Method for peptide oxidation and folding

Abstract

The present disclosure relates to a method for oxidizing and folding a peptide having the amino acid sequence SEQ ID No.: 1 and a peptide prepared by said method. The method comprises the steps of a) providing a compound C comprising or consisting of amino acid sequence SEQ ID No.: 2; b) providing an aqueous medium comprising an oxidizing agent, and a buffer system; and c) contacting the compound C provided in step a) with the aqueous medium provided in step b) to provide compound A.

Description

[0001] selectlON Therapeutics GmbH

[0002] S12906WO2

[0003] METHOD FOR PEPTIDE OXIDATION AND FOLDING

[0004] Technical field

[0005] The present disclosure relates to a method for oxidizing and folding a peptide having the amino acid sequence SEQ ID No.: 1 and a peptide prepared by said method.

[0006] Background

[0007] The voltage-gated Kv1 .3 K+channel is one of 76 potassium channels in the human genome and has been found to be present in human T lymphocytes. All human T cells express the Kv1.3 channel as well as the calcium-activated KCa3.1 , which together provide the counter-balancing potassium efflux for the calcium influx that is necessary for T cell activation and proliferation. The number of channels expressed by a given cell depends on its state of activation and differentiation. Antigen or mitogen stimulated CD4+ and CD8+ TE cells exhibit an approximately 4- to 5- fold increased expression of Kv1 .3, while human naive or TCM cells up-regulate the calcium-activated KCa3.1 channel to regulate membrane potential and Ca2+signaling in the activated state.

[0008] In view of this differential overexpression in TEM cells, the Kv1 .3 channel constitutes a promising new TEM-cell-specific therapeutic target for the treatment of autoimmune diseases, whose pathogenesis involves autoreactive TEM cells such as e.g. multiple sclerosis, rheumatoid arthritis, psoriasis and type-1 diabetes, but also for other chronic inflammatory diseases, such as e.g. parodontitits. The FDA-approved drug clofazimine inhibits Kv1.3 and is used for the treatment of psoriasis and graft-versus-host disease.

[0009] Furthermore, there is indication that Kv1 .3 channels play a role in the regulation of body weight. The Kv1 .3 channel thus also constitutes a promising target for the treatment of obesity.

[0010] WO 2015 / 169901 A1 relates to peptides, such as Si-544, capable of selectively binding to and inhibiting the activity of the potassium channel Kv1 .3.

[0011] However, the synthesis of peptides might turn out challenging.

[0012] After solid-phase peptide synthesis (SPPS), folding peptides and forming intramolecular disulfide bonds encounter several specific difficulties and problems. These issues arise from both the nature of the peptide synthesis process and the intrinsic properties of peptides.

[0013] Unlike in vivo conditions, in vitro folding after SPPS lacks cellular machinery such as chaperones, which assist in proper folding and prevent aggregation. Thus, peptides potentially following multiple folding pathways, and identifying and achieving the correct pathway without cellular assistance is challenging.

[0014] DJB:MHO ln that, achieving correct disulfide bond pairing is critical. Incorrect pairing can lead to non-functional or misfolded peptide, and resulting in a loss of yield.

[0015] Furthermore, disulfide bond formation requires an oxidative environment, which must be carefully controlled. Over-oxidation can lead to incorrect or excessive disulfide bonds, while insufficient oxidation prevents bond formation thus resulting in wrong folding.

[0016] Also, peptides tend to aggregate during folding, particularly if they contain hydrophobic regions. Aggregation competes with proper folding and can lead to insoluble aggregates. Thus, some peptides are poorly soluble in aqueous solutions, making folding and disulfide bond formation difficult. Finding the right solvent or buffer conditions often requires extensive optimizing.

[0017] Folding and disulfide bond formation are also sensitive to pH and temperature. Optimal conditions need to be precisely controlled, which can vary for different peptides. In that, the choice of buffer and its components can significantly impact folding and disulfide bond formation. Finding the right buffer conditions and pH often requires extensive optimization.

[0018] In conclusion, achieving a high yield of correctly folded peptides with the proper disulfide bonds is often difficult due to competing side reactions and aggregation.

[0019] In summary, the main difficulties during peptide folding and forming intramolecular disulfide bonds after SPPS arise from the need to replicate the precise conditions required for correct folding, the challenges of achieving specific disulfide bonds, managing solubility and aggregation issues, ensuring high purity and yield, and the inherent complexities of peptide sequences and structures.

[0020] Summary of the invention

[0021] In view of the high therapeutic potential of inhibiting Kv1 .3 channels, there is an ongoing need for Kv1 .3 channel specific therapeutic compounds that exhibit a strong and specific interaction with the Kv1 .3 channel and are capable of blocking or reducing its activity, as well as for methods of preparing such compounds with high process yield and efficiency.

[0022] Therefore, it is an object of the present invention to improve the synthesis of Kv1 .3 inhibitor compound A comprising or consisting of amino acid sequence SEQ ID No.: 1 , wherein compound A has four intramolecular disulfide bonds, wherein the four intramolecular disulfide bonds are between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38, such as Si-544, by providing a method for peptide folding and oxidative disulfide bond formation, in particular with improved process yield. These and other objectives as they will become apparent from the ensuing description and claims are attained by the subject matter of the independent claims. Some of the preferred embodiments are defined by the dependent claims.

[0023] In a first aspect, the present invention relates to a method for preparing compound A comprising or consisting of the amino acid sequence SEQ ID No.: 1 , wherein compound A has four intramolecular disulfide bonds, wherein the four intramolecular disulfide bonds are between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38(such as Si-544), the method comprising the steps of: a) providing compound C comprising or consisting of amino acid sequence SEQ ID No.: 2, wherein compound C has no intramolecular disulfide bonds; b) providing an aqueous medium comprising an i) oxidizing agent, and II) a buffer system; and c) contacting the compound C provided in step a) with the aqueous medium provided in step b) to provide compound A.

[0024] According to a second aspect, the present invention relates to a compound A comprising or consisting of amino acid sequence SEQ ID No.: 1 , wherein compound A has four intramolecular disulfide bonds, wherein the four intramolecular disulfide bonds are between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38(such as Si-544) prepared by the method according to the second aspect of the present disclosure.

[0025] Detailed description of the invention

[0026] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. One and three letter amino acid abbreviations used herein correspond to IUPAC nomenclature (see e.g. European Journal of Biochemistry, 13829-37, 1984).

[0027] Method of folding and disulfide bond forming

[0028] According to a first aspect, the present disclosure relates to a method for preparing compound A comprising or consisting of the amino acid sequence SEQ ID No.: 1 , the method comprising the steps of: a) providing compound C comprising or consisting of amino acid sequence SEQ ID No.: 2; b) providing an aqueous medium comprising i) an oxidizing agent, and ii) a buffer system; and c) contacting the compound C provided in step a) with the aqueous medium provided in step b) to provide compound A. The inventors of the present disclosure have surprisingly found that the yield of the process of folding and oxidizing can be considerably improved by providing an aqueous medium comprising a buffer system and an oxidizing agent. In this, not only the yield is increased but also the process is more stable, more reproducible, and offers improved controlling than oxidizing using atmospheric oxygen.

[0029] In other words, the present disclosure in a first aspect relates to a method for preparing compound A comprising or consisting of the amino acid sequence SEQ ID No.: 1 , and wherein compound A has four intramolecular disulfide bonds, wherein the four intramolecular disulfide bonds are between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38; the method comprising the steps of: a) providing compound C comprising or consisting of amino acid sequence SEQ ID No.: 2, wherein compound C has no intramolecular disulfide bonds; b) providing an aqueous medium comprising i) an oxidizing agent, and II) a buffer system; and c) contacting the compound C provided in step a) with the aqueous medium provided in step b) to provide compound A.

[0030] According to a preferred embodiment of the present disclosure, the method according to the first aspect provides for a higher yield of compound A comprising or consisting of the amino acid sequence SEQ ID No.: 1 .

[0031] According to a preferred embodiment of the present disclosure, the method according to the first aspect yields compound A comprising or consisting of the amino acid sequence SEQ ID No.: 1 in an amount of at least about 30 % (n / n) in relation to the amount of substance of compound C provided in step a). According to another preferred embodiment of the present disclosure, the method according to the first aspect yields compound A comprising or consisting of the amino acid sequence SEQ ID No.: 1 in an amount of at least about 35 % (n / n) in relation to the amount of substance of compound C provided in step a). According to a further preferred embodiment of the present disclosure, the method according to the first aspect yields compound A comprising or consisting of the amino acid sequence SEQ ID No.: 1 in an amount of at least about 45 % (n / n) in relation to the amount of substance of compound C provided in step a). According to a yet further preferred embodiment of the present disclosure, the method according to the first aspect yields compound A comprising or consisting of the amino acid sequence SEQ ID No.: 1 in an amount of at least about 50 % (n / n) in relation to the amount of substance of compound C provided in step a). According to a most preferred embodiment of the present disclosure, the method according to the first aspect yields compound A comprising or consisting of the amino acid sequence SEQ ID No.: 1 in an amount of at least about 55 % (n / n) in relation to the amount of substance of compound C provided in step a). Providing an aqueous medium

[0032] According to a preferred embodiment of the present disclosure, the buffer system is selected from the group consisting of an acetate buffer, an ammonium acetate buffer, an ammonium triflate buffer, an ammonium carbonate buffer, a phosphate buffer, a guanidine HCI buffer, and mixtures thereof.

[0033] The inventors of the present disclosure have surprisingly found that the selection of buffer system improves the yield of peptide folding and disulfide bond formation. As shown in example 2, the selection of the buffer system may improve the yield of the folding and oxidizing method. This may be due to the difference of solubility of compound A in different buffers which may influence the folding of the peptides in solution.

[0034] According to a preferred embodiment of the present disclosure, the buffer system is an ammonium acetate buffer or an ammonium triflate buffer. According to a further preferred embodiment of the present disclosure, the buffer system is an ammonium acetate buffer.

[0035] As shown in Example 5, the inventors of the present disclosure surprisingly found that the yield of a method of folding and disulfide bond formation / oxidation can be considerably improved when using an ammonium based buffer system, in particular an ammonium acetate buffer.

[0036] According to a preferred embodiment of the present disclosure, the aqueous medium comprises the buffer system in a concentration in the range of from about 4.0 mol / L to about 10.0 mol / L. According to another preferred embodiment of the present disclosure, the aqueous medium comprises the buffer system in a concentration in the range of from about 4.0 mol / L to about 9.0 mol / L. According to another preferred embodiment of the present disclosure, the aqueous medium comprises the buffer system in a concentration in the range of from 5.0 mol / L to about 8.0 mol / L. According to a further preferred embodiment of the present disclosure, the aqueous medium comprises the buffer system in a concentration in the range yet more preferably of from about 5.0 mol / L to about 7.0 mol / L. According to a further preferred embodiment of the present disclosure, the aqueous medium comprises the buffer system in a concentration in the range of from about 5.5 mol / L to about 6.5 mol / L. According to a still further preferred embodiment of the present disclosure, the aqueous medium comprises the buffer system in a concentration in the range of from about 5.8 mol / L to about 6.2 mol / L. According to a most preferred embodiment of the present disclosure, the aqueous medium comprises the buffer system in a concentration of about 6.0 mol / L.

[0037] As shown in Example 6, the inventors of the present disclosure have surprisingly found that the concentration of buffer in the above range improve the yield of the folding and oxidation method according to the present disclosure. Peptide folding is highly sensitive to pH. Proper folding requires a stable pH environment because the ionization states of amino acid side chains affect the peptide's secondary and tertiary structures. Incorrect pH can lead to misfolding or aggregation. If the concentration of buffer is too low, the pH is not sufficiently stable. For peptides containing cysteine residues, controlled oxidation to form disulfide bonds is essential. A buffer of suitable concentration provides the necessary environment to maintain a balance between oxidized and reduced forms of cysteine.

[0038] According to preferred embodiment of the present disclosure, the pH of the aqueous medium is in the range of from about 7.5 to about 9.5. According to another preferred embodiment of the present disclosure, the pH of the aqueous medium is in the range of from about 7.8 to about 9.0. According to another preferred embodiment of the present disclosure, the pH of the aqueous medium is in the range of from about 8.0 to about 9.0. According to another preferred embodiment of the present disclosure, the pH of the aqueous medium is in the range of from about 8.2 to about 8.8. According to a further preferred embodiment of the present disclosure, the pH of the aqueous medium is in the range about 8.3 to about 8.7. According to a still further preferred embodiment of the present disclosure, the pH of the aqueous medium is in the range of from about 8.4 to about 8.6. According to a most preferred embodiment of the present disclosure, the pH of the aqueous medium is about 8.5.

[0039] As shown in Example 3, the pH of the solution plays a crucial role for the solubility of the peptide and hence for peptide folding and oxidation method according to the present disclosure. The peptide (compound A) needs to be in a buffer at a specific pH to ensure the cysteine residues are correctly positioned to form disulfide bonds. The inventors of the present disclosure have surprisingly found that slightly basic conditions as disclosed hereinabove are particularly advantageous for the correct folding of the peptides and hence for successful formation of disulfide bonds thus resulting in a high yield of compound A.

[0040] According to a preferred embodiment of the present disclosure, the pH of the aqueous medium is adjusted. According to a preferred embodiment of the present disclosure, the pH of the aqueous medium is adjusted with aqueous ammonia. According to another preferred embodiment of the present disclosure, the pH of the aqueous medium is adjusted with aqueous ammonia, wherein the concentration of ammonia in said aqueous ammonia is in the range of from about 15 % (VAX) to about 30 % (V / V). According to another preferred embodiment of the present disclosure, the pH of the aqueous medium is adjusted with aqueous ammonia, wherein the concentration of ammonia in said aqueous ammonia is in the range of from about 20 % (V / V) to about 30 % (V / V). According to another preferred embodiment of the present disclosure, the pH of the aqueous medium is adjusted with aqueous ammonia, wherein the concentration of ammonia in said aqueous ammonia is in the range of from about 22 % (V / V) to about 28 % (V / V). According to another preferred embodiment of the present disclosure, the pH of the aqueous medium is adjusted with aqueous ammonia, wherein the concentration of ammonia in said aqueous ammonia is in the range of from about 24 % (V / V) to about 26 % (V / V). According to a further preferred embodiment of the present disclosure, the pH of the aqueous medium is adjusted with aqueous ammonia, wherein the concentration of ammonia in said aqueous ammonia is about 25 % (V / V).

[0041] According to a preferred embodiment of the present disclosure, the oxidizing agent is selected from the group consisting of cystine, glutathione disulfide (GSSG), cystamine, the cyclic disulfide of dithiothreitol (DTT), CuSC , KatFeCNe], Na2S40e, and a mixture thereof. According to a further preferred embodiment of the present disclosure, the oxidizing agent is cystine.

[0042] As shown in Example 7, the inventors of the present disclosure have surprisingly found that the selection of the oxidizing agent may improve the yield of the folding and oxidation reaction. In particular, cystine is suitable for mediating the oxidation reaction and formation of the desired disulfide bonds in compound A.

[0043] According to a preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of at least about 4.0 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of at least about 4.3 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of at least about 4.5 ep. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of at least about 4.8 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of at least about 4.9 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a further preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of at least about 5.0 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a most preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of at least about 5.1 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a).

[0044] According to a preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of about 15 eq. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of about 13 eq. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of about 11 ep. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a further preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of about 10 ep. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a further preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of about 8 eq. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to yet further preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of about 7 eq. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a most preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount of about 6 eq. or less; in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a).

[0045] According to a preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount in the range of from about 4.0 eq. to about 15 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount in the range of from about 4.3 eq. to about 13 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount in the range of from about 4.5 eq. to about 11 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a further preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount in the range of from about 4.8 eq. to about 10 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a further preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount in the range of from about 4.9 eq. to about 8 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a yet further preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount in the range of from about 5.0 eq. to about 7 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a).

[0046] According to a most preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount in the range of from about 5.1 eq. to about 6 eq. in relation to the amount of compound C comprising or consisting of the amino acid sequence SEQ ID No.: 2 provided in step a). According to a most preferred embodiment of the present disclosure, the aqueous medium comprises the oxidizing agent in an amount in the range most preferably of from about 5.2 eq. to about 5.3 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a).

[0047] As shown in Example 7, the inventors have found that the above amounts of oxidizing agent improve the yield of the oxidizing reaction according to the present disclosure. It is understood that the oxidizing reaction of the present disclosure requires the formation of four disulfide bonds. Therefore, if less than 4 eq. of oxidizing agent are added, the reaction is not complete. If the amount of oxidizing agent is too high, undesired side reactions may occur.

[0048] According to a preferred embodiment of the present disclosure, the aqueous medium provided in step b) further comprises a reducing agent.

[0049] According to a preferred embodiment of the present disclosure, the reducing agent is selected from the group consisting of cysteine, glutathione (GSH), cystemine, tris(2- carboxyethyl)phosphine (TCEP), [3-mercaptoethanol, Dithiothreitol (DTT), and mixtures thereof.

[0050] According to a preferred embodiment of the present disclosure, when the oxidizing agent is cystine, the reducing agent is cysteine. According to another preferred embodiment of the present disclosure, when the oxidizing agent is glutathione disulfide (GSSG), the reducing agent is glutathione (GSH). According to another preferred embodiment of the present disclosure, when the oxidizing agent is cystamine, the reducing agent is cystemine. According to another preferred embodiment of the present disclosure, when the oxidizing agent is cyclic disulfide of dithiothreitol (DTT), the reducing agent is dithiothreitol (DTT). According to another preferred embodiment of the present disclosure, when the oxidizing agent is CuSO4, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP). According to another preferred embodiment of the present disclosure, when the oxidizing agent is K3[FeCNe], the reducing agent is p-mercaptoethanol. According to another preferred embodiment of the present disclosure, when the oxidizing agent is Na2S4O6, the reducing agent is Na2SO3.

[0051] According to a further preferred embodiment of the present disclosure, the oxidizing agent is cystine and the reducing agent is cysteine.

[0052] According to a preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of at least about 3.0 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of at least about 4.0eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of at least about 5.0 ep. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of at least about 6.0 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a further preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of at least about 7.0 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a yet further preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of at least about 8.0 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a most preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of at least about 9.0 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a).

[0053] According to another preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of about 30 eq. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of about 25 eq. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of about 20 ep. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of about 17 ep. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a further preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of about 15 eq. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a yet further preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of even more preferably about 13 eq. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a most preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of about 11 eq. or less in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a).

[0054] According to another preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount in the range of from about 3.0 eq. to about 30 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of in the range of from about 4.0 eq. to about 25 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of in the range of from about 5.0 eq. to about 20 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to another preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of in the range of from about 6.0 eq. to about 17 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). According to a further preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of in the range of from about 7.0 eq. to about 15 eq. in relation to the amount of compound C comprising or consisting of the amino acid sequence SEQ ID No.: 2 provided in step a). According to a yet further preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of in the range of from about 8.0 eq. to about 13 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence

[0055] SEQ ID No.: 2 provided in step a). According to a most preferred embodiment of the present disclosure, the aqueous medium provided in step b) comprises a reducing agent in an amount of in the range of from about 9.0 eq. to about 11 .0 eq. in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a).

[0056] Providing compound C comprising or consisting of amino acid sequence SEQ ID No.: 2

[0057] The method according to the first aspect of the present disclosure comprises a step of providing compound C comprising or consisting of amino acid sequence SEQ ID No.: 2.

[0058] Compound C comprising or consisting of the amino acid sequence SEQ ID No.: 2 may be prepared by any method known to the skilled person. A method for preparing the compound C comprising or consisting of the amino acid sequence SEQ ID No.: 2 is disclosed in WO 2015 / 169901 A1 . For instance, the compound C comprising or consisting of the amino acid sequence SEQ ID No.: 2 may be prepared by solid-phase peptide synthesis (SPPS). Alternatively, compound C comprising or consisting of the amino acid sequence SEQ ID No.: 2 may be prepared by expression in cells.

[0059] According to another preferred embodiment of the present disclosure, step a) comprises a step of dissolving compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 in water to provide an aqueous solution of compound C.

[0060] The inventors of the present disclosure have found that the compound C as disclosed herein can be added to the aqueous medium according to the present disclosure in a more controlled manner. Thereby, it is avoided that the temperature is considerably increased upon adding the compound C to the aqueous medium. Therefore, less undesired side reactions occur which in turn improves the yield of the folding and disulfide bond forming / oxidation reaction according to the present disclosure.

[0061] According to another preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of at least about 0.5 g / L. According to another preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of at least about 1 .0 g / L. According to another preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of at least about 1 .5 g / L. According to a further preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of at least about 1 .7 g / L. According to a further preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of at least about 1 .8 g / L. According to a yet further preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of at least about 1.9 g / L. According to a most preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of at least about 2.0 g / L; and / or According to a preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of about 6.0 g / L or less. According to another preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of about 5.0 g / L or less. According to another preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of about 4.0 g / L or less. According to a further preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of about 3.0 g / L or less. According to a further preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of about 2.5 g / L or less. According to a yet further preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of about 2.2 g / L or less. According to a most preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration of about 2.1 g / L or less.

[0062] According to another preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration in the range of from about 0.5 g / L to about 6.0 g / L. According to another preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration in the range of from about 1 .0 g / L to about 5.0 g / L. According to another preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration in the range of from about 1 .5 g / L to about 4.0 g / L. According to another preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration in the range of from about 1 .7 g / L to about 3.0 g / L. According to a further preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration in the range of from about 1 .8 g / L to about 2.5 g / L. According to a yet further preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration in the range of from about 1 .9 g / L to about 2.2 g / L. According to a yet further preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration in the range of from about 1 .9 g / L to about 2.1 g / L. According to a most preferred embodiment of the present disclosure, the aqueous solution of compound C comprises compound C in a concentration in the range of from about 2.0 g / L to about 2.1 g / L.

[0063] The inventors of the present disclosure have surprisingly found that compound C can be conveniently added into the aqueous medium according to the present disclosure provided in step b). Thereby, the compound C can be added to the aqueous medium at a rate so that the temperature does not increase significantly. Thus, undesired side-reactions are prevented and the overall yield of compound A is increased.

[0064] According to another preferred embodiment of the present disclosure, the ratio between the volume of the aqueous solution of compound C provided in step a) and the volume of the aqueous medium provided in step b) is in the range of from about 0.2 to about 5. According to another preferred embodiment of the present disclosure, the ratio between the volume of the aqueous solution of compound C provided in step a) and the volume of the aqueous medium provided in step b) is in the range of from about 0.4 to about 4 . According to another preferred embodiment of the present disclosure, the ratio between the volume of the aqueous solution of compound C provided in step a) and the volume of the aqueous medium provided in step b) is in the range of from about 0.5 to about 3. According to a further preferred embodiment of the present disclosure, the ratio between the volume of the aqueous solution of compound C provided in step a) and the volume of the aqueous medium provided in step b) is in the range of from about 0.5 to about 2. According to a further preferred embodiment of the present disclosure, the ratio between the volume of the aqueous solution of compound C provided in step a) and the volume of the aqueous medium provided in step b) is in the range of from about 0.7 to about 1 .5. According to a yet further preferred embodiment of the present disclosure, the ratio between the volume of the aqueous solution of compound C provided in step a) and the volume of the aqueous medium provided in step b) is in the range of from about 0.8 to about 1 .2. According to a yet further preferred embodiment of the present disclosure, the ratio between the volume of the aqueous solution of compound C provided in step a) and the volume of the aqueous medium provided in step b) is in the range of from about 0.9 to about 1 .1 . According to a most preferred embodiment of the present disclosure, the ratio between the volume of the aqueous solution of compound C provided in step a) and the volume of the aqueous medium provided in step b) is about 1 .

[0065] Contacting the compound C with the aqueous medium

[0066] According to another preferred embodiment of the present disclosure, step c) is performed by adding the compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a) into the aqueous medium provided in step b). According to a preferred embodiment of the present disclosure, compound C is slowly added into the aqueous medium provided in step b). According to another preferred embodiment of the present disclosure, compound C is added into the aqueous medium provided in step b) in a stepwise manner. According to another preferred embodiment of the present disclosure, compound C is added into the aqueous medium provided in step b) in a stepwise manner.

[0067] According to a preferred embodiment of the present disclosure, step c) is performed at a temperature of more than about 20 °C. According to another preferred embodiment of the present disclosure, step c) is performed at a temperature of at least about 22 °C. According to a further preferred embodiment of the present disclosure, step c) is performed at a temperature of at least about 25 °C. According to another preferred embodiment of the present disclosure, step c) is performed at a temperature of at least about 26 °C. According to a further preferred embodiment of the present disclosure, step c) is performed at a temperature of at least about 27 °C. According to a yet further preferred embodiment of the present disclosure, step c) is performed at a temperature of at least about 28 °C. According to a most preferred embodiment of the present disclosure, step c) is performed at a temperature of at least about 29 °C.

[0068] According to a preferred embodiment of the present disclosure, step c) is performed at a temperature of less than about 45 °C. According to a preferred embodiment of the present disclosure, step c) is performed at a temperature of about 40 °C or less. According to another preferred embodiment of the present disclosure, step c) is performed at a temperature of about 38 °C or less. According to a further preferred embodiment of the present disclosure, step c) is performed at a temperature of about 35 °C or less. According to a yet further preferred embodiment of the present disclosure, step c) is performed at a temperature of 33 °C or less. According to a yet further preferred embodiment of the present disclosure, step c) is performed at a temperature of about 32 °C or less. According to a most preferred embodiment of the present disclosure, step c) is performed at a temperature of about 31 °C or less.

[0069] According to another preferred embodiment of the present disclosure, step c) is performed at a temperature of in the range of from more than about 20 °C to less than about 45 °C. According to another preferred embodiment of the present disclosure, step c) is performed at a temperature of in the range of from about 22 °C to about 40 °C. According to another preferred embodiment of the present disclosure, step c) is performed at a temperature of in the range of from about 25 °C to about 38 °C. According to another preferred embodiment of the present disclosure, step c) is performed at a temperature of in the range of from about 26 °C to about 35 °C. According to a further preferred embodiment of the present disclosure, step c) is performed at a temperature of in the range of from about 27 °C to about 33 °C. According to a yet further preferred embodiment of the present disclosure, step c) is performed at a temperature of in the range of from about 28 °C to about 32 °C. According to a most preferred embodiment of the present disclosure, step c) is performed at a temperature of in the range of from about 29 °C to about 31 °C.

[0070] If the temperature is lower than disclosed above, the speed of the reaction is reduced. If the temperature is higher than disclosed above, the compound C and / or compound A may denaturate which reduces the reaction yield.

[0071] According to a preferred embodiment of the present disclosure, step c) comprises adding compound C provided in step a) to the aqueous medium provided in step b) over a course of about 10 min or more. According to another preferred embodiment of the present disclosure, step c) comprises adding compound C provided in step a) to the the aqueous medium provided in step b) over a course of about 20 min or more. According to a further preferred embodiment of the present disclosure, step c) comprises adding compound C provided in step a) to the aqueous medium provided in step b) over a course of about 30 min or more.

[0072] According to a preferred embodiment of the present disclosure, step c) comprises adding compound C provided in step a) to the aqueous medium provided in step b) over a course of about 180 min or less. According to another preferred embodiment of the present disclosure, step c) comprises adding compound C provided in step a) to the the aqueous medium provided in step b) over a course of about 120 min or less. According to a further preferred embodiment of the present disclosure, step c) comprises adding compound C provided in step a) to the aqueous medium provided in step b) over a course of about 90 min or less.

[0073] According to a preferred embodiment of the present disclosure, step c) comprises adding compound C provided in step a) to the aqueous medium provided in step b) over a course of from about 10 min to about 180 min. According to another preferred embodiment of the present disclosure, step c) comprises adding compound C provided in step a) to the the aqueous medium provided in step b) over a course of from about 20 min to about 120 min. According to a further preferred embodiment of the present disclosure, step c) comprises adding compound C provided in step a) to the aqueous medium provided in step b) over a course of from about 30 min to about 90 min.

[0074] In other words, the compound C as disclosed herein is added in a portion wise or stepwise manner into the composition comprising the oxidizing agent and a buffer system as disclosed herein. Thus, only small portions of the compound C are added subsequently.

[0075] If the time of addition is shorter than the above, the temperature within the reaction mixture may increase thus resuling in the formation of side products and reduced reaction yield. If the time of addition is longer than the above, the addition is very slow which results in an inefficient process. Furthermore, the reaction time is increased which results in more side reactions and lower yield. As is shown in example 6 of the present disclosure, the above times of addition are optimal to achieve a high yield.

[0076] According to a preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is at least about 1 h. According to another preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is at least about 1 .5 h. According to another preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is at least about 2.0 h. According to another preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is at least about 2.5 h. According to further preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is at least about 3.0 h. According to a yet further preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is at least about 3.5 h. According to a most preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is at least about 4.0 h.

[0077] According to a preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is about 10 h or less. According to another preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is about 9 h or less. According to another preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is about 8 h or less. According to a further preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is about 7 h or less. According to another preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is about 6 h or less. According to a yet further preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is 5 h or less. According to a most preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is about 4 h or less.

[0078] According to another preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is in the range of from about 1 .0 h to about 10 h. According to another preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is in the range of from 1 .5 h to about 9 h. According to another preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is in the range of from about 2.0 h to about 8 h. According to another preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is in the range of from about 2.5 h to about 7 h. According to a further preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is in the range of from about 3.0 h to about 6 h. According to a yet further preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is in the range of from about 3.5 h to about 5 h. According to a most preferred embodiment of the present disclosure, step c) is performed for a reaction time, wherein the reaction time is about 4 h.

[0079] Further steps

[0080] According to a preferred embodiment of the present disclosure, the method further comprises of a step d) of quenching after step c) by adjusting the pH to 3.0 or less, preferably to about 2.5.

[0081] According to a preferred embodiment of the present disclosure, step d) is performed at a temperature of about 10 °C or less. According to another preferred embodiment of the present disclosure, step d) is performed at a temperature of about 5 °C or less. According to a further preferred embodiment of the present disclosure, step d) is performed at a temperature of about 5 °C.

[0082] According to a preferred embodiment of the present disclosure, adjusting the pH in step d) comprises adding trifluoroacetic acid (TFA).

[0083] According to a preferred embodiment of the present disclosure, the method further comprises a step e) of filtering. According to a preferred embodiment of the present disclosure, the method further comprises a step e) of filtering through a filter having a pore size of about 0.45 pm ± 0.2 pm.

[0084] According to a preferred embodiment of the present disclosure, the method further comprises a step f) of purifying by preparative chromatography.

[0085] Compound

[0086] A compound A comprising or consisting of amino acid sequence SEQ ID No.: 1 , wherein compound A has four intramolecular disulfide bonds, wherein the four intramolecular disulfide bonds are between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38, prepared by the method according to the first aspect of the present disclosure

[0087] Definitions and general embodiments

[0088] As used herein, the term “compound A” refers to a peptide comprising or consisting of an amino acid sequence SEQ ID No.: 1 , that is represented by the following formula:

[0089] H-Thr1-lle2-lle3-Asn4-Val5-Lys6-Cys7-Thr8-Ser9-Pro10-Lys11-Gln12-Cys13-Leu14-Pro15- Pro16-Cys17-Lys18-Ala19-Gln20-Thr21-Gly22-Cys23-Pro24-Tyr25-Gly26-Lys27-Cys28-Met29-Asn30- Arg31-Lys32-Cys33-Lys34-Cys35-An s36-Arg37-Cys38-O H wherein compound A has four intramolecular disulfide bonds, wherein the four intramolecular disulfide bonds are between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38, and wherein Cys38may be L-cysteine or D-cysteine, preferably L-cysteine. According to a preferred embodiment of the present disclosure, compound A consists of an amino acid sequence SEQ ID No.: 1 as disclosed herein. As used herein, the terms “compound A consisting of an amino acid sequence SEQ ID No.: 1 ”, “Si-544”, and “cgtx-544” are used interchangeably.

[0090] Si-544 as disclosed herein has a molecular formula C173H297N55O49S9 (net) and the same molecular mass of 4212.2 g / mol (net).

[0091] As used herein, the term “compound C” refers to a peptide comprising or consisting of an amino acid sequence SEQ ID No.: 2, that is represented by the following formula:

[0092] H-Thr1-lle2-lle3-Asn4-Val5-Lys6-Cys7-Thr8-Ser9-Pro10-Lys11-Gln12-Cys13-Leu14-Pro15- Pro16-Cys17-Lys18-Ala19-Gln20-Thr21-Gly22-Cys23-Pro24-Tyr25-Gly26-Lys27-Cys28-Met29-Asn30- Arg31-Lys32-Cys33-Lys34-Cys35-Ans36-Arg37-Cys38-OH, wherein compound C has no intramolecular disulfide bonds, and wherein Cys38may be L-cysteine or D-cysteine, preferably L-cysteine.

[0093] According to a preferred embodiment of the present disclosure, compound C consists of an amino acid sequence SEQ ID No.: 2 as disclosed herein. As used herein, the terms “compound C consisting of an amino acid sequence SEQ ID No.: 2", “Si-544-linear", and “cgtx-544-linear” are used interchangeably.

[0094] Si-544-linear as disclosed herein has a molecular formula C173H297N55O49S9 (net) and the same molecular mass of 4220.2 g / mol (net).

[0095] As used herein, the term “pharmaceutically acceptable salt" refers to a salt of the compound having amino acid sequence of SEQ ID No.: 1 that retains the desired biological activity of the above-identified compound and include pharmaceutically acceptable acid addition salts. Suitable pharmaceutically acceptable acid addition salts of the compound having amino acid sequence of SEQ ID No.: 1 may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, trifluoroacetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic, and arylsulfonic acid.

[0096] As used herein, the term “oxyma” relates to ethyl cyanohydroxyiminoacetate, also known under CAS number 3849-21-6.

[0097] As used herein, the term “DIC” relates to N,N- diisopropylcarbodiimide, also known under CAS number 693-13-0. As used herein, the term “TBTU” relates to 2-(1 / 7-benzotriazole-1 -yl)-1 ,1 ,3,3- tetramethylaminium tetrafluoroborate, also known under CAS number 125700-67-6.

[0098] As used herein, the term “DIPEA" relates to A / ,A / -diisopropylethylamine, also known as Hunig’s base and under CAS number 7087-68-5.

[0099] As used herein, the term “Fmoc-protected” refers to a compound in which at least one, preferably all, amine groups are protected with a fluorenylmethoxycarbonyl protection group. In other words, an Fmoc-protected compound comprises the following structural element:

[0100] As used herein, the term “buffer system” relates to a pair of weak acid and its conjugate base. Buffer systems are usually added to water or an aqueous solution to form buffer solutions and are responsible for the buffering seen in these solutions. These buffer systems may be added to an aqueous medium in order to stabilize the pH of said aqueous medium upon addition of acid or base or during the course of a chemical reaction. In other words, a buffer system as used herein is a mixture of weak acid and its conjugated base that maintains the pH of an aqueous medium or composition at a specific value or in a specific range.

[0101] Non-limiting examples of buffer systems are formate buffer (formic acid / formate anion), acetate buffer (acetic acid / acetate anion), citrate buffer (citric acid / citrate anion), ammonium buffer (NH^ / NHa), ammonium acetate buffer (acetic acid&NHzT / acetate anion&NHa), ammonium triflate buffer (trifluoroacetic acid&NH4+ / triflate anion&NHa), ammonium carbonate buffer (HaCCh&NH^ / HCO3 &CO32&NH3), phosphate buffer (H3PO4 / H2PO47HPO427PO43), fumarate buffer (fumaric acid / fumarate anion), carbonate buffer (H2CO3 / HCO3 / CO32), sulfite buffer (HSO3 / SO32), maleate buffer (maleic acid / maleate anion), malonate buffer (malonic acid / malonate anion), HEPES buffer (2-[4-(2- Hydoxyethyl)piperazin-1-yl]ethane-1 -sulfonic acid / anion thereof), MES buffer (2-(N- morpholino)ethanesulfonic acid / anion thereof), TRIS buffer (Tris(hydroxymethyl) aminommethan / cation thereof), propionate buffer (propionic acid / propionate anion), succinate buffer (succinic acid / succinate anion), glycolate buffer (glycolic acid / glycolate anion), gluconate buffer (gluconic acid / gluconate anion), lactate buffer (lactic acid / lactate anion), tartrate buffer (tartaric acid / tartrate), glutamate buffer (glutamic acid / glutamate anion), barbitone buffer (HCI / barbitone anion), barbiturate buffer (barbitone / barbitone anion), borax buffer (NazBziOy / NaOH), boric acid buffer (HaBC / NazB^?), glycine buffer (HCI / glycine or glycine / NaOH), and mixtures thereof.

[0102] As used herein, a “weak acid” is a substance that partially dissociates when it is dissolved water. In solution there is an equilibrium between the acid “HA” and the products of dissociation H+and A . As such, a weak acid has a pKa at 25 °C in the range of from 3 to 10. Non-limiting examples of weak acids are formic acid, acetic acid, citric acid, NH4+, H2PO4-, fumaric acid, HCC ’, H2CO3, HSO3; maleic acid, malonic acid, HEPES, MES, TRIS, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, tartaric acid, glutamic acid, alkyl sulfonic acid, arylsulfonic acid, and the like.

[0103] As used herein, the concentration of the buffer system in the aqueous medium may be determined by dividing the amount of substance (such as the amount of substance of weak acid HA and the amount of substance of the conjugated base A ) by the volume of aqueous medium: fl_ „ n(A") + n(HA) concentration of buffer system = — - - - -

[0104] Vfaqueous medium) wherein n(HA) is the amount of substance of weak acid dissolved in the aqueous medium during preparation, n(A ) is the amount of substance of conjugated base dissolved in the aqueous medium during preparation, and V(aqueous medium) is the volume of the aqueous medium.

[0105] For example, if an acetate buffer is prepared from acetic acid and sodium acetate, the above formula may be adapted to n (acetate) + n (acetic acid) concentration of acetate buffer = - - - - - - -

[0106] V(aqueous medium) wherein n(acetate) is the amount of substance of acetate anions dissolved in the aqueous medium during preparation, n(acetic acid) is the amount of substance of acetic acid dissolved in the aqueous medium during preparation, and V(aqueous medium) is the volume of the aqueous medium.

[0107] In an alternative embodiment, the concentration of the buffer system in the aqueous medium may be determined by dividing the amount of substance of the buffer agent by the volume of aqueous medium: n (buffer agent) concentration of buffer system = — - - - -

[0108] V(aqueous medium) wherein n(buffer agent) is the amount of substance of the buffer agent (such as ammonium triflate, ammonium acetate, ammonium carbonate, or the like) dissolved in the aqueous medium during preparation, and V(aqueous medium) is the volume of the aqueous medium. For the case that the buffer system comprises more than one weak acid and / or more than one conjugated base, the concentration of buffer system is determined on the basis of the amount of substance added during preparation, not taking into account further amounts of acid or base added for adjusting the pH. In other words, the amount of substance added during preparation is the amount dissolved in the aqueous medium before adjusting the pH.

[0109] Alternatively, the concentration of a buffer system comprising more than one weak acid (such as NHzF and acetic acid) and / or more than one conjugated base (such as NH3 and acetate) may be calculated taking into account only one of said pairs of weak acid and weak base.

[0110] If the buffer system is an ammonium acetate buffer, the above formula may be adapted to n (ammonium acetate) concentration of ammonium acetate buffer = — - - - - —

[0111] V(aqueous medium) wherein n(ammonium acetate) is the amount of substance of ammonium acetate added during preparation of the buffer system without pH adjustment, and V(aqueous medium) is the volume of the aqueous medium.

[0112] As used herein, the amount of oxidizing agent comprised in the aqueous medium is given in “eq.” in relation to the amount of compound C comprising or consisting of the amino acid sequence SEQ ID No.: 2 provided in step a) unless specified otherwise. As used herein, the amount of oxidizing agent in “eq.” is calculated as n (oxidizing agent) amount of oxidizing agent in eq. = — - - — — ■ 1 eq.

[0113] 5 5 4n(compound C)4wherein “n(oxidizing agent)” is the amount of substance of oxidizing agent comprised in the aqueous medium provided in step b), and “n(compound C)" is the amount of substance of compound C comprising or consisting of the amino acid sequence SEQ ID No.: 2 provided in step a).

[0114] As used herein, the amount of reducing agent comprised in the aqueous medium is given in “eq.” in relation to the amount of compound C comprising or consisting of the amino acid sequence SEQ ID No.: 2 provided in step a) unless specified otherwise. As used herein, the amount of reducing agent in “eq.” is calculated as n (reducing agent) amount of oxidizing agent in eq. = — - — — — ■ 1 eq.

[0115] 5 5 4n(compound C)4wherein “n(reducing agent)” is the amount of substance of oxidizing agent comprised in the aqueous medium provided in step b), and “n(compound C)” is the amount of substance of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present disclosure, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising”. If a group is defined to comprise at least a certain number of embodiments herein, this is also to be understood to disclose a group, which preferably consists only of these embodiments. Furthermore, if a composition is defined using the term “comprising”, it may additionally comprise other elements not explicitly listed, however, not further amounts of an element listed. As such, if, e.g., an aqueous medium comprises cystine in an amount 5.25 eq. based on the amount of compound C provided in step a), said aqueous medium may comprise elements other than cystine, such as cysteine, however, not additional amounts of cystine thereby exceeding the amount of 10 eq..

[0116] The term “about" and “approximately” in conjunction with a numerical value refers to normal deviations of said numerical value. It is to be understood that the terms “about” and “approximately” can each mean a deviation of ± 10 %, preferably ± 5 %, more preferably ± 2.5 % of said numeric value as indicated.

[0117] As used herein, the term “room temperature” means 25 °C.

[0118] As used in this specification and in the intended claims, the singular forms of “a” and “an” also include the respective plurals unless the context clearly dictates otherwise.

[0119] Assay (I)

[0120] The yield of the method of the present disclosure was calculated from a chromatogram measured by UHPLC:

[0121] UHPLC is carried out on a Dionex Ultimate 3000 RS (Dionex / Thermo Scientific) equipped with a ACQUITY CSH C18 UPLC column (1 .7 pm; 2.1 x 150 mm; Waters Corp.) maintained at 80 °C. For elution, a gradient of eluent A (0.15 % (V / V) of pentafluorobutyric acid (PFBA) and 0.15 % (VAX) of heptafluorobutyric acid (HFBA) in H2O / ACN (99:1 VAX)) and eluent B (0.08 % (V / V) of pentafluorobutyric acid (PFBA) and 0.08 % (V / V) of heptafluorobutyric acid (HFBA) in H2O / ACN (5:95 V / V)) was used at the following program:

[0122] The flow rate is kept at 0.37 mL / min. Peaks are detected at 220 nm. Injection volume is 1 .0 to 10.0 pL. The amount of Si-544 in the reaction solution was determined from peak area and using a purified Si-544 as internal standard.

[0123] Assay (II)

[0124] The conversion of the method of the present disclosure was calculated from a chromatogram measured by UHPLC:

[0125] UHPLC is carried out on a Dionex Ultimate 3000 RS (Dionex / Thermo Scientific) equipped with a Waters ACQUITY UPLC BEH C18 UPLC column (1 .7 pm; 2.1 x 150 mm; Waters Corp.) maintained at 50 °C. For elution, a gradient of eluent A (0.05 % (V / V) of trifluoroactic acid (TFA) and in H2O / ACN (99:1 V / V)) and eluent B (0.05 % (V / V) of trifluoroacetic acid (TFA) in ACN was used at the following program:

[0126] The flow rate is kept at 0.4 mL / min. Peaks are detected at 220 nm. Injection volume is 1.0.

[0127] The amount of Si-544 in the reaction solution was determined from peak area and using a purified Si-544 as internal standard.

[0128] Examples

[0129] In the following section, particular examples illustrating various embodiments and aspects of the invention are presented. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description.

[0130] All chemicals and materials used herein were obtained from commercial suppliers unless stated otherwise. All reactions were carried out at room temperature unless indicated otherwise.

[0131] Abbreviations

[0132] The following abbreviations may appear in the example section:

[0133] ACN acetonitrile

[0134] AcOH acetic acid approx. approximately aq. aqueous c concentration

[0135] CR Characterized range DMF N,N-dimethylformamide

[0136] GMP good manufacturing practice

[0137] L Liter

[0138] LC liquid chromatography

[0139] LOD limit of detection

[0140] LOQ limit of quantification

[0141] M molar; mol / L m mass max. maximum min minute min. minimum

[0142] PFPA Pentafluoropropionic acid

[0143] SPPS solid-phase peptide synthesis temp. temperature

[0144] TFA Trifluoroacetic acid

[0145] UHPLC ultra high-performance liquid chromatography

[0146] UV ultraviolet

[0147] V volume vs versus

[0148] Example 1 - Comparative

[0149] Peptide folding and oxidation was performed in accordance with example 7 of WO 2015 / 169901 A1.

[0150] In essence, the method was based on bond formation in phosphate buffer at pH 8.2 in the presence of atmospheric oxygen.

[0151] For that, Si-544-linear was dissolved at a concentration of 0.2 mg / mL in a 20 mM phosphate buffer under air atmosphere for 72 h.

[0152] HPLC analysis of the reaction mixture displayed that the peptide exists as a mixture of folded and partially folded variants.

[0153] The conversion of Si-544-linear as determined according to assay (II) was 19 %.

[0154] Example 2

[0155] An aqueous 9.0 M NH4OAC solution (500 mL per 1 .0 g Si-544-linear (TFA salt, purified)) in a vessel equipped with a mechanical stirrer was prepared and the temperature was set to 30 °C. The pH of the solution was adjusted to 6.8 by addition of 6.0 M HCI (aq.).

[0156] In parallel, a solution of 5.0 eq. cystine in 1 .0 M aq. HCI (40 mg / mL of cystine) was prepared and half of said cystine solution was added to the NH4OAc solution. The other half of the cystine solution was mixed with the aq. solution of Si-544-linear (TFA salt, purified) (c = 2 g / L).

[0157] 10 eq. of cysteine was added to the residual NH4OAC solution containing cystine as solid in one portion and the peptide solution was slowly added to the NH4OAC solution comprising cystine and cysteine over a course of 20 min. During addition of the peptide, precipitation was observed. After addition of the peptide solution was completed, the pH of the resulting solution was increased to 8.5 using 25 % (V / V) NH4OH (aq.) solution and the reaction mixture was stirred for further 2 h.

[0158] The reaction mixture was next quenched by TFA addition to p ~ 2 and purified by preparative HPLC.

[0159] They yield as determined according to assay (I) was 31 %.

[0160] Example 3

[0161] In view of the susceptibility of Si-544-linear to precipitation, the inventors of the present disclosure investigated into the solubility of Si-544 in different buffer systems and at different pH.

[0162] The solubility of 2.0 g / L Si-544-linear (TFA salt, purified) in aqueous media comprising different buffer systems at different molarities and pH was investigated by visual testing. In that, a clear solution indicates sufficient solubility of Si-544-linear, whereas a visual turbid mixture indicates that Si-544-linear is not soluble in the aqueous medium.

[0163] The pH was adjusted with AcOH or aq. 25 % (V / V) NH4OH, respectively. Visual inspections were made after 30 and 60 min at ambient temperature.

[0164] Table 1 Solubility test at 2.0 g / L si-544 (linear, TFA salt, purified) in NH4OAC at different molarity and pH.

[0165] Generally, Si-544-linear shows some differences in the solubility in different buffer systems, both as regards pH and molarity. However, a pH of more than 6 was favorable for solubility.

[0166] In particular, Si-544-linear has a particularly high solubility in ammonium acetate buffer systems, both in high and low molarities.

[0167] Example 4 Based on the solubility tests performed in Example 3, the influence of different buffer systems on the yield of the folding and disulfide bond forming / oxidation of Si-544-linear to Si-544 was studied. Experiments were carried out as indicated in Example 2 unless specified otherwise.

[0168] Additionally, the mode of contacting Si-544-linear with the aqueous medium was modified in that the aqueous medium was added to the peptide solution (Table 2, entries 3 and 4).

[0169] The yield was determined by assay (I): Table 2 Yield of reaction in different buffer systems

[0170] * After complete addition of cystine solution

[0171] In a first set of experiments (Table 2, entries 1 and 2), the NH4OAC concentration was reduced from 4.5 M to 1 .0 M. A 1 .0 M aq. NH4OAc concentration led to lower yield for the formation of si-544 at pH 6.8 compared to the 4.5 M conditions.

[0172] Next, the mode of addition was investigated (Table 2, entries 3 and 4) for potential scale-up. Upon adding the solution of cysteine / cystine to the solution of Si-544-linear in a 4.5 M NH4OAC solution yield of si-544 decreased (Table 2, entries 1 and 3) at pH = 6.8. However, by increasing the pH to 8.5 with the same mode of addition, the oxidation took place with a similar yield as in Example 2.

[0173] Example 5

[0174] Based on the solubility tests performed in Example 3 and insights on mode of addition and pH of Example 4, the influence of different buffer systems on the yield of the folding and disulfide bond forming / oxidation of Si-544-linear to Si-544 was studied. For that, different aqueous media were prepared as indicated in Table 3. Said aqueous media each comprise cystine (10 eq.) and cystine (5.25 eq.). A solution comprising Si-544-linear (2.0 g / L before addition into the aqueous medium; 1 .0 g / L after complete addition of peptide solution into the aqueous medium) was added to the aqueous medium over a course of several minutes. Temperature was kept at 30 °C. The yield was determined by assay (I):

[0175] Table 3 Yield of reaction in different buffer systems

[0176] * buffer system and molartity before addition of peptide solution

[0177] #buffer system and molartity after complete addition of peptide solution

[0178] Changing the buffer from NH4OAC to NaOAc ot NH4TFA did not significantly change the reaction yield (Table 3, entries 1 , 2 and 3).

[0179] However, using a NH4HCO3 buffer that may be commonly used in cysteine / cystine oxidation in the prior art showed poor yield. A lower molarity (0.1 M and 0.5 M after complete addition) was chosen in order to avoid excessive exothermic reaction and bubbling during the quench with TFA at the end of the reaction and thereby keeping the reaction under control.

[0180] In conclusion, the inventors of the present disclosure found that the buffer system is to be used in high molarity to enable optimal folding and oxidation of Si-544-linear peptide.

[0181] Example 6

[0182] Based on the solubility tests in Example 3 and the promising results for NH4OAC buffer systems in Example 5, the influence of the concentration of the buffer system was investigated.

[0183] For that, different aqueous media were prepared. Said aqueous media (pH 8.5) each comprise cystine (10 eq.) and cystine (5.25 eq.). A solution comprising Si-544-linear (2.0 g / L before addition into the aqueous medium; 1 .0 g / L after complete addition of peptide solution into the aqueous medium) was added to the aqueous medium over a course of several minutes (Table 4, entries 4-7) or the aqueous medium was added to the solution comprising Si-544-linear (2.0 g / L) over a course of several minutes (Table 4, entries 1 -3). Temperature was kept at 30 °C. The yield was determined by assay (I): Table 4 Investigation of the buffer concentration and mode of addition for the oxidation of si-544-linear

[0184] * buffer system and molartity before addition of peptide solution

[0185] # buffer system and molartity after complete addition of peptide solution

[0186] As shown in Table 4, adding the solution of Si-544-linear to the aqueous medium was generally beneficial for the yield of oxidative folding. The yield was comparable over a large scope of molarities.

[0187] Example 7 After assessing the buffer agents for the oxidative folding, the effect of different oxidizing agents and reducing agents was investigated.

[0188] For that, different aqueous media were prepared. Said aqueous media (pH 8.5;

[0189] 6.0 M NH4OAC before addition of peptide solution; 3.0 M NH4OAC after complete addition of peptide solution) each comprise an oxidizing agent and optionally a reducing as indicated in Table 5. A solution comprising Si-544-linear (2.0 g / L before addition into the aqueous medium; 1 .0 g / L after complete addition of peptide solution into the aqueous medium) was added to the aqueous medium over a course of 30 minutes. Temperature was kept at 30 °C. The yield was determined by assay (I):

[0190] Table 5 Investigation of the reagent system for the oxidation of si-544-linear

[0191] Glutathione reduced (GSH)ZGIutathione oxidized (GSSG) or Cysteamine / Cystamine, commonly used as alternative redox systems to cysteine / cystine, lead to slightly lower yield than the cystine based oxidation reactions. Interestingly, the use of cystine as sole oxidant gave higher yield for the oxidation of Si-544-linear.

[0192] This result indicates that the oxidation of the linear peptide precursor does not essentially need a reducing agent to occur. Increasing the equivalents of cystine from 5.25 eq. to 10 eq. led to comparable yield.

[0193] In conclusion, the cystine shows superior yield in comparison to other oxidants. In addition, cystine as sole oxidant is more cost effective compared to a mixture of cysteine and cystine, thus becoming a valid point in manufacturing.

[0194] Example 8

[0195] Additionally, the influence of the concentration of Si-544-linear added into the aqueous medium was investigated.

[0196] For that, an aqueous medium (6.0 M NH4OAC before addition of peptide solution;

[0197] 3.0 M NH4OAC after complete addition of peptide solution; pH 8.5, 5.25 eq. of cystine) was prepared. A solution comprising Si-544-linear as indicated in Table 6 was added to the aqueous medium over a course of 30 minutes. Temperature was kept at 30 °C. The yield was determined by assay (I): Table 6 Investigation of the peptide concentration for the oxidation of Si-544-linear

[0198] * Concentration of peptide solution before addition into the aqueous medium

[0199] #Concentration of peptide in solution after complete addition into the aqueous medium

[0200] A peptide concentration of 1.0 g / L after complete addition of the peptide solution into the aqueous medium seems to be optimal for the reaction outcome.

[0201] Example 9

[0202] Additionally, the influence of the temperature during folding and oxidation was investigated.

[0203] For that, an aqueous medium (6.0 M NH4OAC before addition of peptide solution;

[0204] 3.0 M NH4OAC after complete addition of peptide solution , pH 8.5, 5.25 eq. of cystine) was prepared. A solution comprising Si-544-linear (2.0 g / L before addition into the aqueous medium; 1 .0 g / L after complete addition of peptide solution into the aqueous medium) was added to the aqueous medium over a course of 30 minutes. Temperature was varied as indicated in Table 7. The yield was determined by assay (I) after 1 h:

[0205] Table 7 Investigation of the temperature for the oxidation of si-544-linear

[0206] For the oxidation of si-544-linear, the yield is increased at a temperature of more than

[0207] 20 °C: At 20 °C, the calculated yield drops to 38% and reaches up to 57 % at 30 °C.

[0208] Embodiment

[0209] 1 . A method for preparing compound A comprising or consisting of amino acid sequence SEQ ID No.: 1, the method comprising the steps of: a) Providing compound C comprising or consisting of amino acid sequence SEQ ID No.: 2; b) Providing an aqueous medium comprising i) an oxidizing agent, and ii) a buffer system; and c) Contacting the compound C provided in step a) with the aqueous medium provided in step b) to provide compound A.

[0210] 2. The method according to embodiment 1 , wherein the buffer system comprises selected from the group consisting of an acetate buffer, an ammonium acetate buffer, an ammonium triflate buffer, an ammonium carbonate buffer, phosphate buffer, a guanidine HCI buffer, and mixtures thereof, preferably wherein the buffer system is an ammonium acetate buffer or an ammonium triflate buffer, more preferably an ammonium acetate buffer.

[0211] 3. The method according to any one of embodiments 1 or 2, wherein the aqueous medium comprises the buffer system in a concentration in the range of from about 4.0 mol / L to about 10.0 mol / L, preferably of from about 4.0 mol / L to about 9.0 mol / L, more preferably of from 5.0 mol / L to about 8.0 mol / L, yet more preferably of from about 5.0 mol / L to about 7.0 mol / L, even more preferably of from about 5.5 mol / L to about 6.5 mol / L, still more preferably of from about 5.8 mol / L to about 6.2 mol / L, most preferably of about 6.0 mol / L.

[0212] 4. The method according to any one of embodiments 1 to 3, wherein the pH of the aqueous medium is in the range of from about 7.5 to about 9.5, preferably in the range of from about 7.8 to about 9.0, more preferably in the range of from about 8.0 to about 9.0, yet more preferably in the range of from about 8.2 to about 8.8, even more preferably in the range of from about 8.3 to about 8.7, still more preferably in the range of from about 8.4 to about 8.6, most preferably wherein the pH of the aqueous medium is about 8.5.

[0213] 5. The method according to embodiment 4, wherein the pH of the aqueous medium is adjusted with aqueous ammonia, preferably wherein the concentration of ammonia in said aqueous ammonia is in the range of from about 15 % (V / V) to about 30 % (V / V), more preferably in the range of from about 20 % (V / V) to about 30 % (V / V), yet more preferably in the range of from about 22 % (V / V) to about 28 % (V / V), still more preferably in the range of from about 24 % (V / V) to about 26 % (V / V), most preferably wherein the concentration of ammonia in said aqueous ammonia is about 25 % (V / V). The method according to any one of embodiments 1 to 5, wherein the oxidizing agent is selected from the group consisting of cystine, glutathione disulfide (GSSG), cystamine, and a mixture thereof, preferably wherein the oxidizing agent is cystine. The method according to any one of embodiments 1 to 6, wherein the aqueous medium comprises the oxidizing agent in an amount a) of at least about 4.0 eq., preferably at least about 4.3 eq., more preferably at least about 4.5 ep., yet more preferably at least about 4.8 eq., still more preferably at least about 4.9 eq., even more preferably at least about 5.0 eq., most preferably at least about 5.1 eq.; and / or b) of about 15 eq. or less, preferably about 13 eq. or less, more preferably about 11 ep. or less, yet more preferably about 10 ep. or less, still more preferably about 8 eq. or less, even more preferably about 7 eq. or less, most preferably about 6 eq. or less; and / or c) in the range of from about 4.0 eq. to about 15 eq., preferably of from about 4.3 eq. to about 13 eq., more preferably of from about 4.5 eq. to about 11 eq., yet more preferably of from about 4.8 eq. to about 10 eq., still more preferably of from about 4.9 eq. to about 8 eq., even more preferably of from about 5.0 eq. to about 7 eq., most preferably of from about 5.1 eq. to about 6 eq. each in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a). The method according to any one of embodiments 1 to 7, wherein the aqueous medium further comprises a reducing agent, preferably wherein the reducing agent is selected from the group consisting of cysteine, glutathione (GSH), cystemine, and mixtures thereof, more preferably wherein a) when the oxidizing agent is cystine, the reducing agent is cysteine; or b) when the oxidizing agent is glutathione disulfide (GSSG), the reducing agent is glutathione (GSH), or c) when the oxidizing agent is cystamine, the reducing agent is cystemine. The method according to claim 8, wherein the oxidizing agent is cystine and the reducing agent is cysteine. The method according to any one of embodiments 8 or 9, wherein the aqueous medium comprises the reducing agent in an amount a) of at least about 3.0 eq., preferably at least about 4.0eq., more preferably at least about 5.0 ep., yet more preferably at least about 6.0 eq., still more preferably at least about 7.0 eq., even more preferably at least about 8.0 eq., most preferably at least about 9.0 eq.; and / or b) of about 30 eq. or less, preferably about 25 eq. or less, more preferably about 20 ep. or less, yet more preferably about 17 ep. or less, still more preferably about 15 eq. or less, even more preferably about 13 eq. or less, most preferably about 11 eq. or less; and / or c) in the range of from about 3.0 eq. to about 30 eq., preferably of from about 4.0 eq. to about 25 eq., more preferably of from about 5.0 eq. to about 20 eq., yet more preferably of from about 6.0 eq. to about 17 eq., still more preferably of from about 7.0 eq. to about 15 eq., even more preferably of from about 8.0 eq. to about 13 eq., most preferably of from about 9.0 eq. to about 11 .0 eq. each in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a).

[0214] 11 . The method according to any one of embodiments 1 to 10, wherein step a) comprises a step of dissolving compound C comprising or consisting of the amino acid sequence SEQ ID No.: 2 in water to provide an aqueous solution of compound C.

[0215] 12. The method according to embodiment 11 , wherein the aqueous solution of compound C comprises compound C in a concentration a) of at least about 0.5 g / L, preferably at least about 1 .0 g / L, more preferably at least about 1 .5 g / L, yet more preferably at least about 1.7 g / L, still more preferably at least about 1 .8 g / L, even more preferably at least about 1 .9 g / L, most preferably at least about 2.0 g / L; and / or b) of about 6.0 g / L or less, preferably about 5.0 g / L or less, more preferably about 4.0 g / L or less, yet more preferably about 3.0 g / L or less, still more preferably about 2.5 g / L or less, even more preferably about 2.2 g / L or less, most preferably about 2.1 g / L or less; and / or c) in the range of from about 0.5 g / L to about 6.0 g / L, preferably of from about 1 .0 g / L to about 5.0 g / L, more preferably of from about 1 .5 g / L to about 4.0 g / L, yet more preferably of from about 1.7 g / L to about 3.0 g / L, still more preferably of from about 1 .8 g / L to about 2.5 g / L, even more preferably of from about 1 .9 g / L to about 2.2 g / L or of from about 1.9 g / L to about 2.1 g / L, most preferably of from about 2.0 g / L to about 2.1 g / L.

[0216] 13. The method according to any one of embodiments 1 to 12, wherein step c) is performed by adding the compound C comprising or consisting of amino acid sequence

[0217] SEQ ID No.: 2 provided in step a) into the aqueous medium provided in step b).

[0218] 14. The method according to any one of embodiments 1 to 13, wherein step c) is performed at a temperature a) of more than about 20 °C, preferably at least about 22 °C, more preferably at least about 25 °C, yet more preferably at least about 26 °C, still more preferably at least about 27 °C, even more preferably at least about 28 °C, most preferably at least about 29 °C; and / or b) of less than about 45 °C, preferably about 40 °C or less, more preferably about

[0219] 38 °C or less, yet more preferably about 35 °C or less, still more preferably 33 °C or less, even more preferably about 32 °C or less, most preferably about 31 °C or less; and / or c) in the range of from more than about 20 °C to less than about 45 °C, preferably of from about 22 °C to about 40 °C, more preferably of from about 25 °C to about

[0220] 38 °C, yet more preferably of from about 26 °C to about 35 °C, still more preferably of from about 27 °C to about 33 °C, even more preferably of from about 28 °C to about 32 °C, most preferably of from about 29 °C to about 31 °C.

[0221] 15. The method according to any one of embodiments 1 to 14, wherein step c) is performed for a reaction time, wherein the reaction time is a) at least about 1 h, preferably at least about 1 .5 h, more preferably at least about 2.0 h, yet more preferably at least about 2.5 h, even more preferably at least about 3.0 h, still more preferably at least about 3.5 h, most preferably at least about 4.0 h; and / or b) about 10 h or less, preferably about 9 h or less, more preferably about 8 h or less, yet more preferably about 7 h or less, still more preferably about 6 h or less, even more preferably 5 h or less, most preferably about 4 h or less; and / or c) in the range of from about 1 .0 h to about 10 h, preferably of from 1 .5 h to about 9 h, more preferably of from about 2.0 h to about 8 h, yet more preferably of from about

[0222] 2.5 h to about 7 h, still more preferably of from about 3.0 h to about 6 h, even more preferably of from about 3.5 h to about 5 h, most preferably for about 4 h.

[0223] 16. The method according to any one of embodiments 1 to 15, wherein the buffer system is diluted during step c) to a concentration in the range of from about 2.0 mol / L to about 5.0 mol / L, preferably of from about 2.0 mol / L to about 4.5 mol / L, more preferably of from

[0224] 2.5 mol / L to about 4.0 mol / L, yet more preferably of from about 2.5 mol / L to about

[0225] 3.5 mol / L, even more preferably of from about 2.7 mol / L to about 3.3 mol / L, still more preferably of from about 2.9 mol / L to about 3.1 mol / L, most preferably of about

[0226] 3.0 mol / L.

[0227] 17. The method according to any one of embodiments 1 to 16, wherein the method further comprises of a step d) of quenching after step c) by adjusting the pH to 3.0 or less, preferably to about 2.5.

[0228] 18. The method according to embodiment 16, wherein step d) is performed at a temperature of about 10 °C or less, preferably at about 5 °C. The method according to embodiments 16 or 17, wherein adjusting the pH comprises adding trifluoroacetic acid (TFA). The method according to any one of embodiments 1 to 18, wherein the method further comprises a step e) of filtering, preferably filtering through a filter having a pore size of about 0.45 pm ± 0.2 pm. The method according to any one of embodiments 1 to 19, wherein the method further comprises a step f) of purifying by preparative chromatography. A compound A comprising or consisting of amino acid sequence SEQ ID No.: 1 prepared by the method according to any one of embodiments 1 to 20.

Claims

Claims1 . A method for preparing compound A comprising or consisting of amino acid sequence SEQ ID No.: 1 , wherein compound A has four intramolecular disulfide bonds, wherein the four intramolecular disulfide bonds are between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38, the method comprising the steps of: a) Providing compound C comprising or consisting of amino acid sequence SEQ ID No.: 2, wherein compound C has no intramolecular disulfide bonds; b) Providing an aqueous medium comprisingI) an oxidizing agent, and ii) a buffer system; and c) Contacting the compound C provided in step a) with the aqueous medium provided in step b) to provide compound A.

2. The method according to claim 1 , wherein the buffer system is selected from the group consisting of an acetate buffer, an ammonium acetate buffer, an ammonium triflate buffer, an ammonium carbonate buffer, phosphate buffer, a guanidine HCI buffer, and mixtures thereof, preferably wherein the buffer system is an ammonium acetate buffer or an ammonium triflate buffer, more preferably an ammonium acetate buffer.

3. The method according to any one of claims 1 or 2, wherein the aqueous medium comprises the buffer system in a concentration in the range of from about 4.0 mol / L to about 10.0 mol / L, preferably of from about 4.0 mol / L to about 9.0 mol / L, more preferably of from 5.0 mol / L to about 8.0 mol / L, yet more preferably of from about 5.0 mol / L to about 7.0 mol / L, even more preferably of from about 5.5 mol / L to about 6.5 mol / L, still more preferably of from about 5.8 mol / L to about 6.2 mol / L, most preferably of about 6.0 mol / L.

4. The method according to any one of claims 1 to 3, wherein the pH of the aqueous medium is in the range of from about 7.5 to about 9.5, preferably in the range of from about 7.8 to about 9.0, more preferably in the range of from about 8.0 to about 9.0, yet more preferably in the range of from about 8.2 to about 8.8, even more preferably in the range of from about 8.3 to about 8.7, still more preferably in the range of from about 8.4 to about 8.6, most preferably wherein the pH of the aqueous medium is about 8.5.

5. The method according to any one of claims 1 to 4, wherein the oxidizing agent is selected from the group consisting of cystine, glutathione disulfide (GSSG), cystamine, and a mixture thereof, preferably wherein the oxidizing agent is cystine.

6. The method according to any one of claims 1 to 5, wherein the aqueous medium comprises the oxidizing agent in an amounta) of at least about 4.0 eq., preferably at least about 4.3 eq., more preferably at least about 4.5 ep., yet more preferably at least about 4.8 eq., still more preferably at least about 4.9 eq., even more preferably at least about 5.0 eq., most preferably at least about 5.1 eq.; and / or b) of about 15 eq. or less, preferably about 13 eq. or less, more preferably about 11 ep. or less, yet more preferably about 10 ep. or less, still more preferably about 8 eq. or less, even more preferably about 7 eq. or less, most preferably about 6 eq. or less; and / or c) in the range of from about 4.0 eq. to about 15 eq., preferably of from about 4.3 eq. to about 13 eq., more preferably of from about 4.5 eq. to about 11 eq., yet more preferably of from about 4.8 eq. to about 10 eq., still more preferably of from about 4.9 eq. to about 8 eq., even more preferably of from about 5.0 eq. to about 7 eq., most preferably of from about 5.1 eq. to about 6 eq. each in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a).

7. The method according to any one of claims 1 to 6, wherein the aqueous medium further comprises a reducing agent, preferably wherein the reducing agent is selected from the group consisting of cysteine, glutathione (GSH), cystemine, and mixtures thereof, more preferably wherein a) when the oxidizing agent is cystine, the reducing agent is cysteine; or b) when the oxidizing agent is glutathione disulfide (GSSG), the reducing agent is glutathione (GSH), or c) when the oxidizing agent is cystamine, the reducing agent is cystemine.

8. The method according to claim 7, wherein the aqueous medium comprises the reducing agent in an amount a) of at least about 3.0 eq., preferably at least about 4.0eq., more preferably at least about 5.0 ep., yet more preferably at least about 6.0 eq., still more preferably at least about 7.0 eq., even more preferably at least about 8.0 eq., most preferably at least about 9.0 eq.; and / or b) of about 30 eq. or less, preferably about 25 eq. or less, more preferably about 20 ep. or less, yet more preferably about 17 ep. or less, still more preferably about 15 eq. or less, even more preferably about 13 eq. or less, most preferably about 11 eq. or less; and / or c) in the range of from about 3.0 eq. to about 30 eq., preferably of from about 4.0 eq. to about 25 eq., more preferably of from about 5.0 eq. to about 20 eq., yet more preferably of from about 6.0 eq. to about 17 eq., still more preferably of from about7.0 eq. to about 15 eq., even more preferably of from about 8.0 eq. to about 13 eq., most preferably of from about 9.0 eq. to about 11 .0 eq. each in relation to the amount of compound C comprising or consisting of amino acid sequence SEQ ID No.: 2 provided in step a).

9. The method according to any one of claims 1 to 8, wherein step a) comprises a step of dissolving compound C comprising or consisting of the amino acid sequenceSEQ ID No.: 2 in water to provide an aqueous solution of compound C.

10. The method according to claim 9, wherein the aqueous solution of compound C comprises compound C in a concentration a) of at least about 0.5 g / L, preferably at least about 1 .0 g / L, more preferably at least about 1 .5 g / L, yet more preferably at least about 1.7 g / L, still more preferably at least about 1 .8 g / L, even more preferably at least about 1 .9 g / L, most preferably at least about 2.0 g / L; and / or b) of about 6.0 g / L or less, preferably about 5.0 g / L or less, more preferably about 4.0 g / L or less, yet more preferably about 3.0 g / L or less, still more preferably about 2.5 g / L or less, even more preferably about 2.2 g / L or less, most preferably about 2.1 g / L or less; and / or c) in the range of from about 0.5 g / L to about 6.0 g / L, preferably of from about 1 .0 g / L to about 5.0 g / L, more preferably of from about 1 .5 g / L to about 4.0 g / L, yet more preferably of from about 1.7 g / L to about 3.0 g / L, still more preferably of from about1 .8 g / L to about 2.5 g / L, even more preferably of from about 1 .9 g / L to about 2.2 g / L or of from about 1.9 g / L to about 2.1 g / L, most preferably of from about 2.0 g / L to about 2.1 g / L.11 . The method according to any one of claims 1 to 10, wherein step c) is performed at a temperature a) of more than about 20 °C, preferably at least about 22 °C, more preferably at least about 25 °C, yet more preferably at least about 26 °C, still more preferably at least about 27 °C, even more preferably at least about 28 °C, most preferably at least about 29 °C; and / or b) of less than about 45 °C, preferably about 40 °C or less, more preferably about38 °C or less, yet more preferably about 35 °C or less, still more preferably 33 °C or less, even more preferably about 32 °C or less, most preferably about 31 °C or less; and / or c) in the range of from more than about 20 °C to less than about 45 °C, preferably of from about 22 °C to about 40 °C, more preferably of from about 25 °C to about38 °C, yet more preferably of from about 26 °C to about 35 °C, still more preferablyof from about 27 °C to about 33 °C, even more preferably of from about 28 °C to about 32 °C, most preferably of from about 29 °C to about 31 °C.

12. The method according to any one of claims 1 to 11 , wherein the buffer system is diluted during step c) to a concentration in the range of from about 2.0 mol / L to about 5.0 mol / L, preferably of from about 2.0 mol / L to about 4.5 mol / L, more preferably of from 2.5 mol / L to about 4.0 mol / L, yet more preferably of from about 2.5 mol / L to about 3.5 mol / L, even more preferably of from about 2.7 mol / L to about 3.3 mol / L, still more preferably of from about 2.9 mol / L to about 3.1 mol / L, most preferably of about 3.0 mol / L.

13. The method according to any one of claims 1 to 12, wherein the method further comprises a step e) of filtering, preferably filtering through a filter having a pore size of about 0.45 pm ± 0.2 pm.

14. The method according to any one of claims 1 to 13, wherein the method further comprises a step f) of purifying by preparative chromatography.

15. A compound A comprising or consisting of amino acid sequence SEQ ID No.: 1 prepared by the method according to any one of claims 1 to 14.

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