminus to their N-terminus. Thus, the amino acid or peptide group close to the C-terminus of a particular fragment is added to the resin first. This is done 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 desirably also includes side chain protection. Subsequent amino acids or peptide groups are then coupled to the support-bound peptide material until the peptide of interest is formed. Most of these also include side chain protection according to conventional practice. During each subsequent coupling, the masking group at the N-terminus of the resin-bound peptide material is removed. This is then reacted with the C-terminus of the next amino acid whose N-terminus is masked. The product of solid phase synthesis is thus a peptide bound to the resin support. The support-bound peptide is then typically cleaved from the support and subjected to further processing and / or purification.

[0035] Any type of support suitable for carrying out solid phase peptide synthesis may be used. In some embodiments, the support comprises a resin that may be made of one or more polymers, copolymers or combinations of polymers, such as polyamides, polysulfamides, substituted polyethylenes, polyethylene glycols, phenolic resins, polysaccharides, or polystyrenes. The polymeric support may also be any solid that is sufficiently insoluble and inert to the solvents used in peptide synthesis. The solid support typically includes a linking moiety that is coupled to the growing peptide during synthesis and can be cleaved to release the peptide from the support under desired conditions. Suitable solid supports may have photocleavable, TFA cleavable, HF cleavable, fluoride ion cleavable, reductive cleavable, Pd(O) cleavable, nucleophilic cleavable, or radical cleavable linkers. Preferred linking moieties are cleavable under conditions in which the side chain groups of the cleaved peptide are still substantially entirely protected.

[0036] In some embodiments, fluorenylmethoxycarbonyl (Fmoc)-based solid phase peptide synthesis (SPPS) is performed, in which the Fmoc group is used for temporary protection of the alpha amino group. The Fmoc protecting group can be selectively cleaved from the peptide compared to the side chain protecting groups, so that the side chain protection remains intact when the Fmoc is cleaved. This type of selectivity is important to minimize side chain reactions during amino acid coupling. Furthermore, the side chain protecting groups can be selectively cleaved compared to the Fmoc so that they are removed, leaving the Fmoc intact.

[0037] In some embodiments, coupling of Fmoc-AA is carried out using about 2.5 equivalents of the amino acid with N,N-diisopropylcarbodiimide (DIC) and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) in dimethylformamide (DMF).

[0038] In some embodiments, the coupling of Fmoc-AA comprises 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 about 3 hours of coupling time. In some embodiments, 12 of the 28 single couplings are performed with about 2.5 equivalents of amino acid with DIC and Oxyma in DMF for 3 hours. In some embodiments, 16 of the 28 single couplings use about 6 hours of coupling time. In some embodiments, 16 of the 28 single couplings are performed with about 2.5 equivalents of amino acid with DIC and Oxyma in DMF for 6 hours.

[0039] In some embodiments, about 70 equivalents of amino acids are required to perform coupling (28 single couplings, each coupling using about 2.5 equivalents of amino acids). On the other hand, the conventional method for producing the peptide molecule shown in SEQ ID NO:1 requires 27 couplings using 4 equivalents of amino acids (18 single couplings in 45 minutes and 9 double couplings in 45 minutes), thus requiring 144 equivalents to perform coupling. Compared to the conventional method for forming the peptide molecule shown in SEQ ID NO:1, the method described herein advantageously uses about 50% less raw material than the conventional method, for example, 70 equivalents of Fmoc-AA in the improved method vs. 144 equivalents of Fmoc-AA in the conventional method.

[0040] After the coupling is determined to be 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. To couple the next amino acid, removal of the N-terminal protecting group (e.g., Fmoc group) from the resin-bound material is typically accomplished by treatment with a reagent comprising 20-50% (by weight) piperidine in a solvent such as N-methylpyrrolidone (NMP) or dimethylformamide (DMF). For example, in a conventional method for forming the peptide molecule shown in SEQ ID NO:1, deprotection of Fmoc is performed with 20% piperidine in DMF, which is performed twice with 10 volumes for 20 minutes each.

[0041] In some embodiments, Fmoc deprotection is advantageously carried out using about 50% less piperidine compared to conventional methods. In some embodiments, Fmoc deprotection is carried out using 10% piperidine in DMF with 0.15M Oxyma. In some embodiments, Fmoc deprotection is carried out three times using 6.5 volumes, each for 10 minutes.

[0042] After removal of the Fmoc protecting group, washing is performed to remove residual piperidine and Fmoc by-products (e.g., dibenzofulvene and its piperidine adduct). In some embodiments, washing of 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 with 6.5 volumes for 5 minutes each. 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 (28 cycles with 59 volumes of solvent).

[0043] The conventional method for forming the peptide molecule shown in SEQ ID NO:1 requires 20 washes using 5 volumes, thus 100 volumes per cycle. In total, the conventional method uses 700 volumes of solvent. Advantageously, in the improved method described herein, about 40% less volume of solvent (e.g., DMF) is required for the wash steps than the conventional method.

[0044] Furthermore, conventional methods for forming peptides using solid-phase peptide synthesis typically require a capping step with a capping reagent for a certain period of time (e.g., 10 minutes). In some embodiments, forming a peptide by solid-phase peptide synthesis on a resin advantageously does not require a capping step to block the termini of unreacted amino acids from reacting after coupling of Fmoc-AA, thereby eliminating one step.

[0045] In some embodiments, after completion of the synthesis, the next step is a global 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), HO, and triisopropylsilane (TIS). In some embodiments, treating the resin with this solution comprises treating with 7 volumes of the same solution comprising trifluoroacetic acid (TFA), HO, and triisopropylsilane (TIS) for 2.5 hours.

[0046] In some embodiments, the resin can then be removed by filtration and rinsed twice with TFA, thereby providing a filtrate containing the peptide. In some embodiments, the two resin rinses are performed with 0.5 volumes of TFA. In some embodiments, cleavage of the peptide from the resin requires only 8 volumes of solution (cleavage with 7 volumes of cleavage solution and two rinses with 0.5 volumes of TFA).

[0047] Conventional methods for forming the peptide molecule shown 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 3 rinses with 2.5 volumes of TFA). Thus, the peptide cleavage described herein achieves about a 65% volume reduction in cleavage and wash solutions.

[0048] Additionally, in some embodiments, the cleavage solution is advantageously free of ethane-1,2-dithiol (EDT). Conventional methods for forming the peptide molecule shown in SEQ ID NO:1 typically require EDT in their cleavage solution, which is problematic because it is a pungent scavenger. Without being bound to any particular theory, it has been found that EDT is unnecessary in the cleavage solution in the methods described herein, and can remove the pungent scavengers from the cleavage cocktail.

[0049] After cleavage and deprotection of the peptide on the resin, which provides the peptide in solution, the peptide can be precipitated to form a crude product. In some embodiments, the precipitation of the crude product includes the stepwise addition of methyl tert-butyl ether (MTBE) (6 volumes), heptane (8 volumes), and MTBE (8 volumes). In some embodiments, the precipitation of the crude product includes the stepwise addition of 6 volumes of MTBE, 8 volumes of heptane, and 8 volumes of MTBE. Without being bound to any particular theory, the stepwise addition of antisolvents improves filterability, whereas the mixing of antisolvents causes the particles to gel and slows down filtration.

[0050] 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 is performed four times with two volumes of a solution of MTBE and heptane.

[0051] In some embodiments, precipitation of the crude product requires 30 volumes of anti-solvent (stepwise addition of 6 volumes of MTBE, 8 volumes of heptane, 8 volumes of MTBE, and 4 washes with 2 volumes of a solution of MTBE and heptane after filtration). Conventional methods for purifying the peptide molecule shown in SEQ ID NO:1 typically require about 59 volumes of anti-solvent (e.g., precipitation with 45.3 volumes of MTBE and 3 washes with 4.8 volumes of MTBE). Thus, the precipitation described herein advantageously provides about 50% anti-solvent volume reduction compared to conventional methods.

[0052] Conventional methods for forming peptide molecules as shown in SEQ ID NO:1 typically require further powdering of the solid obtained from precipitation with another anti-solvent (e.g., MTBE). For example, conventional powdering of the solid obtained from precipitation can be performed with 15 volumes of MTBE for 15 hours, isolated by filtration, washed three times with 5 volumes of MTBE after filtration, and then redried. Advantageously, powdering of the precipitate is not required in the improved method for purifying peptide molecules as shown in SEQ ID NO:1 described herein. As a result, in some embodiments, the total volume of reactive moieties during global deprotection is 38 volumes (8 volumes during cleavage and 30 volumes during precipitation), which is about 66% reduction of reactive moieties required in conventional methods (23 volumes for cleavage, 59 volumes for precipitation, and 30 volumes for powdering).

[0053] In some embodiments, the next step after cleavage and deprotection of the resin-bound peptide is purification of the crude peptide. In some embodiments, the crude peptide is purified by column chromatography to collect eluate fractions. In some embodiments, purification of the crude product by column chromatography includes the use of reversed-phase high performance liquid chromatography and a C4 reversed-phase column.

[0054] 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 reversed phase column is 100 Å, 120 Å, 200 Å, or 300 Å. Preferably, the pore size of the C4 reversed phase column is 120 Å. In some embodiments, the particle size of the C4 reversed phase column is 3 μm, 5 μm, or 10 μm. Preferably, the particle size of the C4 reversed phase column is 10 μm.

[0055] In some embodiments, purification of the crude product by column chromatography further comprises loading a C4 reverse phase column to a concentration ranging from about 20 mg to about 35 mg of crude product per mL of stationary phase, or about 23 mg to about 30 mg of crude product per mL of stationary phase, or about 25 mg of crude product per mL of stationary phase. In some embodiments, the column bed height is about 10 cm to about 30 cm, about 15 cm to about 25 cm, about 20 cm to about 30 cm, or about 25 cm. In some embodiments, the column bed height is 25 cm.

[0056] In contrast, conventional methods for purifying peptide molecules as shown in SEQ ID NO:1 typically only allow a maximum loading of 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 may produce a front-eluting shoulder peak, which severely limits loading. The higher loading provided in the improved purification process improves processability compared to the purification process in conventional methods for purifying peptide molecules as shown in SEQ ID NO:1.

[0057] In some embodiments, purification of the crude product by column chromatography comprises at least two purification passes in which eluate fractions are collected. 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 process comprises two purification passes through the chromatography medium, the first of which is performed under an ammonium acetate gradient providing a pH of about 7-8.4, followed by a second chromatographic pass under an ACN gradient.

[0058] In some embodiments, purification of the crude product by column chromatography includes adding a stabilizer to the eluate fraction, such as 10V% tris(hydroxymethyl)aminomethane (Tris) at pH 7. Conventional methods for purifying the peptide molecule set forth in SEQ ID NO:1 do not require the addition of stabilizers to the eluate fraction. Instead, purification in conventional methods results in a gelled fraction, which must be redissolved by pH adjustment after visual inspection. Without being bound to any particular theory, the prevention of gelling provided by the purification step of the improved method may advantageously be less invasive than the breakdown of the gelled fraction required in conventional methods for purifying the peptide molecule set forth in SEQ ID NO:1.

[0059] In some embodiments, purification of the crude product by chromatography further comprises pooling eluate fractions containing product concentrations and purities higher than a desired threshold to form a mixed eluate fraction. In some embodiments, the crude product is purified to >90% (e.g., >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%) and appropriate fractions are pooled. In some embodiments, the purity of the peptide can be verified by reversed-phase HPLC followed by characterization of the purified product (i.e., the identity of the peptide can be verified) by liquid chromatography / mass spectrometry (LC / MS) and / or matrix-assisted laser desorption / ionization (MALDI) mass spectrometry. Conventional methods for synthesizing and purifying the peptide molecule set forth in SEQ ID NO:1 cannot produce products with as high a purity as the improved methods described herein, as evidenced in the examples below.

[0060] In some embodiments, the next step after purification of the crude product by column chromatography to collect eluate fractions is concentrating the eluate fraction (or pooled eluate fractions) to form a concentrated eluate. In some embodiments, concentrating the eluate fraction or combined eluate fractions comprises loading the eluate fraction or combined eluate fraction onto a chromatography column comprising polystyrene divinylbenzene resin or C4 material and eluting with a solution of ACN containing ammonium acetate to form a concentrated eluate. The concentration step can increase the concentration of the desired peptide in the solvent by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 fold.

[0061] In some embodiments, the isolation step follows the concentration step. In some embodiments, the isolation of the peptide molecules from the concentrated eluate is performed by precipitation and filtration. In some embodiments, the isolation of the purified product from the concentrated eluate comprises diluting the concentrated eluate with 0.5x volume of acetic acid (AcOH) premixed with ACN to form a reaction mixture. In some embodiments, the isolation of the peptide molecules from the concentrated eluate further comprises diluting the reaction mixture with MTBE. In some embodiments, the isolation of the peptide molecules from the concentrated eluate comprises aging the reaction mixture for 30 minutes or maintaining at 5°C.

[0062] In some embodiments, the isolation of peptide molecules from the concentrated eluate comprises filtering the reaction mixture through a nylon membrane filter to isolate a precipitate after aging. In some embodiments, the nylon membrane filter is a 10 μm nylon membrane filter. In some embodiments, the isolation of peptide molecules from the concentrated eluate comprises filtering the reaction mixture through a nylon membrane filter and then washing the precipitate with MTBE. In some embodiments, after washing, the precipitate is dried.

[0063] 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 they reach about 90% relative humidity, and drying with a stream of N2.

[0064] Conventional methods for forming the peptide molecule set forth in SEQ ID NO:1 do not use a concentration step. Instead, conventional methods pool and lyophilize fractions containing the desired peptide. Additionally, conventional methods for forming the peptide molecule set forth in SEQ ID NO:1 do not use a humidification step because the peptide is isolated through lyophilization.

[0065] Composition Details Peptide synthesis - general procedure The peptides according to the invention are synthesized in multiple steps. The synthesis utilizes solid phase peptide synthesis (SPPS) in which a first amino acid (AA) is covalently attached onto a solid support material and synthesized step-by-step in a single reaction vessel using selective protecting group chemistry. The first step involves attaching an amino-protected amino acid to a solid phase material or resin (most commonly low cross-linked polystyrene beads) to form a covalent bond between the carbonyl group of the amino acid (AA) and the resin. The covalent bond is an amide or ester bond. The amino group protected by protecting groups such as 9-fluorenylmethyloxycarbonyl (Fmoc) and t-butyloxycarbonyl (Boc) is deprotected by treating the solid support-bound AA with a mixture of 10% piperidine and 0.15M Oxyma in DMF. The solid support-bound AA with the now deprotected amino group is reacted / coupled with the carbonyl group of the next N-protected amino acid. This coupling results in a solid phase bearing dipeptide. This above cycle is repeated to form the desired peptide chain. After all reactions are complete, the synthesized peptide is cleaved from the solid support. EXAMPLES

[0066] Example 1 In this example, the peptide shown in SEQ ID NO: 1 is synthesized. The SEQ ID NO: 1 peptide is synthesized in multiple steps as described in the general procedure.

[0067] Step 1: The first step involves solid phase peptide synthesis (SPPS) in which the carbonyl group / C-terminus of amino acid (AA) 30 - alanine (A), whose N-terminus is protected by an Fmoc group, is covalently attached to the HL-Asp(OtBu)-2CT resin, a solid support to which AA 31, D, is already attached. This step involves treating the 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) for about 2 hours at 23°C to obtain the Fmoc-AD-2CT resin. The covalent attachment of the first AA was not monitored directly. The construction of the correct sequence was periodically confirmed by performing microcleavage and analyzing them by HPLC-MS.

[0068] Experimental procedure: Fmoc-L-Ala-OH (2.5 equiv.) and TBTU (2.44 equiv.) were weighed in an appropriate vial. Prior to the coupling of the amino acid, DMF (c=0.25 mol / L for AA) was added to the vial. After a clear solution was formed, DIEA (4.90 equiv.) was added and the activated AA was stirred for 2-10 min. This pre-activated AA was then transferred to the resin and stirred for 120 min at RT. The resin was washed 4 times with DMF [6.5 ml / g (resin), 5 min each].

[0069] Step 2: This step involved deprotection of the N-terminal nitrogen by removing the Fmoc protecting group. This deprotection step involved treating the solid support-bound dipeptide (AD) with a mixture of 10% piperidine and 0.15M Oxyma in DMF to generate the solid support-bound dipeptide (AD) with a deprotected N-terminus. No direct monitoring of this step was performed. Analysis of the microcleavage by HPLC-MS showed no loss of the next amino acid, which could be a direct impurity resulting from incomplete de-Fmoc in this step. This analysis was performed in the R&D stage and then ignored after it was confirmed that this step did not result in any impurities.

[0070] Experimental procedure: The resin was treated with 10% piperidine in DMF containing 0.15M Oxyma three times [6.5 ml / g resin, 10 min each]. The resin was washed five times with DMF [6.5 ml / g resin, 5 min each].

[0071] Step 3: This step involved treating the solid support-bound dipeptide (AD) containing a deprotected N-terminus with 2.5 equivalents of the amino acid alanine AA (A), whose N-terminus is protected with an Fmoc group, with N,N-diisopropylcarbodiamide (DIC) and ethyl 2-cyano-2-(hydroxyamino)acetate (Oxyma) in DMF at temperatures ranging from 20 to 24 °C for approximately 3 hours. The covalent attachment of this AA was not monitored directly. The construction of the correct sequence was routinely confirmed by performing microcleavage and analyzing them by HPLC-MS.

[0072] Experimental procedure: Fmoc-Ala-OH (2.5 eq.) and OxymaPure (2.5 eq.) were weighed into an appropriate vial and dissolved with DMF (c=0.25 mol / L). Prior to coupling of 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 four times with DMF [6.5 ml / g (resin), 5 min each]. Example conditions used are provided in Tables 1-7 below. SPPS sequences are shown in Table 1 below. Example SPPS protocols are provided in Table 2 and the overall deprotection protocol is shown in Table 3. First and second pass purification conditions are shown in Tables 4 and 5, respectively. Concentration passes are shown in Table 6 and Table 7 provides the sedimentation, filtration, and humidification protocols.

[0073] (Table 1) SPPS sequence TIFF2025506650000005.tif157161

[0074] Table 2: SPPS protocol TIFF2025506650000006.tif181159

[0075] (Table 3) Global deprotection TIFF2025506650000007.tif136158

[0076] (Table 4) First pass purification conditions TIFF2025506650000008.tif188158

[0077] (Table 5) Second pass purification conditions TIFF2025506650000009.tif188158

[0078] (Table 6) Concentration passage TIFF2025506650000010.tif175158

[0079] Table 7. Sedimentation, Filtration, and Humidification TIFF2025506650000011.tif163158

[0080] Comparative Example 1 Comparison of material purity The peptide according to SEQ ID NO:1 of the present invention is synthesized and purified by the conventional method described below. Tables 8-12 below show the conditions of the comparative (conventional) method. The SPPS sequence, SPPS protocol, global deprotection, first pass purification, and concentration pass protocol of the comparative method are shown in Tables 8-12, respectively.

[0081] (Table 8) SPPS sequence of the comparative method TIFF2025506650000012.tif145161

[0082] Table 9. SPPS protocol for the comparative method TIFF2025506650000013.tif98159TIFF2025506650000014.tif221159

[0083] Table 10. Overall deprotection of comparative methods TIFF2025506650000015.tif118163

[0084] Table 11. First pass purification conditions for the comparative method TIFF2025506650000016.tif93163

[0085] Table 12. Concentration pass through of the comparison method TIFF2025506650000017.tif75162Isolation was performed by freeze-drying.

[0086] A chromatogram overlay comparing the peptide from Example 1 and the peptide from Comparative Example 1 is shown in FIG. 1. FIG. 2 is a normalized display of the chromatogram overlay shown in FIG. 1 normalized to the main peak. FIG. 3 is an MS TIC chromatogram of the material made by the conventional method including the peptide and impurities. Table 13 compares the retention time (min) of the impurity and product peaks, the area percentage purity of each peak, and the AUC (area under the curve) of each of the peaks. As used herein, the term "impurity" can refer to process and product-related impurities including generated degradation products, measured by area percentages as illustrated in Table 13 (i.e., percent impurity or purity). Product purity refers to the percentage area of ​​the product peak relative to the total integrated peak area (e.g., Table 13). In this case, the purity of the product through the improved method in this example was 96.51%, while the purity of the product through the conventional method was 89.84%. It is noteworthy in this example that the impurity peak at 17.918 min (5.53 area %) in the conventional method was not present in the improved method, and the peak was previously difficult to remove by preparative chromatography.

[0087] Table 13: Purity of the product of the comparative method TIFF2025506650000018.tif155167

[0088] Table 14, shown below, provides further details regarding the MS peaks seen in the chromatogram of the conventionally produced material (FIG. 3).

[0089] Table 14: MS peaks of the comparative method TIFF2025506650000019.tif73145

[0090] As shown by Example 1 and Comparative Example 1, the material produced by Example 1 advantageously has a high purity of the peptide of interest, i.e., contains fewer impurities. The method of Example 1 also has a number of advantages over the conventional method of Comparative Example 1, including the use of less raw material, less volume of solvent, less volume of cleavage cocktail, less volume of antisolvent, elimination of the trituration step, increased loading during purification to improve processability, and the addition of a concentration step to improve precipitation.

[0091] Comparison Summary The improved method provided surprisingly high purity and purification (column) loading due to the absence of pre-eluting peaks that are difficult to remove by purification. The formula molecular weight of SEQ ID NO:1 was estimated to be 3112.41 Da, with an exact mass of 3110.55 Da (molecular formula C 134 H 215 N 37 O 48 The protected FW was 4754.81 Da (molecular formula C 254 H 351 N 37 O 52 The differences between the examples and the advantages of the method of the present invention over the conventional method are summarized in Tables 15-18 below.

[0092] Table 15: SPPS comparison of examples TIFF2025506650000020.tif173161

[0093] Table 16: Global deprotection of examples TIFF2025506650000021.tif194161

[0094] Table 17. First and second passes of the purification process of the examples TIFF2025506650000022.tif150161

[0095] Table 18: Concentrations and isolation of examples TIFF2025506650000023.tif160161

[0096] Definition: The terms and abbreviations used herein have the meanings provided herein. If a particular term is not defined herein, it has the meaning commonly known to one of ordinary skill in the art.

[0097] The term "DMF" refers to the solvent dimethylformamide.

[0098] The term "elute" refers to an eluant or eluate.

[0099] The term "Oxyma" as used herein refers to ethyl 2-cyano-2-(hydroxyamino)acetate.

[0100] The term "Fmoc" as used herein is intended to represent the protecting group 9-fluorenylmethyloxycarbonyl group, and the terms "Boc" or "t-Boc" are intended to represent the t-butyloxycarbonyl group. The Boc and Fmoc groups are used to protect the amino group / terminus of amino acids during solid phase peptide synthesis (SPPS).

[0101] The term "SPPS" refers to solid phase peptide synthesis, a synthetic method used to synthesize peptides.

[0102] The term "AA" as used herein represents any amino acid.

[0103] The symbol "D" stands for aspartic acid.

[0104] The symbol "A" represents alanine.

[0105] The symbol "G" represents glycine.

[0106] The symbol "S" represents serine.

[0107] The symbol "V" stands for valine.

[0108] The symbol "N" represents asparagine.

[0109] The symbol "K" represents lysine.

[0110] The symbol "E" represents glutamine.

[0111] The symbol "H" represents histidine.

[0112] The symbol "L" represents leucine.

[0113] The symbol "T" represents threonine.

[0114] The symbol "Y" represents tyrosine.

[0115] The term "TIS" stands for triisopropylsilane.

[0116] The term "TFA" stands for trifluoroacetic acid.

[0117] The embodiments of the present disclosure described above are intended to be illustrative only, and many variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be included within the scope of the present disclosure as defined in any appended claims.

[0118] All references cited in this disclosure are incorporated herein by reference in their entirety. Various aspects of the present disclosure may be characterized by potential claims listed in the paragraphs following this paragraph (and prior to the actual claims provided at the end of this application). These potential claims form part of the detailed description of this application. Thus, the subject matter of the following potential claims may be presented as actual claims in a later proceeding of this application or any application claiming priority from this application. The inclusion of such potential claims should not be considered to mean that the actual claims do not cover the subject matter of the potential claims. Thus, a decision not to present these potential claims in a later proceeding should not be considered to provide that subject matter to the public.

[0119] References: TIFF2025506650000024.tif35162

Claims

1. 1. A method for synthesizing a peptide as set forth in SEQ ID NO:1, comprising: (i) coupling an amino acid (AA) to a solid support resin through its C-terminus to form a first solid support-bound AA, the N-terminus of the amino acid being protected to avoid reaction at the N-terminus; (ii) deprotecting the N-terminus of the first solid support-bound AA by removing a protecting group; (iii) coupling a second AA to the first solid support-bound AA to form a second 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, and 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 obtain a mixture of peptides with desired sequence IDs; (vi) separating the peptide mixture from the solid support by filtration to obtain a crude product; (vii) diluting the separated peptide mixture with a solvent to form a precipitate, which is isolated by filtration; (viii) subjecting the isolated precipitate to column chromatography and collecting eluate fractions containing a purified version of the peptide; (ix) concentrating the eluate fraction to form a concentrated eluate containing a purified version of the peptide; and (x) isolating the peptide from the concentrated eluate by precipitation followed by filtration. The method comprises:

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

3. 3. The method of claim 2, wherein the C-terminus of AA alanine (A) is coupled to the resin-bound amine of aspartic acid of the solid support by treating about 2.5 equivalents of the AA (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. 4. The method of claim 3, wherein the Fmoc-protected N-terminus of the dipeptide (AD) is deprotected by three successive treatments of the solid support-bound dipeptide (AD) with a mixture of 10% piperidine and 0.15 M Oxyma in DMF.

5. 5. The method of claim 4, wherein the solid support-bound dipeptide (AD) containing 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 the N-terminus of the second AA (A) is further protected by using Fmoc as a protecting group.

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

7. The method of claim 6, wherein the solid support-bound AA having a deprotected N-terminus is successively treated using the process of any one of claims 5 and 6 until a solid-supported peptide having the structure of SEQ ID NO:1 is formed.

8. The peptide represented by SEQ ID NO:1 is 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 said peptide having SEQ ID NO:1 from said solid support by passing the mixture through a filter; is obtained by wherein the peptide having SEQ ID NO:1 passes through the filter into the filtrate; and wherein the filtered solid is further washed up to eight times with an aqueous solution containing TFA and TIS to obtain a filtrate containing the peptide of SEQ ID NO:1; 8. The method of claim 7.

9. 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 obtain the peptide represented by SEQ ID NO:1 as a precipitate.

10. The SEQ ID NO:1 peptide precipitate was further 4 The method according to claim 9, which is subjected to reverse-phase high performance liquid column chromatography using a reverse-phase column.

11. Said C 4 11. The method of claim 10, wherein the pore size of the reversed phase column is in the range of about 100 to 120 Å.

12. Said C 4 12. The method of claim 11, wherein the particle size of the reverse phase column is about 10 μm.

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

15. C 4 Purifying the peptide precipitate by reversed-phase high performance liquid column chromatography using a reversed-phase column to a concentration of about 23 mg of crude product per mL of stationary phase. 4 15. The method of claim 14, further comprising loading onto a reverse phase column.

16. C 4 16. The method of claim 15, wherein the reverse phase column bed has a height of about 20 cm to about 40 cm.

17. The mobile phase is 4 17. The method of claim 16, wherein the peptides are collected as an eluate after passing through a reverse phase column, and the eluate is further diluted with 10% tris(hydroxymethyl)aminomethane (Tris) in water at a pH of about 7 to obtain the peptides.

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

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

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

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

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

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

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

25. Wet the peptide molecules until they achieve a relative humidity of about 90%. 2 The peptide molecules are then humidified using N 2 25. The method of claim 24, comprising drying with a stream to obtain the peptide in a dry form.

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