Manufacture of disulfide bonded peptides

Abstract

Methods for the manufacture of disulfide bonded peptides are provided. These methods rely on the use of dehydroascorbic acid as oxidizing agent and are advantageous in that they are applicable to aggregation-prone peptides, reduce the formation of unwanted side products, avoid working with highly diluted samples of peptide and oxidizing agent, and thereby allow for economic as well as environmentally friendly production.

Description

[0001] Bachem Holding AG

[0002] Manufacture of disulfide bonded peptides

[0003] The present invention generally relates to the field of peptide manufacture at an industrial or laboratory scale. Improved methods for the chemical synthesis of disulfide bonded peptides are disclosed. In one aspect, the present invention is directed to methods of effectively forming intramolecular disulfide bonds while suppressing the formation of intermolecular disulfide bonds and other unwanted side reactions.

[0004] Chemical peptide synthesis in general is well-known in the art. Two standard approaches can be distinguished, namely Liquid Phase Peptide Synthesis (LPPS) and Solid Phase Peptide Synthesis (SPPS). A variation of these approaches is tag- assisted peptide synthesis, where a solubility modifying tag, also referred to a solubility modifying support or pseudo-solid phase protecting group, is covalently bound to the peptide chain. The tag is used to facilitate isolation of the peptide, e.g. by precipitation, while allowing to perform the synthesis reactions in solution. Hence, tag-assisted peptide synthesis may be considered a type of support-assisted peptide synthesis. In addition, hybrid synthetic approaches can be utilized, where fragments are first synthesized by one of the above techniques and then joined together using the other approach. In a variation of this scheme, chemically synthesized peptide fragments may be joined together by enzymatic reactions. All of the above approaches have in common that there is no translation of nucleic acid encoded sequence information into a peptide sequence, as is the case with biological, e.g. recombinant, peptide synthesis.

[0005] During chemical peptide synthesis, formation of the peptide bond between the alpha amino group of the first amino acid and the alpha carboxyl group of a second amino acid should be favored over unintended side reactions. This is commonly achieved by the use of “permanent” and “temporary” protecting groups. The latter are used to block the alpha amino group of the second amino acid during the coupling step, thereby avoiding, e.g., peptide bond formation between multiple copies of the second amino acid. The former are used to block reactive amino acid side chains and the C-terminal carboxyl group of the growing peptide chain and are typically only removed at the end of the entire synthesis.

[0006] Intermolecular disulfide bonds are formed between two sulfhydryl groups of the same molecule, typically between two cysteine side chains. This normally involves reaction with an oxidizing agent. Depending on the oxidizing agent and the sulfhydryl protecting group used during peptide synthesis, the sulfhydryl groups need to be deprotected before disulfide bond formation.

[0007] Disulfide bond formation may be challenging, as it commonly gives rise to unwanted side reactions such as the oxidation of methionine, histidine, tyrosine and tryptophane moieties. Another unwanted side reaction is so-called overoxidation of sulfhydryl groups to yield (cysteine) sulfinic acid, (cysteine) sulfonic acid, and thiosulfinate species. In addition, the intramolecular reaction is to be favored over an intermolecular reaction, as the latter leads to the formation of high molecular weight impurities and loss of material.

[0008] Various approaches have been explored to optimize disulfide bond formation of chemically synthesized peptides:

[0009] One approach to large scale manufacture of disulfide bonded peptides is iodine oxidation of the crude peptide obtained, e.g., as a salt of trifluoroacetic acid (TFA) after cleavage from the SPPS resin. However, to suppress both the formation of disulfide bonded peptide oligomers and other side reactions, it is often necessary to work in highly diluted solutions or to use pseudo dilution for both the peptide and the oxidation reagent as described in W020202124777. This is, however, disadvantageous in that it is rather time consuming and yields the disulfide bonded peptide at fairly low concentrations, which leads to cumbersome further processing. In addition, side reactions due to the composition of the raw peptide may occur.

[0010] A second approach relies on oxidation on the SPPS resin directly after synthesis and before cleavage of the peptide from the resin. This has been reported, e.g. in US 5656721 using hydrogen peroxide and in US 20160096866 using Thallium (III) trifluoroacetate. The idea here is to suppress intermolecular disulfide bond formation, as each single chain is immobilized. However, this pseudo dilution approach by immobilization limits the maximal resin loading during SPPS, which is detrimental to process economy. In addition, there is the potential for side reactions, e.g. disulfide bond reduction and potential reshuffling, during the following, harsh deprotection and cleavage steps. The on-resin oxidation approach is particularly attractive if using iodine in combination with Acm or Trt protecting groups for the cysteine moieties, as no prior removal of the protecting groups is needed for disulfide bond formation. However, iodine in DMF is an aggressive chemical and not compatible with standard steel reactors.

[0011] Further attempts to suppress unwanted side reactions apply the pseudo dilution principle in that the oxidizing agent is immobilized to a solid support. For example, immobilized Ellman’s reagent is marketed under the trademark of CLEAR-OX™. Green and Bulaj (Protein & Peptide Letters, 2006, 13, 67-70) compared the use of CLEAR-OX™ with on-column refolding of purified conotoxin peptides comprising two or three disulfide bonds. Refolding was catalyzed by flushing a solid-phase extraction column, on which the peptide was adsorbed, with a refolding buffer comprising equimolar amounts of reduced and oxidized glutathione. Of note, the on- column refolding approach was characterized by a low concentration of the peptide on the column.

[0012] In the view of the prior art, there still a need for an improved method for disulfide bond formation: Ideally, such a method should generate product of good purity with high yield, the employed reagents should have a good safety profile and low environmental impact be non-toxic and easily available in large quantities at reasonable price, and the method would be suitable for use with standard large scale equipment and compatible with standard Fmoc-SPPS without a need for unusual protecting groups or specific deprotection protocols. Moreover, an improved method should be suitable for use with aggregation-prone peptides and exhibit good process economy. This latter goal requires the ability to work with rather concentrated peptide compositions.

[0013] Surprisingly, the present inventors found that the use of dehydroascorbic acid (DHA) may suppress the formation of disulfide-bonded oligomers of the peptide to be oxidized, thereby allowing for efficient disulfide bond formation without a need of highly diluting the peptide of interest. Moreover, the disulfide bonded peptide did not undergo side reactions with DHA, even when working with an excess of oxidation reagent. It is therefore possible to add the whole amount of DHA needed into the reaction mixture at once, without a need to titrate it in. In addition, the present inventors combined this approach with peptide purification, by carrying out the oxidation with DHA while the peptide is adsorbed to the stationary phase of a reversed phase chromatography column. This allowed to remove any impurities contained in the raw peptide, which may interfere with the disulfide bond formation, to further suppress the formation of disulfide bonded multimers, and to avoid precipitation of the disulfide bonded peptide, all while yielding a fairly concentrated solution of the disulfide bonded peptide in a buffered solution of choice. The process therefore offers unique improvements in process economy and is characterized by a favorable process mass intensity (PMI), in particular when dealing with peptides having a high propensity to aggregate.

[0014] Ascorbate is known as an important antioxidant in humans, which may alleviate oxidative stress by undergoing oxidation to dehydroascorbic acid (DHA). In human cells, DHA is recycled back to ascorbate by various cysteine compounds such as glutathione, homocysteine and various proteins including the glutaredoxin (Grx) system. The interaction of these compounds has been studied using in vitro model systems by various authors (e.g. Kouakou et al., Free Radical Biology and Medicine 141 (2019) 233-243; Arhuie et al., Rapid Commun Mass Spectrom. 2020;34: e8774; Saaranen et al, Antioxid. Redox Signal. 12, 15-25).

[0015] Beyond these studies on the cell biology of DHA, DHA is occasionally reported as a component of refolding buffers, e.g. in the oxidative refolding of recombinantly expressed antibody constructs in US20210395298. However, the inventors are not aware of prior art describing the use of DHA in oxidative disulfide bond formation for the manufacture of disulfide bonded peptides.

[0016] In general terms, the present invention provides, inter alia, a method for the manufacture of a peptide (Pox) comprising an intramolecular disulfide bond, comprising the following steps: a. Providing a composition comprising a chemically synthesized peptide (Pred), which has at least two free sulfhydryl groups; b. Combining the composition of step a with at least one equivalent of dehydroascorbic acid, relative to the total amount of free sulfhydryl groups contained in the composition of step a, so as to obtain an aqueous reaction composition (C1 ) having a pH of 3.0 to 8.0 and comprising dehydroascorbic acid and the peptide (Pred), wherein the peptide (Pred) is either present in this aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column at an initial amount of at least 0.05 g net peptide per cm2column diameter or is dissolved in the aqueous reaction composition (C1 ) at an initial concentration of at least 1 mg net peptide per ml, or wherein the initial amount of dehydroascorbic acid in the aqueous reaction composition (C1 ) is at least one equivalent, relative to the total amount of free sulfhydryl groups contained in the composition of step a; and c. Maintaining the aqueous reaction composition (C1 ) for a time sufficient to allow conversion of the peptide (Pred) to the disulfide bonded peptide (Pox).

[0017] In step c., the disulfide bonded peptide (Pox) is obtained.

[0018] In some embodiments, the present invention provides a method for the manufacture of a peptide (Pox) comprising an intramolecular disulfide bond, comprising the following steps: a. Providing a composition comprising a chemically synthesized peptide (Pred), which has at least two free sulfhydryl groups; b. Combining the composition of step a with at least one equivalent of dehydroascorbic acid, relative to the total amount of free sulfhydryl groups contained in the composition of step a, so as to obtain an aqueous reaction composition (C1 ) having a pH of 3.0 to 8.0 and comprising dehydroascorbic acid and the peptide (Pred), wherein the peptide (Pred) is either present in this aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column at an initial amount of at least 0.05 g net peptide per cm2column diameter or is dissolved in the aqueous reaction composition (C1 ) at an initial concentration of at least 1 mg net peptide per ml, or wherein the initial amount of dehydroascorbic acid in the aqueous reaction composition (C1 ) is at least one equivalent, relative to the total amount of free sulfhydryl groups contained in the composition of step a., and the final concentration of the net disulfide bonded peptide (Pox) in the aqueous reaction composition (C1 ) is at least 1 mg / ml after step c.; and c. Maintaining the aqueous reaction composition (C1 ) for a time sufficient to allow conversion of the peptide (Pred) to the disulfide bonded peptide (Pox), thereby obtaining the disulfide bonded peptide (Pox).

[0019] In some embodiments, the present invention provides a method for the manufacture of a peptide (Pox) comprising an intramolecular disulfide bond, comprising the following steps: a. Providing a composition comprising at least 1 g, preferably 100 to 50000 g, net weight of a chemically synthesized peptide (Pred), which has at least two free sulfhydryl groups; b. Combining the composition of step a with at least one equivalent of dehydroascorbic acid, relative to the total amount of free sulfhydryl groups contained in the composition of step a, so as to obtain an aqueous reaction composition (C1 ) having a pH of 3.0 to 8.0 and comprising dehydroascorbic acid and the peptide (Pred), wherein the peptide (Pred) is either present in this aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column at an initial amount of at least 0.05 g net peptide per cm2column diameter or is dissolved in the aqueous reaction composition (C1 ) at an initial concentration of at least 1 mg net peptide per ml, or wherein the initial amount of dehydroascorbic acid in the aqueous reaction composition (C1 ) is at least one equivalent, relative to the total amount of free sulfhydryl groups contained in the composition of step a; and c. Maintaining the aqueous reaction composition (C1 ) for a time sufficient to allow conversion of the peptide (Pred) to the disulfide bonded peptide (Pox), thereby obtaining the disulfide bonded peptide (Pox).

[0020] In some embodiments, the peptide (Pred) is either present in the aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column at an initial amount of at least 0.1 g, preferably 0.1 -1.5 g, net peptide per cm2column diameter or is dissolved in the aqueous reaction composition (C1 ) at an initial concentration of at least 3 mg, preferably 3-50 mg, net peptide per ml.

[0021] In some embodiments, the peptide (Pred) is either present in the aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column at an initial amount of at least 0.2 g, preferably 0.2-1.5 g, net peptide per cm2column diameter or is dissolved in the aqueous reaction composition (C1 ) at an initial concentration of at least 10 mg, preferably 10-50 mg, net peptide per ml.

[0022] In some embodiments, the initial amount of dehydroascorbic acid in the aqueous reaction composition (C1 ) is 1 to 10 equivalents relative to the total amount of free sulfhydryl groups contained in the composition of step a and the final concentration of the net disulfide bonded peptide (Pox) in the aqueous reaction composition (C1 ) is at least 3 mg / ml or at least 5 mg / ml, preferably 3-50 mg / ml or 5-50 mg / ml after step c.

[0023] In some embodiments, the initial amount of dehydroascorbic acid in the aqueous reaction composition (C1 ) is 1 to 10 equivalents relative to the total amount of free sulfhydryl groups contained in the composition of step a and the final concentration of the net disulfide bonded peptide (Pox) in the aqueous reaction composition (C1 ) is at least 10 mg / ml, preferably 10-50 mg / ml.

[0024] The method of the present invention may be used at an industrial scale. For example, the method may yield more than 1 g, in particular more than 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 g of net oxidized peptide. The method of the present invention is easily scalable and particularly suited for scale up in that the oxidizing agent is nonhazardous, nonvolatile, and can be easily dosed. Mixing of the reaction composition is not as critical as with oxidation agents such as iodine and hydrogen peroxide, because local maxima in DHA concentration are not expected to increase side product formation; moreover, the progress of oxidation can easily be followed by optical sensors. At a larger scale, the method may yield more than 150 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900g, 1 kg, 2 kg, 3 kg, 4 kg, 5 kg, 6 kg, 8 kg, 9 kg, 10 kg, 20 kg, 30 kg, 40 kg, or 50 kg of net disulfide bonded peptide. For example, the total amount of net disulfide bonded peptide (Pox) adsorbed to a stationary phase of a reversed phase liquid chromatography column obtained in step c. may be 1 -1000, 10-1000, 50-1000, 100-1000, 150-1000, 200- 1000, 1 -2000, 10-2000, 50-2000, 100-2000, 150-2000, 200-2000, 1 -3000, 10-3000, 50-3000, 100-3000, 150-3000, 200-3000, 1-4000, 10-4000, 50-4000, 100-4000, 150-4000, or 200-4000, 1 -30000, 10-30000, 50-30000, 100-30000, 150-30000, or 200-30000 g. As a further example, the amount of net disulfide bonded peptide (Pox) obtained in solution in step c may be 1 -1000, 10-1000, 50-1000, 100-1000, 150- 1000, 200-1000 g, 1 -5000, 10-5000, 50-5000, 100-5000, 150-5000, 200-5000 g, 1 - 5000, 10-5000, 50-5000, 100-5000, 150-5000, 200-5000 g, , 1 -10000, 10-10000, 50-10000, 100-10000, 150-10000, 200-10000 g, 1 -20000, 10-20000, 50-20000, 100-20000, 150-20000, 200-20000 g, 1 -30000, 10-30000, 50-30000, 100-30000, 150-30000, 200-30000 g, 1-40000, 10-40000, 50-40000, 100-40000, 150-40000, 200-40000 g, 1 -50000, 10-50000, 50-50000, 100-50000, 150-50000, or 200- 50000 g.

[0025] As used in the context of the present application:

[0026] - the expression “peptide (Pred)” is to be understood as shorthand for and synonymous with the expression “a chemically synthesized peptide (Pred), which has at least two free sulfhydryl groups”, which is in turn synonymous with “a chemically synthesized peptide, which has at least two free sulfhydryl groups”;

[0027] - the expression “peptide (Pox)” is to be understood as shorthand for and synonymous with the expression “a peptide (Pox) comprising at least one intramolecular disulfide bond”, which is in turn synonymous with the expression “a peptide comprising at least one intramolecular disulfide bond”; and

[0028] - the expression “aqueous reaction composition (C1 )” is to be understood as shorthand for and synonymous with the expression “an aqueous reaction composition (C1 ) as defined in step b of the method according to the invention”, which is in turn synonymous with “an aqueous reaction composition as defined in step b of the invention”.

[0029] In other words: The expression in brackets -i.e. (Pred), (Pox), and (C1 ), respectively- may be understood as a reference mark, which has no meaning by itself and is only used to facilitate reading.

[0030] As used herein and unless specified otherwise, the terms “about” and “around” in combination with a numeric value may indicate that this value may vary by plus or minus 10%.

[0031] As used herein and unless specified otherwise, the term “or” means “and / or”. In other words: the requirement “condition 1 or condition 2” is fulfilled if either “condition 1” is fulfilled, or if “condition 2” is fulfilled, or if “condition 1” and “condition 2” are fulfilled.

[0032] Unless specified otherwise, numeric values will be understood herein as rounded to the last decimal place indicated, as per common rounding rules. For example, the value of “10 g” is considered to encompass all values in the interval from 9.5 g to 10.4 g. As another example, the value “10.0 g” is considered to encompass all values in the interval from 9.95 g to 10.04 g.

[0033] As used herein, the term "peptide" and "polypeptide" may be understood interchangeably and relates to compounds, where at least two amino acids are covalently linked by an amide bond between a carboxyl group of one amino acid moiety and the amino group of another amino acid moiety. Typically, this bond is between the carboxyl group of a first alpha amino acid and the (alpha) amino group of a second alpha amino acid and is referred to as "peptide bond". As used herein, the term "amino acid" refers to an organic acid -i.e. a compound containing at least one carboxyl group (-COOH)-, which further contains at least one amino group (- NH2). Alpha amino acids (RCH(NH2)COOH) are the building blocks, from which peptides and proteins are predominantly constructed. However, in particular artificial peptides may contain building blocks other than alpha amino acids. A peptide may comprise between 2 and 100, preferably between 2 and 60 amino acid moieties.

[0034] As used herein, the name of a peptide is used to specify the peptide’s amino acid sequence. Generally, if the oxidation state of the peptide’s sulfhydryl groups is not specified, its default status will be meant. For example, the expressions “octreotide” or “octreotide peptide” normally refer to the disulfide bonded octreotide molecule. If the reduced state of a peptide’s sulfhydryl groups is to be specified for clarity, it may be referred to as “linear peptide”, “(SH) reduced peptide”, “sulfhydryl reduced peptide” “reduced peptide”, or similar. Likewise, the disulfide bonded state of a peptide’s sulfhydryl groups may be stressed by using the expressions “cyclic peptide”, “disulfide-bonded peptide”, “(SH) oxidized peptide”, “sulfhydryl oxidized peptide”, “oxidized peptide”, or similar.

[0035] The skilled practitioner will readily understand that the peptide (Pred) may be provided by any type of chemical synthesis, i.e. any type of synthesis in vitro, which does not rely on recombinant techniques, i.e. on enzymatic translation of nucleic- acid encoded information into a peptide sequence. In particular, chemical synthesis includes liquid-phase peptide synthesis, solid-phase peptide synthesis, tag-assisted peptide synthesis and hybrid approaches combining the above techniques. Moreover, as used herein, the expression “chemically synthesized peptide” comprises peptides, which are obtained by enzymatic ligation of peptide fragments produced by chemical synthesis and peptides obtained from a combination of chemical and recombinant synthesis. For example, a peptide, which has been recombinantly expressed and then been in vitro modified in its primary sequence or by modification to its side chains may be considered a “chemically synthesized peptide” herein. In one embodiment, the peptide (Pred) has been produced without a step of recombinant peptide synthesis.

[0036] In some embodiments, the peptide (Pred) comprises exactly two free sulfhydryl groups. In some embodiments, the peptide (Pox) comprises exactly one intramolecular disulfide bond. In some embodiments, the peptide (Pox) comprises exactly one intramolecular disulfide bond and is 6 to 100, preferably 6 to 60 amino acids in length.

[0037] In some embodiments, the peptide (Pox) and / or its reduced version (Pred) are prone to aggregation. Typical forms of aggregates are amorphous aggregates, ordered aggregates such as amyloid fibrils, and hydrogels. In some embodiments, the aggregation prone peptide comprises a hydrophobic modification such as an C4 to C24 alkyl moiety. For example, a saturated alkylamine, alkanol, carboxylic acid or dicarboxylic acid having 4 to 24 carbon atoms may be directly or indirectly attached to the peptide via an amide or ester bond. The point of attachment may be an amino group, carboxyl group, or a hydroxyl group either on the peptide itself (direct attachment) or on a linker moiety attached to the peptide chain (indirect attachment).

[0038] In some embodiments, the sequence of the aggregation prone peptide comprises one or more aggregation prone regions (APRs). APRs may comprise successive hydrophobic amino acids with high beta-sheet propensity and a low net charge. APRs may be identified based on prediction programs such as AggreProt 1.0 (Planas-lglesias et al., Nucleic Acids Res. 2024 Jul 5;52(W1 ):W159-W169). In some embodiments, the sequence of the aggregation prone peptide comprises one or more predicted aggregation prone regions (APRs) selected from the group consisting of NLSTCMLG, LSTC, LSTCM, STCML, TCMLG, AIGVGAP, AIGVG, IGVGA, GVGAP, ANFLVHSS, CNTAT, NTAT, ANFLVHSSNNFGAILS, ANFLV, NFLVH, FLVHS, LVHSS, NFGAI, FGAIL, GAILS, EFIAWLVK, EFIAW, FIAWL, IAWLV, AWLVK, FVQWLMN, FVQWL, VQWLMM, QWLMN (SEQ ID NOs: 7-35), and homologs thereof, where one amino acid has been replaced by an amino acid with similar hydrophobicity.

[0039] In some embodiments, the peptide (Pox) has at least 80%, 83%, 86%, 89%, 91 %, 94%, or 97% sequence identity with human amylin over the whole of the human amylin amino acid sequence [Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-GIn-Arg-Leu- Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-lle-Leu-Ser-Ser-Thr-Asn- Val-Gly-Ser-Asn-Thr-Tyr (SEQ ID NO:6)]. Additionally, the peptide (Pox) may comprise exactly one intramolecular disulfide bond and may be 30 to 50 amino acids in length. In some embodiments, the peptide (Pox) has at least 80%, 83%, 86%, 89%, 91 %, 94%, or 97% sequence identity with human amylin over the whole of the human amylin amino acid sequence [Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-GIn- Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-lle-Leu-Ser-Ser- Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr (SEQ ID NO:6)] and is 30 to 37, 31 to 37, 32 to 37, 33 to 37, 34 to 37, 35 to 37, or 36 to 37 amino acids long, respectively.

[0040] In some embodiments, the peptide (Pox) has at least 80%, 83%, 86%, 89%, 91 %, 94%, or 97% sequence identity with pramlintide over the whole of the pramlintide amino acid sequence [Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-GIn-Arg-Leu-Ala-Asn- Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-lle-Leu-Ser-Ser-Thr-Asn-Val-Gly- Ser-Asn-Thr-Tyr (SEQ ID NO:4)]. Additionally, the peptide (Pox) may comprise exactly one intramolecular disulfide bond and may be 30 to 50 amino acids in length. In some embodiments, the peptide (Pox) has at least 80%, 83%, 86%, 89%, 91 %, 94%, or 97% sequence identity with pramlintide over the whole of the pramlintide amino acid sequence [Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-GIn-Arg-Leu-Ala-Asn- Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-lle-Leu-Ser-Ser-Thr-Asn-Val-Gly- Ser-Asn-Thr-Tyr (SEQ ID NO:4)] and is 30 to 37, 31 to 37, 32 to 37, 33 to 37, 34 to 37, 35 to 37, or 36 to 37 amino acids long, respectively.

[0041] In some embodiments, the peptide (Pox) has at 78%, 81 %, 84%, 87%, 90%, 93%, or 96% sequence identity with human calcitonin over the whole of the human calcitonin amino acid sequence [H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly- Thr-Tyr-Thr-GIn-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-GIn-Thr-Ala-lle-Gly-Val- Gly-Ala-Pro-NH2 (Disulfide bond) (SEQ ID NO:3)]. Additionally, the peptide (Pox) may comprise exactly one intramolecular disulfide bond and may be 25 to 50 amino acids in length. In some embodiments, the peptide (Pox) has at least 78%, 81 %, 84%, 87%, 90%, 93%, or 96% sequence identity with human calcitonin over the whole of the human calcitonin amino acid sequence [H-Cys-Gly-Asn-Leu-Ser-Thr- Cys-Met-Leu-Gly-Thr-Tyr-Thr-GIn-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-GIn- Thr-Ala-lle-Gly-Val-Gly-Ala-Pro-NH2 (Disulfide bond) (SEQ ID NO:3)] and is 25 to 32, 26 to 32, 27 to 32, 28 to 32, 29 to 32, 30 to 32, or 31 to 32 amino acids long, respectively. Amino acid identity between a first and a second amino acid sequence over the length of the first amino acid sequence may be calculated by counting the number of identical amino acids between both sequences and dividing this number by the number of amino acids within the first sequence. It follows that the values for sequence identity are no continuum, but that they increase incrementally. Side chain modifications may not be considered in this calculation, i.e. a side chain modified amino acid will be considered as if no modification were present.

[0042] In some embodiments, the peptide (Pox) and / or its reduced version (Pred) are particularly prone to side reactions during disulfide bond formation. Such peptides may be characterized by the presence of at least one methionine, tryptophane, tyrosine, or histidine moiety. Additionally or alternatively, such peptides may comprise at least one aryl group (in particular a phenyl or a naphthyl group), thioalkyl group (in particular a thiomethyl or a thioethyl group), indolyl group, or imidazolyl group.

[0043] In some embodiments, the peptide (Pox) and / or its reduced version (Pred) have an isoelectric point of 6 to 8 or of 2-3.

[0044] In some embodiments, the peptide is selected from the group consisting of atosiban, lanreotide, octreotide, oxytocin, vasopressin, calcitonin, carbetocin, desmopressin, somatostatin, terlipressin, and adrenomedullin.

[0045] In some embodiments, the peptide has a sequence selected from the group consisting of SEQ ID NOs: 1 , 2, 3, 4, 5, and 6, or the peptide has a sequence, which has at least 75% sequence identity with a peptide selected from the group consisting of SEQ ID NOs: 1 , 2, 3, 4, 5, and 6, over the entire length of said peptide’s amino acid sequence.

[0046] The composition comprising the chemically synthesized peptide (Pred) provided in step a according to the methods of the present invention may either comprise the crude peptide (Pred) or a partially or completely purified peptide (Pred). For the purpose of the present application, the terms "raw" and "crude" are used interchangeably to designate preparations of a peptide, which are essentially a direct product of synthesis and isolation processes and have not (yet) been submitted to specific purification steps. Chemical synthesis usually yields crude peptide preparations having a purity of around 20-90%, and commonly 40 to 80%. Moreover, the chemically synthesized peptide (Pred) may be free of all protecting groups or may be partially protected, provided that it comprises at least two free sulfhydryl groups, e.g., two unprotected cysteine side chains. The skilled person executing step a of the methods according to the present invention will choose the degree of (pre-) purification of the peptide (Pred) and of protecting group removal depending on the project at hand. For example, some crude peptides may prove unstable in solution, such that they are advantageously subjected to a purification step prior to disulfide bond formation. As a further example, in case the peptide is destined for conjugation to another moiety after disulfide bond formation, it may be desired to use a partially deprotected peptide (Pred).

[0047] Therefore, step a of the method according to the present invention may comprise the following: a. a. Conducting support-assisted peptide synthesis; a.b. Cleaving the peptide from the support and / or removing at least the protecting groups from two sulfhydryl moieties so as to obtain a cleaved and at least partially deprotected peptide with at least two free sulfhydryl groups (Pred); a.c. Isolating the peptide (Pred); a.d. Dissolving the isolated peptide (Pred) to obtain a solution thereof; a.e. Optionally, flowing the solution of step a.d. through a reversed phase liquid chromatography column, so as to allow adsorption of the peptide (Pred) to the stationary phase of the column; and a.f. Optionally, eluting the peptide (Pred) from the column so as to obtain an eluted solution comprising the peptide (Pred).

[0048] In one embodiment, step a of the method according to the present invention may comprise the following: a.a1 . Conducting solid phase peptide synthesis; a.b1 . Cleaving the peptide from the solid support and removing at least the protecting groups from two sulfhydryl moieties so as to obtain a cleaved and at least partially deprotected peptide with at least two free sulfhydryl groups (Pred); a.c. Isolating the peptide (Pred); a.d. Dissolving the isolated peptide (Pred) to obtain a solution thereof; a.e. Optionally, flowing the solution of step a.d. through a reversed phase liquid chromatography column, so as to allow adsorption of the peptide (Pred) to the stationary phase of the column; and a.f. Optionally, eluting the peptide (Pred) from the column so as to obtain an eluted solution comprising the peptide (Pred).

[0049] In another embodiment, step a of the method according to the present invention may comprise the following: a.a2. Conducting tag-assisted peptide synthesis; a.b2. Removing at least the protecting groups from two sulfhydryl moieties so as to obtain an at least partially deprotected peptide with at least two free sulfhydryl groups (Pred); a.c. Isolating the peptide (Pred); a.d. Dissolving the isolated peptide (Pred) to obtain a solution thereof; a.e. Optionally, flowing the solution of step a.d. through a reversed phase liquid chromatography column, so as to allow adsorption of the peptide (Pred) to the stationary phase of the column; and a.f. Optionally, eluting the peptide (Pred) from the column so as to obtain an eluted solution comprising the peptide (Pred).

[0050] In another embodiment, step a of the method according to the present invention may comprise the following: a.a3. Conducting Fmoc solid phase peptide synthesis; a.b3. Cleaving the peptide from the solid support and removing at least the protecting groups from two sulfhydryl moieties so as to obtain a cleaved and at least partially deprotected peptide with at least two free sulfhydryl groups (Pred); a.c. Isolating the peptide (Pred); a.d. Dissolving the isolated peptide (Pred) to obtain a solution thereof; a.e. Optionally, flowing the solution of step a.d. through a reversed phase liquid chromatography column, so as to allow adsorption of the peptide (Pred) to the stationary phase of the column; and a.f. Optionally, eluting the peptide (Pred) from the column so as to obtain an eluted solution comprising the peptide (Pred). In the methods according to the present invention, cleavage of protecting groups and cleavage from the solid support or tag may be performed according to the procedures known in the art.

[0051] SPPS is commonly carried out on gel phase rather than solid phase supports. Suitable resins may be based on polystyrene, polystyrene-PEG composites, PEG, PEGA, cross-linked ethoxylate acrylate (CLEAR), polyamides, polydimethylacrylamide, or any other support with the desired physical and chemical properties. Resins based on beaded polystyrene with 1 % divinylbenzene are among the routinely used supports, typically having a size distribution of 200-400 mesh or 100-200 mesh. Polystyrene based 2-Chlorotrityl chloride (CTC) resin, CTC Amido-methyl resin, diphenyldiazomethane (PDDM) resin, 4-(2',4'- Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxymethyl-polystyrene (Rink) resin, 2- Methoxy-4-alkoxybenzyl alcohol (Sasrin) resin, and 4-Alkoxybenzyl alcohol (Wang) resin are particularly suitable for use with the methods of the present invention. Fmoc amino acid derivatives are likewise readily available and may be coupled by various protocols. Deprotection and cleavage from the resin are usually performed using an acidic cleavage solution, which may optionally further comprise scavengers. The most common cleavage solutions comprise trifluoroacetic acid (TFA) and one or more carbocation scavenger(s). For example, the cleavage solution may comprise at least 80% of TFA, water, and one or more scavenger(s). The scavengers may be selected from the group consisting of silanes, thiols, and aromatic compounds. Suitable scavengers comprise, e.g., triisopropylsilane (TIPS), triethylsilane (TES), dithiothreitol (DTT), dithioerythriol (DTE), 1 ,2-ethanedithiol (EDT), beta-mercaptoethanol, ethyl methyl sulfide, anisole, thioanisole, m- or p- cresol, 2-Me-indole, Ac-Trp-OMe, and tryptamine.

[0052] In the above methods, step a.c. of isolating the peptide (Pred) may be performed by precipitation, e.g. by mixing with an anti-solvent. For example, partial or complete deprotection of the peptide, and cleavage from the solid support or -optionally- from the tag usually leads to a cleavage composition comprising the (partially) deprotected peptide in an acidic solution, together with residual solvents and reagents from previous synthesis steps, scavengers, and other by-products of the cleavage reaction. After an optional filtration step, the peptide may be precipitated out of this mixture by adding an ether, such as diethyl ether, isopropyl ether, or methyl fe / Y-butyl ether. In the alternative, the mixture may be added into the ether. Precipitation may be allowed to proceed for a certain time, e.g. several hours, often at a temperature between 0 and 15°C, before collecting the peptide precipitate by filtration and optionally drying it. A crude peptide (Pred) thus prepared may be a salt, often a TFA salt, comprising residual scavengers and other solvents or reagents as well as by-products from the cleavage reaction.

[0053] In the above methods, step a.d. of dissolving the isolated peptide (Pred) will be performed depending on the properties of the peptide at hand. For example, a dried crude peptide may be dissolved by gentle stirring in an aqueous phase while adjusting temperature and pH as appropriate. The skilled practitioner may consider that a peptide is usually easier dissolved in aqueous solution if the pH of the solution is at least one unit different from the peptide’s isoelectric point. It may be advantageous to dissolve the crude peptide precipitate in diluted acids, e.g. hydrochloric acid, acetic acid, trifluoroacetic acid, or diluted bases, e.g. aqueous NaOH, ammonia, phosphate, or TEAR. In some cases, the peptide may be dissolved in water without the addition of a buffering agent. In some cases, it may be advantageous for the peptide solution to contain an organic modifier.

[0054] It will be understood that the composition of step a of the method of the present invention may be a solution of the peptide (Pred) or the composition of step a may comprise the peptide (Pred) adsorbed to the stationary phase of a chromatography column. In this latter case, optional step a.e of the above embodiments will be performed to load the peptide onto a chromatographic column. The skilled person will routinely choose a suitable feed composition and will prepare the solution of step a.d accordingly. In the former case, the solution may be either a solution of the crude peptide (Pred) or a solution of a purified or partially purified peptide (Pred). The latter may be obtained by chromatographic purification, e.g., by reversed-phase chromatography. For example, optional steps a.e and a.d of the above embodiments may be performed to elute the peptide (Pred) from a reversed phase column. The skilled person may select the elution conditions such that the peptide (Pred) may be separated from unwanted peptidic components. The eluate may be collected in several fractions and be pooled depending on the purity of the peptide (Pred) contained to obtain the composition of step a according to the present invention.

[0055] The skilled person will understand that the composition of step a of the method of the present invention may be a solution of the peptide (Pred), which may comprise a suitable aqueous buffer, and optionally further additives such as organic solvents, salts, or chaotropic agents. Moreover, in case the composition of step a comprises a crude peptide, it may comprise traces of the reagents used for synthesis and cleavage, by-products of said reactions, and filtration aids. Unless the peptide has been highly purified, the composition of step a may further comprise related peptidic substances. These comprise truncated versions, deletion and insertion variants, other sequence variants, as well as derivatives of the peptide (Pred). As used herein, the expression "truncated variant" refers to continuous fragments, i.e. subsequences without gaps, of a given peptide, which lack one or more amino acids at the N-terminus and / or the C-terminus of the peptide sequence. As used herein, the expression "deletion variant" is used to refer to variants of a specific peptide, which differ from it in that their primary sequence lacks a single or multiple amino acid(s). The "omitted" amino acid(s) may be at any position within the original peptide sequence. Hence, truncation variants can be considered a specific type of deletion variant. As used herein, the expression "insertion variant" is used to refer to variants of a specific peptide, which differ from it in that their primary sequence comprises one or more additional amino acid(s), which may be inserted at any position into the original peptide sequence. The expression “other sequence variants” is used to describe any other related peptide, which differs from the peptide of interest in that it comprises one or more amino acid exchange(s). Such sequence variants may occur through chemical reaction, e.g. by hydrolysis of acid amide groups, or during synthesis due to trace contaminations of the amino acid derivatives used. The term "derivative" or "derivatives" as used herein refers to a compound which can be obtained from a first compound by a chemical reaction. As a result, a derivative may differ from the first compound by the presence or absence of one or more substituent(s). All of the above related peptidic substances may represent an unwanted peptidic component, from which the disulfide bonded peptide (Pox) is to be separated.

[0056] Unless specified otherwise, the concentration of a peptide (Pred) in solution will be indicated herein as the mass of peptide material without any counterions or other components of a solid peptide material, i.e., the net peptide mass, dissolved per volume of the solution. The other components of a solid peptide material may comprise, e.g., precipitation aids, residual solvents and reagents, and non-peptidic side products of previous synthesis steps. The net peptide mass may be routinely determined by quantitative amino acid analysis or by elemental / nitrogen analysis. These methods are well known in the art and commercial devices are available from established producers. Guidelines for protein / peptide analysis by nitrogen analysis can be found in the European Pharmacopoeia 11.0, chapter 2.5.33 (Method 7, Procedure B) and in the U.S. Pharmacopeia official as of 01 -Aug-2019, chapter <1057> (Method 7, Procedure B), herein incorporated by reference. Depending on the purity of the peptide (Pred), the net peptide mass may be approximated by the net API mass, which may be determined by quantitative chromatographic assays. The skilled practitioner will understand that, if the composition of step a is a solution of the peptide peptide (Pred), the concentration of the peptide may be in a similar range or higher as indicated below for the aqueous reaction composition (C1 ) obtained in step b. The peptide (Pred) may be present in the composition of step a at a concentration of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 ,15, 16, 17, 18, 19, or 20 g / l. In one embodiment, the peptide (Pred) is dissolved in the composition of step a at a concentration of 1 to 50 g / l, 2 to 50 g / l, 3 to 50 g / l, 4 to 50 g / l, 5 to 50 g / l, 6 to 50 g / l, 7 to 50 g / l, 8 to 50 g / l, 9 to 50 g / l, 10 to 50 g / l, 11 to 50 g / l, 12 to 50 g / l, 13 to 50 g / l, 14 to 50 g / l, 15 to 50 g / l, 16 to 50 g / l, 17 to 50 g / l, 18 to 50 g / l, 19 to 50 g / l, or 20 to 50 g / l. In one embodiment, the peptide (Pred) is dissolved the in composition of step a at a concentration of 1 to 50, 2 to 50, 3 to 50, 4 to 50, 5 to 50, 6 to 50, 7 to 50, 8 to 50, 9 to 50, 10 to 50, 1 1 to 50, 12 to 50, 13 to 50, 14 to 50, or 15 to 50 g net peptide per liter. In one embodiment, the peptide (Pred) is dissolved in the composition of step a at a concentration of 1 to 40, 2 to 40, 3 to 40, 4 to 40, 5 to 40, 6 to 40, 7 to 40, 8 to 40, 9 to 40, 10 to 40, 11 to 40, 12 to 40, 13 to 40, 14 to 40, or 15 to 40 g net peptide per liter. In one embodiment, the peptide (Pred) is dissolved in the composition of step a at a concentration of 1 to 30, 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 11 to 30, 12 to 30, 13 to 30, 14 to 30, or 15 to 30 g net peptide per liter. In one embodiment, the peptide (Pred) is dissolved in the composition of step a at a concentration of 1 to 25, 2 to 25, 3 to 25, 4 to 25, 5 to 25, 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 1 1 to 25, 12 to 25, 13 to 25, 14 to 25, or 15 to 25 g net peptide per liter. The skilled practitioner will select the concentration of the peptide (Pred) in the composition of step a such that it results in a volume, which is easy to handle, and such that the peptide is sufficiently stable in the composition of step a. Depending on the properties of the specific peptide at hand, parameters like, e.g., temperature, pH, ionic strength, content in organic modifiers will be routinely adjusted.

[0057] Alternatively, the composition of step a may comprise the peptide (Pred) adsorbed to the stationary phase of a reversed phase chromatography column. In this case, the composition of step a may essentially correspond to the content of a reversed phase chromatography column loaded with the peptide (Pred). Unless specified otherwise, the amount of the peptide (Pred) on the column is indicated herein as the loaded mass of peptide material without any counterions or other components of a solid peptide material, i.e., the net peptide mass as explained above, divided by the cross- sectional area of the column [calculated as %*( column radius)2] (also referred to as “g net peptide per cm2column diameter”). This metric is commonly used and advantageous in that it does not depend on column packing and or column length. Hence, this metric does not change in dependence of the scale of a given experiment. As explained above, the net peptide mass may be routinely determined by quantitative amino acid analysis or by Nitrogen analysis. Depending on the purity of the peptide (Pred), the net peptide mass may be approximated by the net API mass, which may be determined by chromatography. The skilled practitioner will understand that the details, e.g. regarding the nature of the stationary phase and the amount of peptide on the column, which are given below for the aqueous reaction composition (C1 ) obtained in step b of the method are likewise applicable to the composition of step a. Hence, the peptide (Pred) in the composition of step a may be adsorbed to the stationary phase of a chromatography column at an initial amount of at least 0.05, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 g / cm2The peptide (Pred) in the composition of step a may be adsorbed to the stationary phase of a chromatography column at an initial amount of 0.05 to 5 g / cm2, 0.1 to 5 g / cm2, 0.15 to 5 g / cm2, 0.2 to 5 g / cm2, 0.25 to 5 g / cm2, 0.3 to 5 g / cm2, 0.35 to 5 g / cm2, 0.4 to 5 g / cm2, 0.45 to 5 g / cm2, 0.5 to 5 g / cm2. In some embodiments, the peptide (Pred) in the composition of step a is adsorbed to the stationary phase of a chromatography column at an initial amount of 0.05 to 2, 0.1 to 2, 0.15 to 2, 0.2 to 2, 0.25 to 2, 0.3 to 2, 0.35 to 2, 0.4 to 2, 0.45 to 2, 0.5 to 2, 0.05 to 1.5, 0.1 to 1.5, 0.15 to 1.5, 0.2 to 1 .5, 0.25 to 1 .5, 0.3 to 1 .5, 0.35 to 1 .5, 0.4 to 1 .5, 0.45 to 1 .5, 0.5 to 1 .5, of 0.05 to 1 , 0.1 to 1 , 0.15 to 1 , 0.2 to 1 , 0.25 to 1 , 0.3 to 1 , 0.35 to 1 , 0.4 to 1 , 0.45 to 1 , or 0.5 to 1 g net peptide per cm2column diameter. The stationary phase may typically be provided in an aqueous solution containing limited amounts of organic modifier and optionally further components such as salts and buffers. The skilled person will routinely determine the composition of this aqueous solution, taking into account the properties of the properties of peptide (Pred) and the reaction conditions envisaged in step b of the method.

[0058] The composition provided in step a. of the method may comprise more than 1 g, in particular more than 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 g of net reduced peptide (Pred). At a larger scale, the composition provided in step a. of the method may comprise more than 150 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900g, 1 kg, 2 kg, 3 kg, 4 kg, 5 kg, 6 kg, 8 kg, 9 kg, 10 kg, 20 kg, 30 kg, 40 kg, or 50 kg of net reduced peptide (Pred). For example, the total amount of net reduced peptide (Pred) adsorbed to a stationary phase of a reversed phase liquid chromatography column may be around 1-1000, 10-1000, 50-1000, 100-1000, 150- 1000, 200-1000, 1 -2000, 10-2000, 50-2000, 100-2000, 150-2000, 200-2000, 1 - 3000, 10-3000, 50-3000, 100-3000, 150-3000, 200-3000, 1 -4000, 10-4000, 50- 4000, 100-4000, 150-4000, or 200-4000, 1-30000, 10-30000, 50-30000, 100-30000, 150-30000, or 200-30000 g. As a further example, the amount of net reduced peptide (Pred) dissolved in the composition of step a may be 1 -1000, 10-1000, 50- 1000, 100-1000, 150-1000, 200-1000 g, 1-5000, 10-5000, 50-5000, 100-5000, 150- 5000, 200-5000 g, 1 -5000, 10-5000, 50-5000, 100-5000, 150-5000, 200-5000 g, , 1 -10000, 10-10000, 50-10000, 100-10000, 150-10000, 200-10000 g, 1 -20000, I Q- 20000, 50-20000, 100-20000, 150-20000, 200-20000 g, 1 -30000, 10-30000, 50- 30000, 100-30000, 150-30000, 200-30000 g, 1 -40000, 10-40000, 50-40000, 100- 40000, 150-40000, 200-40000 g, 1 -50000, 10-50000, 50-50000, 100-50000, 150- 50000, or 200-50000 g.

[0059] Dehydroascorbic acid (DHA) is an oxidized form of ascorbic acid. Although the structure of DHA is often given as formula 1 below, it is present in aqueous solution mainly as a bicyclic monohydrate (formula 2) and possibly as dihydrate (formula 3). Solid DHA can form crystals of symmetrical dimers (formula 4), known to undergo epimerization. While formulae 1 to 3 are drawn to illustrate the configuration of L- threo-dehydroascorbic acid, the terms “dehydroascorbic acid” and “DHA” as used herein are intended to encompass dimeric and monomeric forms as well as stereoisomers, in particular epimers, of the compounds drawn. Formula 4

[0060] DHA can be prepared by numerous chemical and enzymatic methods and is available from various commercial sources. Alternatively, it may be generated in situ by oxidation of ascorbic acid. For example, a solution of ascorbic acid may be reacted with iodine, where ascorbic acid is in slight excess to avoid any residual iodine. As the conversion is complete and the resulting iodide does not hamper the subsequent disulfide bond formation, the resulting solution of DHA may be used without further processing. Another method is oxidation of ascorbic acid in ethanol (or similar alcohols such as isopropanol or methanol) by air or oxygen in the presence of activated charcoal. The resulting DHA and / or DHA-alcohol complexes may be separated from the charcoal by filtration, isolated by evaporation of solvent and lyophilization and optionally be used without further purification.

[0061] One molecule of DHA monomer reacts with two sulfhydryl groups to form one disulfide bridge. As used herein, one equivalent of DHA, relative to the total amount of free sulfhydryl groups, is the molar amount of DHA theoretically needed to completely convert the given amount of free sulfhydryl groups to disulfide bonds. As an example, if 1 mol of sulfhydryl groups is present, one equivalent of DHA corresponds to 0.5 mol of DHA, due to the stoichiometry of the reaction as explained above. When working with crude DHA preparations and / or crude peptide of unknown purity, it may be helpful to use a functional definition of one equivalent: Practically speaking, it is the amount of DHA, which is just sufficient to achieve complete conversion of the total amount of free sulfhydryl groups to disulfide bonds. “Complete conversion” is herein typically assessed by analytical chromatography and may be interpreted to mean that the peptide (Pred) is detectable in an amount, which is less than 5%. 4%, 3%, 2%, 1 %, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1 %. Commonly, the linear peptide (Pred) may not be detectable at all upon complete conversion. As used herein, the amount of a peptide in a given composition may be determined from an analytical chromatogram by dividing the area of the peak corresponding to the peptide of interest by the sum of the areas of all peaks detected. Typically, such an analytical chromatogram may be recorded using an UV detector to record absorption of the eluate at 220 nm. In other settings, e.g., when the composition of step a comprises a purified preparation of the peptide (Pred) and the peptide (Pred) is essentially the only source of sulfhydryl groups in the composition of step a, the purities of DHA and the peptide content may be determined, e.g. by chromatography. Using these data, one may easily calculate the molar amount and the mass of DHA, which correspond to one equivalent of DHA, relative to the total amount of free sulfhydryl groups contained in the composition of step a.

[0062] Step b of the methods according of the invention involves combining the composition of step a with at least one equivalent of DHA. This may be performed by any procedure: For example, a solution of DHA may be mixed in any order with the composition of step a, or the amount of DHA needed may be added as a solid into and dissolved in the composition of step a. The skilled person will understand that for good yields, the whole composition of step a. should be combined with DHA, and will routinely take measures to avoid incomplete transfer and loss of material. In case the composition of step a is a solution of the peptide (Pred), which has been purified by chromatography, step b may be performed by collecting those parts of the eluate, which comprise the peptide (Pred) in the desired form, in a container, which has been prefilled with the necessary amount of DHA. In case the composition of step a comprises the peptide (Pred) adsorbed to the stationary phase of a reversed phase chromatography column, step b may be executed by flowing an aqueous solution comprising the required amount of DHA through the column. The total amount of DHA combined with the peptide (Pred) may be calculated in such a case by multiplying the total volume of DHA containing aqueous solution flowed through the column with the concentration of DHA in said solution. In methods according to the invention, wherein the initial concentration initial amount of dehydroascorbic acid in the aqueous reaction composition (C1 ) is at least 1 equivalent relative to the total amount of free sulfhydryl groups contained in the composition of step a., it is also possible to add the composition of step a. slowly, e.g. dropwise, into a composition, usually a solution, comprising the amount of DHA needed for the reaction. Compared to the prior art, this scheme allows to end up with a fairly concentrated solution of the peptide, because the oxidation agent needs not to be diluted and a high excess over peptide can be tolerated. As has been elaborated above, it is a decisive advantage of the methods according to the present invention that the formation of disulfide bonds may occur in a reaction mixture comprising at least one of the educts of this reaction — i.e. , the peptide (Pred) or DHA, or both- in fairly high concentration.

[0063] Because the peptide (Pred) is converted inside the aqueous reaction composition (C1 ) into a disulfide bonded peptide (Pox), the concentration of the peptide (Pred) [or the initial amount of the peptide (Pred) on the chromatography column] is given as the initial concentration of the peptide (Pred) [or the initial amount of the peptide (Pred) on the chromatography column] at the very beginning of the disulfide bond formation reaction, i.e. at time zero. It is a special feature of the methods according to present invention that, due to the use of DHA as oxidizing agent, the initial concentration of the peptide (Pred) [or the initial amount of the peptide (Pred) on the chromatography column] and / or the initial concentration of DHA can be rather high inside the aqueous reaction composition (C1 ), without provoking the excessive occurrence of unwanted side reactions such as intermolecular disulfide bond formation.

[0064] In one embodiment, the peptide (Pred) is dissolved in the aqueous reaction composition (C1 ) and is present at the initial concentration of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 ,15, 16, 17, 18, 19, or 20 g / l. In one embodiment, the peptide (Pred) is dissolved in the aqueous reaction composition (C1 ) and is present at the initial concentration of 1 to 50 g / l, 2 to 50 g / l, 3 to 50 g / l, 4 to 50 g / l, 5 to 50 g / l, 6 to 50 g / l, 7 to 50 g / l, 8 to 50 g / l, 9 to 50 g / l, 10 to 50 g / l, 11 to 50 g / l, 12 to 50 g / l, 13 to 50 g / l, 14 to 50 g / l, 15 to 50 g / l, 16 to 50 g / l, 17 to 50 g / l, 18 to 50 g / l, 19 to 50 g / l, or 20 to 50 g / l. In one embodiment, the peptide (Pred) is dissolved in the aqueous reaction composition (C1 ) and is present at the initial concentration of 1 to 30, 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 11 to 30, 12 to 30, 13 to 30, 14 to 30, or 15 to 30 g net peptide per liter. In one embodiment, the peptide (Pred) is dissolved in the aqueous reaction composition (C1 ) and is present at the initial concentration of 1 to 25, 2 to 25, 3 to 25, 4 to 25, 5 to 25, 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 11 to 25, 12 to 25, 13 to 25, 14 to 25, or 15 to 25 g net peptide per liter. The skilled practitioner will select the concentration of the peptide (Pred) in the aqueous reaction composition (C1 ) such that it is maximally high while avoiding aggregation such as precipitation, crystallization, gel formation or the excessive formation of side products. For example, the concentration of the peptide (Pred) in the aqueous reaction composition (C1 ) may be 10 to 50% of the maximal concentration achievable for this peptide in a given reaction composition (C1 ) at a given temperature. The maximal concentration achievable of the peptide (Pred) in a given reaction composition (C1 ) may be the concentration at which no aggregation, precipitation, gel formation, and / or crystallization is detectable at a given temperature after incubation for 24 hours in a solution corresponding to the given reaction composition (C1 ), but not containing DHA. The solubility of the peptide and its aggregation propensity are peptide specific parameters. The skilled practitioner will take these into account and identify a suitable concentration in the aqueous reaction composition (C1 ) for a specific peptide as part of the routine work. Likewise, the skilled practitioner will adjust pH, ionic strength, and temperature of the reaction composition (C1 ) according to the properties of the peptide at hand.

[0065] In one embodiment, the peptide (Pred) in the aqueous reaction composition (C1 ) is adsorbed to the stationary phase of a chromatography column at an initial amount of at least 0.05, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 g / cm2The peptide (Pred) in the aqueous reaction composition (C1 ) may be adsorbed to the stationary phase of a chromatography column at an initial amount of 0.05 to 5 g / cm2, 0.1 to 5 g / cm2, 0.15 to 5 g / cm2, 0.2 to 5 g / cm2, 0.25 to 5 g / cm2, 0.3 to 5 g / cm2, 0.35 to 5 g / cm2, 0.4 to 5 g / cm2, 0.45 to 5 g / cm2, 0.5 to 5 g / cm2. In some embodiments, the peptide (Pred) in the aqueous reaction composition (C1 ) is adsorbed to the stationary phase of a chromatography column at an initial amount of 0.05 to 2, 0.1 to 2, 0.15 to 2, 0.2 to 2, 0.25 to 2, 0.3 to 2, 0.35 to 2, 0.4 to 2, 0.45 to 2, 0.5 to 2, 0.05 to 1 .5, 0.1 to 1.5, 0.15 to 1.5, 0.2 to 1.5, 0.25 to 1.5, 0.3 to 1.5, 0.35 to 1.5, 0.4 to 1.5, 0.45 to 1.5, 0.5 to 1.5, of 0.05 to 1 , 0.1 to 1 , 0.15 to 1 , 0.2 to 1 , 0.25 to 1 , 0.3 to 1 , 0.35 to 1 , 0.4 to 1 , 0.45 to 1 , or 0.5 to 1 g net peptide per cm2column diameter. In these embodiments, the fluid or dissolved components of the aqueous reaction composition (C1 ) are present in a mobile phase contained in or flushed through the chromatography column.

[0066] As set out above, in step b according to the methods of the invention, the peptide (Pred) is combined with at least one equivalent of DHA, relative to the total amount of free sulfhydryl groups contained in the composition of step a. In some embodiments, at least 1.1 , 1.2, 1.3, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 equivalents of DHA are used, relative to the total amount of free sulfhydryl groups contained in the composition of step a. In some embodiments, the amount of DHA used is 1.1 to 10 equivalents, 1.2 to 10 equivalents, 1.3 to 10 equivalents, 1.5 to 10 equivalents, 1.8 to 10 equivalents, 2 to 10 equivalents, 2.5 to 10 equivalents, 3 to 10 equivalents, 3.5 to 10 equivalents, 4 to 10 equivalents, 4.5 to 10 equivalents, 5 to 10 equivalents, 5.5 to 10 equivalents, 6 to 10 equivalents, 2 to 6 equivalents, or 3 to 6 equivalents, relative to the total amount of free sulfhydryl groups contained in the composition of step a. In some embodiments, the initial amount of DHA in the aqueous reaction composition (C1 ) is at least 1.1 , 1.2, 1.3, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 equivalents, relative to the total amount of free sulfhydryl groups contained in the composition of step a. In some embodiments, the initial amount of DHA in the aqueous reaction composition (C1 ) is 1.1 to 10 equivalents, 1.2 to 10 equivalents, 1.3 to 10 equivalents, 1.5 to 10 equivalents, 1.8 to 10 equivalents, 2 to 10 equivalents, 2.5 to 10 equivalents, 3 to 10 equivalents, 3.5 to 10 equivalents, 4 to 10 equivalents, 4.5 to 10 equivalents, 5 to 10 equivalents, 5.5 to 10 equivalents, 6 to 10 equivalents, 2 to 6 equivalents, or 3 to 6 equivalents, relative to the total amount of free sulfhydryl groups contained in the composition of step a. While the skilled person would not use such a high excess on a routine basis for reasons of economy, it is anticipated that DHA can be used in an excess of 50 or 100, relative to the total amount of free sulfhydryl groups contained in the composition of step a. Thus, in some embodiments, the amount of DHA used is 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, or 1 to 100 equivalents, relative to the total amount of free sulfhydryl groups contained in the composition of step a. In some embodiments, the amount of DHA used is 2 to 20, 2 to 30, 2 to 40, 2 to 50, 2 to 60, 2 to 70, 2 to 80, 2 to 90, or 2 to 100 equivalents, relative to the total amount of free sulfhydryl groups contained in the composition of step a. In some embodiments, the total amount of DHA used corresponds to the initial amount of DHA in the aqueous reaction composition (C1 ).

[0067] In step b according to the methods of the present invention, an aqueous reaction composition (C1 ) is obtained, which has a pH of 3.0 to 8.0. As used herein, an aqueous composition comprises at least 10% (v / v) of water. In some embodiments, the aqueous reaction composition (C1 ) will comprise at least 40%, 50%, 60%, or 70% (v / v) of water. Depending on the content of organic solvents, it may be difficult to measure directly the pH value of the aqueous reaction composition (C1 ). Therefore, for aqueous reaction compositions comprising more than 30% of organic solvents, the pH of the reaction composition may be approximated by the pH of the aqueous composition before the addition of solvent. As pH values are temperature dependent, the value indicated refers to the temperature, at which the disulfide bond formation is being carried out. The present inventors found that the pH of the reaction composition (C1 ) can be varied between 3.0 and 8.0. This broad range is advantageous in that a pH may be chosen, which is compatible with the peptide at hand. If working at pH values below 5, it may be advantageous to increase the reaction temperature to speed up the reaction kinetics. If working at a pH in the range of 7 to 8, hold times of DHA may be shortened and a larger molar excess of DHA may be used to compensate for losses due to DHA hydrolysis. Additionally or alternatively, a pulse protocol like the protocol of example 18 may be used. This may involve alternatingly flushing the column with a solution comprising DHA at a pH, at which the compound is stable, e.g. at a pH of 3 to 5, and with a buffer having the desired pH in the range of 7 to 8. For example, a solution of DHA in 10 mM NH4OAc pH 4.9 and a 50 mM aqueous NH4OAc buffer at pH 8 may be used. Both solutions may comprise an organic modifier, e.g. 5-15% acetonitrile. The skilled person will adjust the pH values and buffer strengths of both solutions such that the intended pH value can be obtained during the oxidation reaction.

[0068] In the method according to the invention, a buffer may be used to control the pH value of the aqueous reaction composition (C1 ) during the oxidation reaction, i.e. to keep it within a desired range during the formation of disulfide bonds. The pH of the aqueous reaction composition (C1 ) may be 3.0-8.0, preferably 4.0-7.0. Buffers are mixtures comprising a weak acid and its conjugate weak base; their buffer range is defined by the pKa of said acid. For the sake of brevity, a buffer may be described herein by indicating the weak acid only. It is clear, however, that such a buffer will normally comprise both the acid and its conjugated base, preferably in about equimolar amounts, and may further comprise counterions. For example, an acetic acid buffer may comprise acetic acid and sodium acetate. In the alternative, the name of a salt and the pH may be indicated: For example, a buffer described as “sodium phosphate, pH 5.5” will be understood to contain Na+, H2PO4 , and HPCU2' ions, but close to no PCU3' ions. The concentration of the buffering compounds defines the buffer’s capacity. The skilled practitioner is aware of these principles and will choose a suitable buffer substance and define its concentration as part of its routine tasks.

[0069] The skilled practitioner is aware that a suitable buffer for this purpose may generally have a pKa in the range of 3-8, preferably 4-7, and may not tend to form radicals or act as an oxidizing or reducing agent under the reaction conditions. Suitable buffers for use in the aqueous reaction composition (C1 ) [and in the solution (S3), which will be explained below], comprise, but are not limited to, citric acid (pKai 3.13 / pKa2 4.76 / pKas 6.4 at 25°C), formic acid (pKa 3.75 at 25°C), acetic acid (pKa 4.75 at 25°C), carbonic acid (pKa 6.36 at 25°C), or phosphoric acid (Kai 2.16 / pKa2 7.21 / pKas 12.32 at 25°C), ammonium (pKa 9.25) and mixtures thereof.

[0070] In one embodiment, the suitable buffer concentrations may be in the range of 10 mM to 1 M, preferably 20 mM to 200 mM, i.e., the sum of the concentration of the weak acid plus the concentration of its conjugated base falls in the above range.

[0071] In some embodiments of the present invention, the peptide (Pred) is present in the aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column. Historically, the expression “reversed phase liquid chromatography” has been used to differentiate from “normal phase” chromatography using polar, silica based stationary phases. In keeping with this, as used herein, “reversed phase liquid chromatography” refers to any type of chromatography, where the stationary phase used exhibits a largely apolar surface. Separation may occur because the components of a sample adsorb to different degrees to the apolar surface of the stationary phase. This involves various interactions, such as hydrophobic interactions and van der Waals forces, between each component of the sample and both the mobile phase and the stationary phase.

[0072] To perform reversed-phase chromatography, the stationary phase will be filled into a chromatography column, i.e. in a essentially cy I indric vessel adapted to allow flow of the mobile phase through the length of the vessel. The skilled person is well aware of the fact that chromatography columns can vary in their setup, from basic to highly refined columns. An appropriate column will routinely be chosen by the skilled person depending on the specific project at hand. The column diameter may range from 10 mm to 200 cm, e.g. 10 mm, 19 mm, 30 mm, 5 cm, 10 cm, 15, cm, 20 cm, 30 cm, 45 cm, 50 cm, 60 cm, or 160 cm.

[0073] Stationary phases for use in reversed-phase chromatography may be silica modified with apolar ligands / carbohydrate moieties or polymer based stationary phases like styrene or methylacrylate polymers. It is understood that the type of chemical bond between the hydrocarbon moiety and the carrier material as well as the chemical nature of the bonded hydrocarbon moieties may vary. In addition, stationary phases for reversed phase liquid chromatography may comprise polar groups, e.g. within the bonded hydrocarbon moieties, to yield so-called polar-embedded phases. In the context of the present application, the expression “hydrocarbon bonded silica” refers to stationary chromatographic phases made at least in part from silica particles or silica gels and having chemically bonded hydrocarbon moieties at their surface. Silica particles may be porous or not, or may have a solid core with a porous shell.

[0074] For example, a stationary phase for use with the present application may be made from polymers (e.g., polystyrene, methylarcylate), or silica particles or silica gels having chemically bonded hydrocarbon moieties of 4 to 18 carbon atoms. Such hydrocarbon moieties are preferably linear alkyl chains or aromatic moieties. Common types of hydrocarbon bonded silica have hydrocarbon moieties with four (C4), six (C6), eight (C8), or eighteen (C18) carbon atoms. Some types of hydrocarbon bonded silica have unbranched alkyl chains of four (C4), eight (C8), or eighteen (C18) carbon atoms, i.e. butyl, octyl, dodecyl, or octadecyl moieties. Common examples of hydrophobic stationary phases have C18, C8, or C4 alkyl groups or aromatic groups like phenyl, biphenyl, phenyl-hexyl, and fluoro phenyl (PFP) on their surface.

[0075] In some embodiments of the present invention, the reversed phase chromatography column contains a polymeric reversed phase (PRP). In some embodiments of the present invention, the reversed phase chromatography column contains a stationary phase selected from the group consisting of C4 silica, C8 silica, C18 silica, phenyl silica, biphenyl silica, phenyl-hexyl silica, and polar-embedded solid phases. In the context of the present application, the expression “C4 silica” is used to designate stationary chromatographic phases made at least in part from silica particles or silica gels having at their surface chemically bonded C4 hydrocarbon moieties, preferably linear butyl, i.e. n-butyl, moieties. “C8 silica” is used to designate stationary chromatographic phases made at least in part from silica particles or silica gels having at their surface chemically bonded C8 hydrocarbon moieties, preferably linear octyl, i.e. n-octyl, moieties. Likewise, the terms “C18 silica” or“ODS” are used herein interchangeably to refer to stationary chromatographic phases made at least in part from silica particles or silica gels having at their surface chemically bonded C18 hydrocarbon moieties, preferably linear octadecyl, i.e. n-octadecyl, moieties. “Phenyl silica”, “biphenyl silica” and “phenyl-hexyl silica” are used to designate stationary chromatographic phases made at least in part from silica particles or silica gels having at their surface chemically bonded phenyl, biphenyl, or phenyl-hexyl moieties, respectively. As explained above, silica particles may be porous or not or may have a solid core with a porous shell. A wide range of hydrocarbon bonded silica materials is commercially available. Examples of stationary phases which can be used in present invention are DaisogelTM C18 ODS, Daiso ODS-Bio, Daiso-ODS-A-HG C18, DaisogelTM C8- Bio, YMC ODS-A, YMC Triart C8-L, Luna C8, Luna C18, KromasilTM C18, and KromasilTM C8 produced by Daiso, YMC, Phenomenex, and AkzoNobel, respectively.

[0076] The silica particles may be of 2 to 200 micrometer, preferably 2.5 to 20 micrometer, preferably 5-15 micrometer, and most preferably 10 micrometer, in diameter and may have a pore size of 50 to 1000 A, preferably of 80 to 400 A, preferably of 100 to 300 A, most preferably of (about) 100 A.

[0077] In step c according to the methods of the present invention, the aqueous reaction composition (C1 ) is maintained for a time sufficient to allow conversion the of peptide (Pred) to the disulfide bonded peptide (Pox). As the skilled practitioner is well aware, the exact time needed will depend, inter alia, on the temperature of the reaction composition, the pH, and the concentration of the educts. As an example, the reaction may be carried out at room temperature for between 30 and 120 minutes. It is also possible to perform the reaction at lower or slightly elevated temperatures, e.g. at about 10, 20, 30, 40, 50, 60, or 70 °C. In case the reaction composition comprises the peptide adsorbed to the stationary phase of a reversed- phase chromatography column, it is to be understood that the expression “maintaining the aqueous reaction composition (C1 )” may mean to continuously flow a liquid comprising dehydroascorbic acid (DHA) over the column. This may be done while recycling the liquid or unidirectionally, i.e. while continuously supplying fresh DHA containing solution.

[0078] The skilled person is well aware of the fact that the steps b. and c. of the method do not need to occur sequentially but may often occur simultaneously or in part simultaneously. One such example is pumping a DHA containing solution over the column, until all peptide on the column has been oxidized. Another example is a setup where the peptide is added slowly into a solution of DHA.

[0079] The skilled practitioner may use the UV absorbance of ascorbic acid, which is formed during the disulfide bond formation and concomitant reduction of DHA, to follow the progress of the oxidation reaction. Contrary to DHA, ascorbic acid exhibits a strong absorption maximum at around 265 nm. Therefore, by following the absorption at around 265 or 254 nm of the aqueous reaction composition (C1 ) or, in some embodiments, of the liquid phase being flown over the reversed-phase chromatography column in step c., one can determine the kinetics of the oxidation reaction. This may be used as an in process control.

[0080] The time needed for step c. of the present methods may be evaluated by taking samples at different times and analyzing them by HPLC for the content in linear peptide (Pred) and in disulfide bonded peptide (Pox). The reaction may be considered finished, if less than 5%. 4%, 3%, 2%, 1 %, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1 % of linear peptide can be detected. Preferably, step c is carried out for such a time that no linear peptide can be detected. Alternatively, the time needed for step c. of the present methods may be evaluated by following the absorption at around 265 or 254 nm of the aqueous reaction composition (C1 ) or of the liquid phase being flown over the reversed-phase chromatography column in step c. If no change in the absorbance at around 265 or 254 nm is observable anymore, the reaction may be considered finished. The skilled practitioner is well aware of other established test methods for following the progress of disulfide bond formation, such as the Ellmann test for free sulfhydryl groups. Any of these tests may be used to establish the time needed for step c.

[0081] As set out above, the amount of DHA is chosen such that complete conversion of the linear peptide (Pred) to the disulfide bonded peptide (Pox) occurs. The skilled practitioner is aware that the mass of disulfide bonded peptide (Pox) theoretically obtainable from a given mass of linear peptide (Pred) can be calculated as: mass(P0X) = mass(

[0082] = mass(

[0083] For small peptides, the molecular weight of the linear peptide (Pred) will be in the order of 1000 g / mol; for larger peptides, the molecular mass of the linear peptide (Pred) is commonly in the order of 4000-5000 g / mol. Therefore, the theoretically obtainable mass of the oxidized peptide (Pox) will be usually about 99.8% to about 99.9% of the mass of the corresponding reduced peptide (Pred).

[0084] In some embodiments, step c. yields the peptide (Pox) dissolved in the aqueous reaction composition (C1 ) and at a final concentration of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 ,15, 16, 17, 18, 19, or 20 g / l. In some embodiments, step c. yields the peptide (Pox) dissolved in the aqueous reaction composition (C1 ) and at a final concentration of at least 1 to 50 g / l, 2 to 50 g / l, 3 to 50 g / l, 4 to 50 g / l, 5 to 50 g / l, 6 to 50 g / l, 7 to 50 g / l, 8 to 50 g / l, 9 to 50 g / l, 10 to 50 g / l, 11 to 50 g / l, 12 to 50 g / l, 13 to 50 g / l, 14 to 50 g / l, 15 to 50 g / l, 16 to 50 g / l, 17 to 50 g / l, 18 to 50 g / l, 19 to 50 g / l, or 20 to 50 g / l. In some embodiments, step c. yields the peptide (Pox) dissolved in the aqueous reaction composition (C1 ) and at a final concentration of at least 1 to 30, 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 11 to 30, 12 to 30, 13 to 30, 14 to 30, or 15 to 30 g net peptide per liter. In some embodiments, step c. yields the peptide (Pox) dissolved in the aqueous reaction composition (C1 ) and at a final concentration of at least of 1 to 25, 2 to 25, 3 to 25, 4 to 25, 5 to 25, 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 11 to 25, 12 to 25, 13 to 25, 14 to 25, or 15 to 25 g net peptide per liter. As set out above, the total amount of net disulfide bonded peptide (Pox) obtained in solution in step c may be 1 -1000, 10-1000, 50-1000, 100-1000, 150-1000, 200-1000, 1 -5000, 10-5000, 50- 5000, 100-5000, 150-5000, 200-5000, 1-5000, 10-5000, 50-5000, 100-5000, 150- 5000, 200-5000, , 1-10000, 10-10000, 50-10000, 100-10000, 150-10000, 200- 10000 g, 1 -20000, 10-20000, 50-20000, 100-20000, 150-20000, 200-20000, 1- 30000, 10-30000, 50-30000, 100-30000, 150-30000, 200-30000 g, 1 -40000, IQ- 40000, 50-40000, 100-40000, 150-40000, 200-40000, 1 -50000, 10-50000, 50- 50000, 100-50000, 150-50000, or 200-50000 g.

[0085] In some embodiments, step c. yields the peptide (Pox) in the aqueous reaction composition (C1 ) adsorbed to the stationary phase of a chromatography column at a final amount of at least about 0.05, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 g / cm2In some embodiments, step c. yields the peptide (Pox) in the aqueous reaction composition (C1 ) adsorbed to the stationary phase of a chromatography column at a final amount of at least about 0.05, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 g net peptide per cm2column diameter. In some embodiments, step c. yields the peptide (Pox) in the aqueous reaction composition (C1 ) adsorbed to the stationary phase of a chromatography column at a final amount of about 0.05 to 5 g / cm2, 0.1 to 5 g / cm2, 0.15 to 5 g / cm2, 0.2 to 5 g / cm2, 0.25 to 5 g / cm2, 0.3 to 5 g / cm2, 0.35 to 5 g / cm2, 0.4 to 5 g / cm2, 0.45 to 5 g / cm2, 0.5 to 5 g / cm2. In some embodiments, step c. yields the peptide (Pox) in the aqueous reaction composition (C1 ) adsorbed to the stationary phase of a chromatography column at a final amount of about 0.05 to 2, 0.1 to 2, 0.15 to 2, 0.2 to 2, 0.25 to 2, 0.3 to 2, 0.35 to 2, 0.4 to 2, 0.45 to 2, 0.5 to 2, 0.05 to 1.5, 0.1 to 1.5, 0.15 to 1.5, 0.2 to 1.5, 0.25 to 1.5, 0.3 to 1.5, 0.35 to 1.5, 0.4 to 1.5, 0.45 to 1.5, 0.5 to 1.5, of 0.05 to 1 , 0.1 to 1 , 0.15 to 1 , 0.2 to 1 , 0.25 to 1 , 0.3 to 1 , 0.35 to 1 , 0.4 to 1 , 0.45 to 1 , or 0.5 to 1 g net peptide per cm2column diameter. As set out above, the total amount of net disulfide bonded peptide (Pox) adsorbed to a stationary phase of a reversed phase liquid chromatography column obtained in step c. may be, e.g., 1 -1000, 10-1000, 50-1000, 100-1000, 150-1000, 200-1000, 1 - 2000, 10-2000, 50-2000, 100-2000, 150-2000, 200-2000, 1 -3000, 10-3000, 50- 3000, 100-3000, 150-3000, 200-3000, 1-4000, 10-4000, 50-4000, 100-4000, 150- 4000, 200-4000, 1-30000, 10-30000, 50-30000, 100-30000, 150-30000, or 200- 30000 g.

[0086] In one embodiment of the present invention, the method for the manufacture of a peptide (Pox) comprising an intramolecular disulfide bond, comprises the following steps: a.1. Providing an aqueous solution (S1 ) comprising a chemically synthesized peptide (Pred), which has at least two free sulfhydryl groups; a.2 Flowing the solution (S1 ) over a reversed phase liquid chromatography column, so as to allow adsorption of the peptide (Pred) to the stationary phase of the column; b.1 Contacting the adsorbed peptide on the stationary phase with a solution (S3) having a pH between 3.0 and 8.0, and comprising at least one equivalent of dehydroascorbic acid, relative to the amount of free sulfhydryl groups on the stationary phase; c.1 Maintaining the adsorbed peptide on the stationary phase in contact with the solution (S3) for a time sufficient to allow conversion of the adsorbed peptide (Pred) to an adsorbed disulfide bonded peptide (Pox); and d.1 . Eluting the adsorbed disulfide bonded peptide (Pox) from the stationary phase with a solution (S5) containing an organic modifier.

[0087] In some embodiments, the above method is used for the manufacture of an aggregation prone protein.

[0088] In some embodiments, the above method may further comprise one or both of the following steps: a.3 washing the chromatography column after step a.2 with an aqueous liquid (S2); and c.2 washing the chromatography column after step c.1 with an aqueous liquid (S4). The meaning of the expressions “peptide (Pox)” and “peptide (Pred)” as used herein has been explained above. Further, as used in the context of the present application:

[0089] - the expression “solution (S1 )” is to be understood as synonymous with the expression “the solution (S1 ) as defined in step a.1 of some embodiments of the invention”, which is in turn synonymous with “the solution as defined in step a.1 of some embodiments of the invention”;

[0090] - the expression “liquid (S2)” is to be understood as shorthand for and synonymous with the expression “the liquid (S2) as defined in step a.3 of some embodiments of the invention”, which is in turn synonymous with the expression “the liquid as defined in step a.3 of some embodiments of the invention”;

[0091] - the expression “solution (S3)” is to be understood as shorthand for and synonymous with the expression “the solution (S3) as defined in step b.1 of some embodiments of the invention”, which is in turn synonymous with “the solution as defined in step b.1 of some embodiments of the invention”;

[0092] - the expression “liquid (S4)” is to be understood as shorthand for and synonymous with the expression “the liquid (S4) as defined in step c.2 of some embodiments of the invention”, which is in turn synonymous with “the liquid as defined in step c.2 of some embodiments of the invention”; and

[0093] - the expression “solution (S5)” is to be understood as shorthand for and synonymous with the expression “the solution (S5) as defined in step d.1 of some embodiments of the invention”, which is in turn synonymous with “the solution as defined in step d.1 of some embodiments of the invention”.

[0094] In other words: The expression in brackets -i.e. (S1 ), (S2), (S3), (S4), and (S5), respectively- may be understood as a reference mark, which has no meaning by itself and is only used to facilitate reading.

[0095] Ways to provide an aqueous solution (S1 ) comprising a chemically synthesized peptide, which has at least two free sulfhydryl groups (Pred), have been described above in respect of step a of the inventive methods and are likewise applicable to step a1 of the inventive methods. In particular, step a.1 may involve carrying out steps a. a, a.b, a.c, and a.d, or steps a.a1 , a.b1 , a.c, and a.d, or steps a.a2, a.b2, a.c, and a.d, or steps a.a3, a.b3, a.c, and a.d as outlined above.

[0096] Step a.2 involves flowing the solution (S1 ) over a reversed phase liquid chromatography column, so as to allow adsorption of the peptide (Pred) to the stationary phase of the column. The skilled practitioner will do so routinely and will select the amount and the composition of the solution (S1 ) depending on whether the peptide is simply to be loaded for immobilization in a “solid phase extraction” setup, or whether a subsequent purification step is intended. If the latter is the case, it is desirable for the peptide to adhere to a rather narrow zone on top of the column. This may be fine-tuned by adjusting the amount of peptide loaded and the amount of organic modifier contained in the solution (S1 ) and / or by the optional washing step a.3.

[0097] The optional step a.3 of washing the chromatography column after step a.2 with an aqueous liquid (S2) allows to remove some components of the solution (S1 ), which might otherwise be detrimental to the disulfide bond formation reaction. Further, the solution (S2) may comprise a certain amount of organic modifier. Moreover, the aqueous liquid (S2) may comprise a buffer to adjust the pH value to a desired range, e.g. to the range suitable for carrying out the disulfide bond reaction. The same buffer may or may not be comprised in the solution (S3). In some embodiments, the optional step a.3 comprises subsequent washes with two or more different solutions.

[0098] In some embodiments, the liquid (S2) is selected from water, water comprising 1- 40% of an organic modifier, an aqueous buffer, or an aqueous buffer comprising 1- 40% of an organic modifier. The skilled practitioner will routinely select the concentration of the organic modifier so as to remove unwanted components from the stationary phase, without disturbing adsorption of the peptide (Pred) to the stationary phase, i.e. without eluting the peptide P(red) from the column. The aqueous buffer may comprise components selected from, but not limited to, the group consisting of citric acid, formic acid, acetic acid, carbonic acid, phosphoric acid, and ammonium (NH4+). In some embodiments, the liquid (S2) comprises 0.01 to 1 M ammonium acetate (NH4OAC) at a pH of between pH 5.0 and 7.0 and an organic modifier. The organic modifier may be selected from the group consisting of acetonitrile, methanol, ethanol, isopropanol, and mixtures thereof. The organic modifier may be acetonitrile.

[0099] As explained above, the peptide (Pred) may be adsorbed at an upper zone of the reversed phase liquid chromatography column. For example, for a column of 1 to 100 cm diameter, preferably 10, 20, 30, or 60 cm diameter, the peptide (Pred) may adhere to a zone of less than 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4 cm, 3 cm, 2 cm, or 1 cm thickness at the beginning of step b. In step b.1 , the sulfhydryl reduced peptide (Pred) is contacted on the solid phase with a solution (S3) having a pH between 3.0 and 8.0 and comprising at least one equivalent of dehydroascorbic acid (DHA), relative to the amount of free sulfhydryl groups on the stationary phase. The amount of DHA contained in the solution (S3) may be calculated by multiplying the concentration of DHA in said solution (S3) with the total volume of the solution (S3), which is contacted with the stationary phase in step b.1 . The solution (S3) may comprise an aqueous buffer to control the pH within the desired range. This buffer may comprise components selected from, but not limited to, the group consisting of citric acid, formic acid, acetic acid, carbonic acid, phosphoric acid, and ammonium. Moreover, the solution (S3) may comprise an organic modifier. In some embodiments, the solution (S3) comprises of sodium acetate at a pH of 4.0 to 7.0, ammonium acetate at a pH of 4.0 to 7.0, sodium phosphate at a pH of 4.0 to 7.0, ammonium phosphate at a pH of 4.0 to 7.0, sodium carbonate at a pH of 4.0 to 7.0, or ammonium carbonate at a pH of 4.0 to 7.0. The concentration of the buffer may be 10 to 200 mM.

[0100] Practically, step b.1 may be performed by flowing the solution (S3) through the reversed phase liquid chromatography column. To maintain the peptide (Pred) in contact with the solution (S3) for a sufficient time, a continuous flow of the solution

[0101] (53) may be maintained in step c.1. This may be done by continuously supplying fresh solution (S3) and discarding the column flow-through, or by recirculating a certain amount of liquid through the column. In the alternative, the flow may be stopped and the column held with the solution (S3) inside for the time needed. Regarding the time and temperature suitable to allow conversion of the adsorbed peptide (Pred) to an adsorbed disulfide bonded peptide (Pox), the above general considerations for step c of the methods of the present invention apply.

[0102] In optional step c.2, the chromatography column is washed with an aqueous liquid

[0103] (54) after the disulfide bond formation to remove any unwanted components from the column and / or to condition the peptide on the column. For example, step c.2 may be used to convert the peptide to a salt form of choice, to remove excess salt, or to adjust the composition of the mobile phase to achieve good resolution from unwanted peptidic components in the subsequent elution step d.1. Hence, in some embodiments, the aqueous liquid (S4) and the solution (S5) contain no buffer, the same buffer as contained in the solution (S3), or a buffer different from the buffer contained in solution (S3). In some embodiments, the aqueous liquid (S4) and the solution (S5) contain the same buffer, which is different from the buffer contained in the solution (S3). In some embodiments, the aqueous liquid (S4) and the solution (S5) contain essentially the same components, but the concentration of organic modifier in the aqueous liquid (S4) is lower than in the solution (S5). In some embodiments, the liquid (S4) is selected from water, water comprising 1-40% of an organic modifier, an aqueous buffer, or an aqueous buffer comprising 1 -40% of an organic modifier. The skilled practitioner will routinely select the concentration of the organic modifier so as to remove unwanted components from the stationary phase, without disturbing adsorption of the peptide (Pred) to the stationary phase, i.e. without eluting the peptide P(red) from the column. The aqueous buffer may comprise components selected from, but not limited to, the group consisting of citric acid, formic acid, acetic acid, carbonic acid, phosphoric acid, and ammonium (NH4+). In some embodiments, the liquid (S4) comprises 0.01 to 1 M ammonium acetate (NH4OAC) at a pH of between pH 5.0 and 7.0 and an organic modifier. The organic modifier may be selected from the group consisting of acetonitrile, methanol, ethanol, isopropanol, and mixtures thereof. The organic modifier may be acetonitrile. In some embodiments, the optional step c.2 comprises subsequent washes with two or more different solutions.

[0104] Step d.1 of the above embodiments involves eluting the adsorbed disulfide bonded peptide (Pox) from the stationary phase with a solution (S5) containing an organic modifier. The skilled person will adjust the concentration of the organic modifier such that it disrupts the interactions between the solid phase and the adsorbed disulfide bonded peptide (Pox) and thereby causes the disulfide bonded peptide (Pox) to elute. This step may be performed as a chromatographic separation step: The elution conditions may be tailored such that the adsorbed disulfide bonded peptide (Pox) elutes different than at least one unwanted peptidic component and thereby gets separated. This may be achieved, e.g., by a linear or stepwise gradient in the concentration of the organic modifier during elution and by carefully adapting the chromatographic system (i.e., the choice of the stationary phase and of the components of the mobile phase).

[0105] The organic modifier may be an organic solvent capable to modulate adhesion of the peptide to the stationary phase of the reversed phase chromatography column. It may be selected from, e.g., the group consisting of nitriles, alcohols, or ethers. In some embodiments, the organic modifier is selected from the group consisting of acetonitrile, methanol, ethanol, isopropanol, and mixtures thereof. The skilled person is aware that it may be beneficial to include a small amount of organic modifier in each of the solution (S1 ), the solution (S3), and, if used, the liquid (S2) and the liquid (S4). For example, the solution (S1 ), the solution (S3), and, if used, the liquid (S2) and the liquid (S4) may comprise between 1 % and 40% of an organic modifier. This may help to maintain the surface of the solid phase and to elute unwanted components from the column. The skilled person may routinely adjust the concentration of the organic modifier such that these targets are achieved without causing unwanted elution of the peptide of interest.

[0106] The disulfide bonded peptide (Pox) obtained may further conjugated to another moiety, which may be peptidic or non-peptidic. For example, the disulfide bonded peptide (Pox) may be conjugated to a carrier compound such as polyethylene glycol, to a toxin, a dye, a vitamin, a fatty acid, a carbohydrate, an amino acid, another peptide, or to a protein.

[0107] The disulfide bonded peptide (Pox) or the conjugate of the disulfide bonded peptide (Pox) may subsequently be subjected to one or more further purification steps, e.g. by crystallization, precipitation, dialysis, diafiltration, or chromatography, in particular reversed phase high performance liquid chromatography.

[0108] Further, the disulfide bonded peptide (Pox) or the conjugate of the disulfide bonded peptide (Pox) may subsequently be subjected to drying, preferably freeze-drying.

[0109] In addition to the methods detailed above, the present invention relates to an aqueous reaction composition (C1 ) having a pH of 3.0 to 8.0 comprising dehydroascorbic acid and a chemically synthesized peptide (Pred), which has at least two free sulfhydryl groups, wherein the peptide (Pred) is either present in this aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column at an initial amount of at least 0.05 g net peptide per cm2column diameter or is dissolved in the aqueous reaction composition (C1 ) at an initial concentration of at least 1 mg net peptide per ml, or wherein the initial amount of dehydroascorbic acid in the reaction composition (C1 ) is at least one equivalent, relative to the total amount of disulfide bonds to be formed.

[0110] One molecule of DHA monomer reacts with two sulfhydryl groups to form one disulfide bridge. As used herein, one equivalent of DHA, relative to total amount of disulfide bonds to be formed, is the molar amount of DHA theoretically needed to convert free sulfhydryl groups to a certain total amount of disulfide bonds. As an example, if 1 mol of disulfide groups is to be formed, one equivalent of DHA corresponds to 1 mol of DHA, due to the stoichiometry of the reaction as explained above.

[0111] In some embodiments, the present invention relates to an aqueous reaction composition (C1 ) having a pH of 3.0 to 8.0 comprising dehydroascorbic acid and a chemically synthesized peptide (Pred), which has at least two free sulfhydryl groups, wherein the peptide (Pred) is either present in this aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column at an initial amount of at least 0.1 g net peptide per cm2column diameter or is dissolved in the aqueous reaction composition (C1 ) at an initial concentration of at least 3 mg net peptide per ml, or wherein the initial amount of dehydroascorbic acid in the reaction composition (C1 ) is 1.5 to 5 equivalents, relative to the total amount of disulfide bonds to be formed, wherein the composition comprises a buffer selected from the group consisting of acetic acid, citric acid, formic acid, phosphoric acid, carbonic acid, and ammonium. In some embodiments, the initial amount of DHA in the aqueous reaction composition (C1 ) is at least 1.1 , 1.2, 1.3, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 equivalents, relative to the total amount of disulfide bonds to be formed. In some embodiments, the initial amount of DHA in the aqueous reaction composition (C1 ) is 1.1 to 10 equivalents, 1.2 to 10 equivalents, 1.3 to 10 equivalents, 1.5 to 10 equivalents, 1.8 to 10 equivalents, 2 to 10 equivalents, 2.5 to 10 equivalents, 3 to 10 equivalents, 3.5 to 10 equivalents, 4 to 10 equivalents, 4.5 to 10 equivalents, 5 to 10 equivalents, 5.5 to 10 equivalents, 6 to 10 equivalents, 2 to 6 equivalents, or 3 to 6 equivalents, relative to the total amount of disulfide bonds to be formed.

[0112] Mutatis mutandis, all of the above embodiments and explanations with respect to the methods of the invention are likewise applicable to the composition of the present invention and vice versa.

[0113] The following Figures and Examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and are not to be construed as limiting the scope of the claims. The methods disclosed in the examples may be used analogously to prepare other peptides comprising an intramolecular disulfide bond. Brief Description of the Figures

[0114] Figure 1

[0115] Degradation of dehydroascorbic acid in aqueous solution in dependence of time and pH at room temperature. Experimental details are given in Example 3: Stability of DHA in solution.

[0116] Figure 2

[0117] Kinetics of octreotide disulfide bond formation at different pH values at room temperature. Experimental details are given in Example 4: pH dependent kinetics of oxidation reaction.

[0118] Figure 3

[0119] Representative HPLC-chromatogram of the oxidation reaction at pH 5.1 , reaction time 10 min at room temperature. Asterisk (*) denotes cyclic, i.e. disulfide bonded, octreotide; two asterisks (**) denote linear, i.e. sulfhydryl-reduced, octreotide. Experimental details are given in Example 4: pH dependent kinetics of oxidation reaction.

[0120] Figure 4

[0121] Octreotide peptide (disulfide bonded) was incubated with iodine or DHA as described in Example 5: Comparison of side reactions iodine vs. DHA. Analytical chromatograms of sample 22e (incubation with iodine, shown on top) and sample 22i (incubation with DHA, shown on bottom). Asterisk (*) denotes cyclic, i.e. disulfide bonded, octreotide.

[0122] Figure 5

[0123] Analytical chromatograms of octreotide oxidized by various methods as described in Example 7: Octreotide oxidation in solution; comparison iodine vs. DHA of different origins.

[0124] 1 : linear octreotide, no oxidation (oligomers 6.0%)

[0125] 2: octreotide oxidized with DHA, cf. Table 4 entry 21 q (oligomers 5.8%)

[0126] 3: octreotide oxidized with iodine, cf. Table 4 entry 21 r (Oligomers 18.2%)

[0127] 4: octreotide oxidized with iodine, cf. Table 4 entry 21 s (Oligomers 9.8%)

[0128] Asterisk (*) denotes cyclic, i.e. disulfide bonded, octreotide; two asterisks (**) denote linear, i.e. sulfhydryl reduced, octreotide; three asterisks (***) denote oligomers. Figure 6

[0129] Representative chromatogram for the entire oxidation and purification process of octreotide described as described in Example 13: Oxidation of octreotide with DHA on column and Example 14: Oxidation of octreotide with DHA on column. Hashtag (#) denotes oxidation phase; Asterisk (*) denotes cyclic, i.e. disulfide bonded, octreotide.

[0130] Figure 7

[0131] Representative chromatogram for the entire oxidation and purification process of atosiban described in Example 15: Oxidation of atosiban with DHA on column. Hashtag (#) denotes oxidation phase; Cross (+) denotes disulfide bonded atosiban.

[0132] Figure 8

[0133] Analytical chromatograms of pramlintide oxidized by various methods as described in Example 8: Pramlintide oxidation in solution; comparison iodine vs. DHA vs. hydrogen peroxide

[0134] 1 : linear pramlintide, no oxidation

[0135] 2: pramlintide oxidized with iodine, cf. Table 7 entry 1 a

[0136] 3: pramlintide oxidized with DHA, cf. Table 7 entry 1 e

[0137] 4: pramlintide oxidized with hydrogen peroxide, cf. Table 7 entry 1 m

[0138] Asterisk (*) denotes linear, i.e. sulfhydryl reduced, pramlintide; two asterisks (**) denote cyclic, i.e. disulfide bonded, pramlintide; three asterisks (***) denote side products.

[0139] Figure 9

[0140] Analytical chromatograms of lanreotide free acid oxidized by various methods as described in Example 9: Lanreotide free acid oxidation in solution; comparison iodine vs. DHA vs. hydrogen peroxide.

[0141] 1 : linear lanreotide free acid, no oxidation

[0142] 2: lanreotide free acid oxidized with iodine, cf. Table 8 entry 1 a

[0143] 3: lanreotide free acid oxidized with hydrogen peroxide, cf. Table 8 entry 1 m

[0144] 4: lanreotide free acid oxidized with DHA, cf. Table 8 entry 1 u

[0145] Asterisk (*) denotes linear, i.e. sulfhydryl-reduced, lanreotide free acid; two asterisks (**) denote cyclic, i.e. disulfide bonded, lanreotide free acid.

[0146] Figure 10 Representative chromatogram (254 nm) for the entire oxidation and purification process of calcitonin described in Example 17: Oxidation of human calcitonin with DHA on column. One star (*) denotes oxidation phase; Two stars (**) denote product elution of disulfide bonded calcitonin.

[0147] Figure 11

[0148] Representative chromatogram (254 nm) for the entire oxidation and purification process of calcitonin described in Example 18: Oxidation of human calcitonin on column with DHA pulse protocol. One star (*) denotes oxidation phase, where reaction kinetics can be monitored by the change in pulse peak height. In the case depicted, oxidation was essentially complete after 4 pulses; Two stars (**) denote product elution of disulfide bonded calcitonin.

[0149] Figure 12

[0150] Lanreotide free acid peptide (disulfide bonded) was incubated with iodine, DHA, or hydrogen peroxide as described in Example 6: Comparison of side reactions iodine vs. DHA vs. H2O2. Analytical chromatograms: 1 Lanreotide free acid sample without oxidant; 2: sample 2a (incubation with iodine 1 h); 3: sample 2b (incubation with DHA 1 h); 4: sample 2c (incubation with H2O2, 1 h). Asterisk (*) denotes cyclic, i.e. disulfide bonded, lanreotide free acid.

[0151] Examples

[0152] 1. General methods

[0153] 1.1. Materials

[0154] DHA (Sigma-Aldrich Article number 261556, batch BCCH9233; Carbon Content 40.5%), ascorbic acid (DKSH Switzerland Ltd; Article number 5300066415; Batch DY02612200054; Purity 99.7%); iodine (DKSH Switzerland Ltd; Article number 5300066977; batch 536658, Purity 100%).

[0155] 1.2. Peptide synthesis

[0156] All peptides were synthesized by SPPS on 2-chlorotrityl or Rink amide AM resins using standard methods and Fmoc-amino acid derivatives. The sequence of octreotide (SEQ ID NO:1 ) is H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-L-threoninol (Disulfide bond), the sequence of atosiban (SEQ ID NO:2) is 3-Mercaptopropionyl- D-Tyr(Et)-lle-Thr-Asn-Cys-Pro-Orn-Gly-NH2 (Disulfide bond), the sequence of human calcitonin (SEQ ID NO:3) is H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly- Thr-Tyr-Thr-GIn-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-GIn-Thr-Ala-lle-Gly-Val- Gly-Ala-Pro-NH2 (Disulfide bond), the sequence of pramlintide (SEQ ID NO:4) is H- Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-GIn-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser- Ser-Asn-Asn-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH2 (Disulfide bond), and the sequence of lanreotide free acid (SEQ ID NO:5) is H-D-2- Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-OH (Disulfide bond). Peptide cleavage and concomitant cleavage of protecting groups was performed using cleavage cocktails comprising at least 80% TFA, water, and scavengers. The raw peptides were precipitated from the cleavage cocktail using diisopropyl ether as anti-solvent. Crude linear peptides were used for all experiments. Octreotide linear crude purity 86%, TFA content 24.5%, net octreotide content 63.8%. Atosiban purity 82.1 %; TFA content 23.1 %; net atosiban content 57.8%.

[0157] 1.3. HPLC analysis of dehydroascorbic acid (DHA)

[0158] DHA samples were analyzed by reversed phase analytical high-performance chromatography (HPLC) using an ACQUITY UPLC HSS T3 (C18 silica, 100 A, 1 .8 pm, 2.1x150 mm) column at 30°C on a Waters Acquity UPLC System equipped with a photo diode array (PDA) UV detector (220 nm). An isocratic elution program with 0.2 % KH2PO4 pH 3 at flow rate of 0.3 ml / min was applied for chromatographic analysis. 1.4. HPLC analysis of Octreotide

[0159] Octreotide samples were analyzed by HPLC using an C18 column at 60°C on a LIPLC System equipped with a PDA UV detector (220 nm). Eluent A: 0.05% trifluoroacetic acid (TFA) 1 % acetonitrile (ACN) and eluent B: 0.05% TFA in ACN were used and elution was according to Table 1 below.

[0160] Table 1

[0161] The purity of the linear or oxidized octreotide was expressed as the percentage of the area of the peak corresponding to linear or oxidized octreotide divided by the total area of all peaks detected.

[0162] 1.5. HPLC analysis of Atosiban

[0163] Atosiban samples were analyzed by HPLC using an C18 column at 60°C on a UPLC System equipped with an UV diode array detector (DAD) (220 nm). Eluent A: 0.02%

[0164] TFA 1 % ACN and eluent B: 0.02% TFA in ACN were used and elution was according to Table 2 below.

[0165] Table 2

[0166] The purity of the linear or oxidized atosiban was expressed as the percentage of area of the peak corresponding to linear or oxidized atosiban divided by the total area of all peaks detected.

[0167] 1.6 HPLC analysis of pramlintide

[0168] Pramlintide samples were analyzed by HPLC using a C18 column at 50°C on a LIPLC System equipped with a DAD UV detector (220 nm). Eluents A: 0.05% TFA 1 % ACN and eluent B: 0.05% TFA in ACN were used. A gradient elution program was applied for chromatographic analysis.

[0169] The purity of the linear or oxidized pramlintide was expressed as the percentage of area of the peak corresponding to linear or oxidized pramlintide divided by the total area of all peaks detected.

[0170] 1.7. HPLC analysis of calcitonin

[0171] Calcitonin samples were analyzed by HPLC using a C18 column at 60°C on a UPLC System equipped with a DAD UV detector (220 nm). Eluents A: 0.05% TFA 1 % ACN and eluent B: 0.05% TFA in ACN were used. A gradient elution program was applied for chromatographic analysis.

[0172] The purity of the linear or oxidized calcitonin was expressed as the percentage of area of the peak corresponding to linear or oxidized calcitonin divided by the total area of all peaks detected.

[0173] 1.8 HPLC analysis of lanreotide free acid

[0174] Lanreotidie free acid samples were analyzed by HPLC using a C18 column at 45°C on a UPLC System equipped with a DAD UV detector (220 nm). Eluents A: 0.025% TFA in H2O and eluent B: 0.1 % TFA in ACN were used. A gradient elution program was applied for chromatographic analysis.

[0175] The purity of the linear or oxidized lanreotide free acid was expressed as the percentage of area of the peak corresponding to linear or oxidized lanreotide free acid divided by the total area of all peaks detected.

[0176] 2. Example 1: Synthesis of dehydroascorbic acid (DHA) by oxidation of ascorbic acid (AA) with iodine Ascorbic acid was dissolved in water or appropriate buffer (pH<7) and maximal 0.99 molar equivalents of iodine were added. Reaction was mixed until disappearance of yellow color (usually less than 5 min). The pH was then adjusted to the corresponding values. The resulting DHA was used without further purification.

[0177] 3. Example 2: Synthesis of DHA by oxidation of ascorbic acid with oxygen in presence of active coal

[0178] 5 g ascorbic acid was dissolved in 150 ml ethanol and 7 g activated charcoal (carbon - Carbopal Gn) was added. Air was bubbled through the mixture at room temperature. The reaction was controlled by HPLC (see general methods). After the completion of reaction (usually after 6h), the active charcoal was filtered off, and the solvent evaporated under reduced pressure. To the residual viscous slurry, 10 ml H2O was added, and the mixture was frozen and lyophilized. The solid obtained (4.4 g) was stored at -20°C and used without further purification. The analogous procedure was successfully carried out using isopropanol or methanol as the solvent.

[0179] 4. Example 3: Stability of DHA in solution

[0180] DHA was dissolved in 50 mM phosphate buffer containing 10% ACN (6 mg / ml) and pH was adjusted to corresponding values. At given times, DHA mixtures were analyzed using HPLC (see general methods). Time-dependent degradation of DHA is shown in Figure 1.

[0181] 5. Example 4: pH dependent kinetics of oxidation reaction

[0182] Linear octreotide peptide was dissolved at a concentration of 20 mg / ml in 50 mM NH4OAC buffer comprising 15 % ACN. 25% NH4OH was used to adjust the pH to different values in parallel 1 ml samples of the peptide solution. 88pl of DHA solution (80 mg / ml) in ACN was added to each of the samples. The reaction mixtures were mixed at room temperatures and reaction progress was measured using analytical HPLC at given times (see general methods). A representative chromatogram is shown in Figure 3. The kinetics of the oxidation reaction at different pH values is shown in Figure 2. 6. Example 5: Comparison of side reactions iodine vs. DHA

[0183] Octreotide peptide, i.e. sulfhydryl-oxidized peptide with the intramolecular disulfide bond formed between both Cys moieties, was dissolved at a concentration of 20 mg / ml in 50 mM NH4OAC buffer pH 6.5 comprising 15 % ACN. Parallel 1 ml samples of the peptide solution were mixed with either iodine (0.75 mg iodine per mg octreotide, i.e., 2.95 pmol iodine per mg octreotide [3 equivalents]; addition of I2 (40 mg / ml) solution in I to the peptide sample) or DHA (1.7 mg / mg octreotide, i.e., <= 11 .56 pmol DHA per mg octreotide[roughly 4 equivalents]; addition of DHA solution (80 mg / ml) in ACN to the peptide sample). The reaction mixtures were incubated for 1 h at either 20°C or 40°C, iodine was quenched with ascorbic acid, and subsequently analyzed using analytical HPLC (see general methods). As reference, octreotide solution without oxidants was used. Results of analytical HPLC are summarized in Table 3 and example chromatograms are given in Figure 4.

[0184] Table 3: HPLC purity of octreotide samples incubated with and without oxidants (iodine and DHA)

[0185] It was concluded that iodine undergoes extensive side reactions with the sulfhydryl- oxidized octreotide peptide, while DHA does not.

[0186] 7. Example 6: Comparison of side reactions iodine vs. DHA vs. H2O2

[0187] Lanreotide free acid, i.e. sulfhydryl-oxidized peptide with the intramolecular disulfide bond formed between both Cys moieties, was dissolved at a concentration of 20 mg / ml in 50% ACN. Parallel 2 ml samples of the peptide solution were mixed with either iodine (0.69 mg iodine per mg lanreotide free acid , i.e., 2.71 pmol iodine per mg lanreotide free acid [3 equivalents]; addition of I2 (40 mg / ml) solution in 90% ACN to the peptide sample) or DHA (0.47 mg / mg lanreotide free acid, i.e., <= 2.72 pmol DHA per mg lanreotide free acid [3 equivalents]; addition of DHA solution (25 mg / ml) in 90% ACN to the peptide sample), or H2O2 (0.09 mg / mg lanreotide free acid, i.e., <= 2.71 pmol H2O2 per mg lanreotide free acid [3 equivalents]; addition of H2O2 solution (10 mg / ml) in H2O to the peptide sample). The reaction mixtures were incubated for 1 h at 20°C, iodine was quenched with ascorbic acid. All reaction mixtures were analyzed using analytical HPLC (see general methods). As reference, lanreotide free acid solution without oxidants was used. Results of analytical HPLC are summarized in Table 6 and chromatograms are shown in Figure 12.

[0188] Table 6: HPLC purity of lanreotide free acid samples incubated with and without oxidants (iodine, hydrogen peroxide, and DHA)

[0189] It was concluded that iodine and hydrogen peroxide undergo significant side reactions with the sulfhydryl-oxidized lanreotide free acid peptide, while DHA does not.

[0190] 8. Example 7: Octreotide oxidation in solution; comparison iodine vs. DHA of different origins

[0191] A set of experiments were performed to screen for optimal oxidation conditions. Generally, a solution of oxidant was added to a solution of linear octreotide peptide. The reaction mixture was mixed at RT for the time indicated and analyzed with analytical HPLC (see general methods). Results are given in Table 4.

[0192] The impurity patterns of samples 21 q (DHA oxidation at high peptide concentration), 21 r (iodine oxidation at high peptide concentration using same buffer as in sample 21 q) and 21 s (iodine oxidation at high peptide concentration with optimized buffer conditions for iodine) are shown in Figure 5. Highest quantities of oligomers were found by oxidation with iodine in ammonium acetate buffer (18.2%; chromatogram 3), followed by oxidation with iodine in acetic acid (9.8%, chromatogram 4). By contrast, comparable quantities of oligomers were found by oxidation with DHA in ammonium acetate buffer (5.8%, chromatogram 2) as in non-oxidized sample (6.0%; chromatogram 1 ).

[0193] Table 4: Oxidation of octreotide linear precursor with different quantities of DHA and iodine.

[0194] Conclusion: DHA allows to use concentrated octreotide peptide solution; a similar peptide concentration gives around 10% less purity when oxidized with iodine (compare entries 21 r, 21s to 211, 21 m). At least a part of the improvement in purity obtained with DHA appears to be due to the suppression of disulfide bonded oligomers. No influence of the origin of the DHA used could be observed. 9. Example 8: Pramlintide oxidation in solution; comparison iodine vs. DHA vs. hydrogen peroxide

[0195] Example 8a: Product purity in dependence of peptide concentration

[0196] Pramlintide linear peptide precursor, i.e. reduced peptide with no intramolecular disulfide bond formed between both cysteine moieties, was dissolved in H2O at various concentrations as indicated in Table 7. Parallel 2 ml samples of peptide solution were mixed with either iodine (0.25 pmol / mg peptide [>=1 equivalent]; addition of I2 (40 mg / ml) solution in 90% ACN to the peptide samples) or DHA (0.25 pmol / mg peptide [>= 1 equivalent]; addition of DHA solution (27 mg / ml) in 50% ACN to the peptide solution) or H2O2 (0.25 pmol / mg peptide [>= 1 equivalent]; addition of H2O2 solution (10 mg / ml) in H2O to the peptide sample). For H2O2 and DHA oxidations, the pH was adjusted to 6-7 with 5% NH4OH. The reaction mixtures were stirred at room temperature until the oxidation reactions were completed (20 min for I2; 1 h for H2O2 and 2 h for DHA). The iodine was quenched with ascorbic acid. All reaction mixtures were analyzed with analytical HPLC (see general methods). Representative chromatograms are shown in Figure 8 and results are given in Table 7. Moreover, it was observed that the peptide in sample 1 n formed a gel, while sample 1v remained a clear solution.

[0197] It was concluded that oxidation with DHA gave higher purities of oxidized pramlintide compared to both iodine and hydrogen peroxide. As observed with octreotide in example 7, DHA oxidation of pramlintide was not sensitive to peptide concentration and compatible with using a rather concentrated peptide solution of about 19 mg / ml.

[0198] Table 7 Oxidation of pramlintide linear precursor at different concentrations with iodine, DHA and hydrogen peroxide.

[0199] Example 8b: Comparison of DHA and H2O2 at acidic pH

[0200] As pramlintide is known to be more stable in acidic solution and the gel formation in sample 1 n pointed to a lack of robustness of the process at pH 6-7, it was investigated whether pramlintide can be oxidized at acidic pH with either DHA or hydrogen peroxide.

[0201] Pramlintide linear peptide precursor, i.e. reduced peptide with no intramolecular disulfide bond formed between both cysteine moieties, was dissolved in H2O (10 g / L). To the pramlintide solution was added either DHA (0.5 pmol / mg peptide [>=2 equivalent]; or H2O2 (0.25 pmol / mg peptide [>= 1 equivalent]; The pH was adjusted to around 5 with 5% NH4OH and reactions were mixed at room temperature for 120 or 300 min, respectively, before analysis with analytical HPLC (see general methods).

[0202] For the DHA sample, the reaction was somewhat slower at acidic pH, in line with the observations of example 4. Nevertheless, the linear peptide was completely consumed and highly pure oxidized peptide formed at pH 4.8 within 120 min (HPLC purity of oxidized peptide: 77.5%; no linear peptide detectable). In contrast, a significant amount of linear peptide was still detectable after 5 hours in the sample oxidized with H2O2 at pH 5.1 and the formation of side products could be observed (HPLC purity of oxidized peptide: 55%; HPLC purity of linear peptide: 26%).

[0203] 10. Example 9: Lanreotide free acid oxidation in solution; comparison iodine vs. DHA vs. hydrogen peroxide

[0204] Lanreotide free acid, i.e. reduced peptide with no intramolecular disulfide bond formed between both cyteine moieties, was dissolved at different concentrations in 50% aqueous ACN. Parallel 2 ml samples of the peptide solution were mixed with either iodine (0.23 mg iodine per mg lanreotide free acid , i.e., 0.91 pmol iodine per mg lanreotide free acid [>= 1 equivalent]; addition of I2 (40 mg / ml) solution in 90% ACN to the peptide sample) or DHA (0.16 mg / mg lanreotide free acid, i.e., <= 0.90 pmol DHA per mg lanreotide free acid [>= 1 equivalent]; addition of DHA solution (15 mg / ml) in 49%ACN to the peptide sample), or H2O2 (0.03 mg / mg lanreotide free acid, i.e. , <= 0.90 pmol H2O2 per mg lanreotide free acid [>= 1 equivalent]; addition of H2O2 solution (10 mg / ml) in H2O to the peptide sample). pH of reaction mixtures was adjusted to 6-6.5 for reactions with DHA and H2O2 with 5% NH4OH. The reaction mixtures were incubated until reactions were completed (20 min at 20°C for iodine; 60 min at 20°C for H2O2 and 180 min at 40°C for DHA. Iodine was quenched with ascorbic acid. All reactions mixtures were analyzed using analytical HPLC (see general methods). As reference, lanreotide free acid reduced solution without oxidants was used. Results of analytical HPLC are summarized in Table 8 and example chromatograms are given in Figure 9.

[0205] Table 8 Oxidation of lanreotide free acid linear precursor at different concentrations with iodine, DHA and hydrogen peroxide.

[0206] Conclusion: DHA allowed for high purity of the oxidized product independently on the peptide concentration. Oxidation with hydrogen peroxide produced almost exclusively side-products; for the oxidation with iodine, high cyclic peptide purity could only be achieved at low peptide concentration. 11. Comparative Example 10: Octreotide oxidation in solution with iodine under high-dilution conditions

[0207] Solutions (8 ml) of octreotide linear crude peptide in 30 % AcOH (50 g / l) and 8.4 ml iodine / KI solution (8.6 g / l iodine, 16.2 g / l KI) in H2O were separately added, via a dual syringe pump (8 ml / h) to the reaction vessel containing 9 ml 30% AcOH. After addition reaction mixture was stirred for 10 minutes and the iodine excess quenched with 4% ascorbic acid (20 pl). The product purity in the reaction mixture was 84.2%.

[0208] The cyclized product was purified using an C18 column (10x250 mm) column on a UHPLC-System equipped with an automated fraction collector. Eluents A: 3% ACN 0.1 % TFA and B: 60% ACN 0.1 % TFA were used. At first, the column was equilibrated with eluent A. Then, the reaction mixture (22.1 ml) was loaded and washed with Eluent A, 1 M NH4OAC 10% ACN, and Eluent A. The product was eluted using a gradient program. Collected fractions were analyzed with analytical HPLC (see general methods) and all fractions with octreotide purity >99% were pooled together and lyophilized. Using this protocol 220 mg (63% of the initially loaded 347 mg) octreotide with HPLC purity 99.6% was obtained.

[0209] Conclusion: The optimized, high dilution protocol with iodine as an oxidant gave a lower purity (84.2%) as compared to the purities obtained with the simpler, high concentration protocols developed with DHA as an oxidant (purity 90.2% and 89.9%, see entries 211, 21 m of example 7 above).

[0210] 12. Example 11: Octreotide oxidation in solution with DHA under high- concentration conditions

[0211] DHA (105.2 mg [>= 1 .5 equivalents]) was added to a solution (20 ml) of octreotide linear crude peptide (20 g / l) in 50 mM NH4OAC, 15% ACN pH 6.7. The reaction mixture was stirred for 95 min. The product purity in the reaction mixture was 87.4%. The cyclized product was diluted with 10 ml H2O and purified (25.9 ml loading) using the same equipment and method as described in example 10. Using this protocol, 224 mg (65% of the initially loaded 345 mg) octreotide with HPLC purity 99.6% was obtained.

[0212] Conclusion: The simpler, high concentration protocol with DHA as an oxidant again gave a slightly higher purity of 87.4% compared to the optimized, high dilution protocol with iodine as an oxidant (84.2%, see comparative example 10 above). For the overall process comprising oxidation and subsequent peptide purification, the high concentration protocol with DHA as an oxidant not only required less time, but also resulted in slightly increased total yield (65% vs. 63%). It was estimated that at industrial scale, the processing time could be reduced by at least 14% as compared to the process of example 10. Hence, this protocol allows to improve process economy in two aspects.

[0213] 13. Example 12: Pramlintide oxidation in solution with DHA under high- concentration conditions

[0214] 1.76 ml DHA solution (15 mg / ml) was added to 30 ml Pramlintide solution (10 mg / ml). The pH was adjusted to 6 with 5% NH4OH. The oxidation reaction was mixed at RT and was completed after 20 min. The molar ratio of total DHA divided by pramlintide peptide was estimated to be at least 2.0. The oxidized product had a HPLC purity of 79.1 % at a concentration of about 10mg / ml and was purified using a C18 column (10x250 mm) column on a UHPLC-System equipped with an automated fraction collector. Eluents A: 3% ACN 0.1 % TFA and B: 80% ACN 0.1 % TFA were used. At first, the column was equilibrated with 2 CV of eluent A. After loading the cyclized pramlintide, the column was washed with eluent A (1 CV) and the product was eluted using a gradient program 0%B - T - 20%B - 128' - 100%B. Collected fractions were analyzed with analytical HPLC (see general methods) and all fractions with pramlintide purity >92% were pooled together and lyophilized. Using this protocol, 99 mg (gross yield 44%) pramlintide with HPLC purity 94.2% was obtained. The PMI of the process was calculated as 2’002 kg / kg.

[0215] By comparison, a conventional high dilution iodine oxidation protocol for pramlintide yielded an oxidized product with HPLC purity of 52-58% at a concentration of 2 mg / ml. This product required a two-dimensional HPLC purification to achieve adequate product purities, which resulted in a gross yield of 22% pramlintide with a HPLC purity of 98.2%. The PMI of the process was calculated as 14’878 kg / kg.

[0216] Conclusion: Compared to oxidation with iodine, the in-solution oxidation with DHA resulted in higher purity of oxidized product (79.1 % vs. 52-58% HPLC purity) at higher concentration (10 mg / ml vs. 2 mg / ml). When considering the oxidation together with the subsequent purification steps, a significant increase of yield (44%) for in-solution oxidation with DHA relative to in-solution oxidation (22%) with iodine with similar final product purity was observed. Processes based on DHA oxidation are therefore expected to have a significantly reduced PMI, compared to in-solution oxidation with iodine. 14. Example 13: Oxidation of octreotide with DHA on column

[0217] For the on-column oxidation experiments, the same equipment and eluents like in Example 10 were used. At first, the column was equilibrated with eluent A. Then, a solution (22.1 ml) of linear crude octreotide peptide in 50 mM NH4OAC 15% ACN pH 6 (16 g / l) was loaded on the column and washed with 1.5 CV 50 mM NH4OAC 15% ACN pH 6.7. The column load was 0.45 g / cm2The loaded linear peptide was oxidized by pumping a solution of DHA (2.4 g / l) in 50 mM NH4OAC 10% ACN pH 6 for 50 min (3 CV). The molar ratio of total DHA divided by octreotide peptide was estimated to be at least 2.4. Afterwards, the column was washed with 50 mM NH4OAC 10% ACN and eluent A and the product was eluted using a gradient program. Collected fractions were analyzed with analytical HPLC (see general methods) and all fractions with octreotide purity >99% were pooled together and lyophilized. Using this protocol, 209 mg (59%) octreotide with HPLC purity 99.4% was obtained.

[0218] Conclusion: The on-column oxidation and purification protocol delivered octreotide of comparable purity, but with slightly lower overall yield (59%) as compared to the comparative example 10 (63 %). However, the protocol is significantly faster as compared to the process of example 10, thereby improving process economy. It was estimated that at industrial scale, the processing time could be reduced by at least 27% as compared to the process of example 10.

[0219] 15. Example 14: Oxidation of octreotide with DHA on column

[0220] Comparable results to those of example 11 were obtained in analogous experiments with commercial DHA (Sigma-Aldrich®), DHA prepared by oxidation of ascorbic acid with iodine (Example 1 ) and with DHA obtained by ascorbic acid oxidation with O2 (Example 2). Phosphate, and carbonate buffers with Na+and NH4+counter ions were also used with comparable results as shown in Table 5. A representative chromatogram for the entire oxidation and purification process is given in Figure 6.

[0221]

[0222] Table 5: On column oxidation of octreotide with DHA of various sources and different buffer conditions. 16. Example 15: Oxidation of atosiban with DHA on column

[0223] On-column oxidation experiments were performed on a C8 column (4.6x250 mm) column on a UHPLC-System equipped with an automated fraction collector. Eluent A: 10% ACN 0.1 % TFA and eluent B: 50% ACN 0.1 % TFA were used. At first, the column was equilibrated with eluent A. Then, a solution (7 ml) of linear crude atosiban precursor in Eluent A (15 g / l) was loaded and washed with 0.5 M NF OAc 10% ACN pH 5. The column load was 0.6 g / cm2The loaded linear peptide was oxidized by pumping a solution of DHA (7.3 g / l) in 0.5 M NH4OAC 10% ACN pH 6 for 50 min (3 CV). The molar ratio of total DHA divided by atosiban peptide was estimated to be at least 5. Afterwards, the column was washed with 50 mM NH4OAC 10% ACN pH 5 and eluent A and the product was eluted using a gradient of eluent B. Collected fractions were analyzed with analytical HPLC (see general methods). Fractions contained cyclized product of 94.5%-97.2% purity and recovery 87%- 99%. A representative chromatogram is given in Figure 7.

[0224] 17. Example 16: Oxidation of pramlintide with DHA on column

[0225] For the on-column oxidation experiments, the same equipment and eluents like in Example 12 were used. At first, the column was equilibrated with 2 CV of eluent A. Then, the solution of linear pramlintide precursor (10 g / l; 24.1 ml) was loaded on the column and washed with 1.3 CV 50 mM NH4OAC 5% ACN pH 7. The column load was 0.31 g / cm2. The molar ratio of total DHA divided by pramlintide peptide was estimated to be at least 3.7. The loaded linear peptide was oxidized by pumping a solution of DHA (0.62 g / l) in 50 mM NH4OAc 15% ACN pH 5.4 for 50 min (3 CV). Afterwards, the column was washed with eluent A (1 CV) and the product was eluted using a gradient program 0%B - T - 20%B - 128' - 100%B. Collected fractions were analyzed with analytical HPLC (see general methods) and all fractions with pramlintide purity >97% were pooled together and lyophilized. Using this protocol, 97 mg (gross yield 241 mg / 97 mg *100 = 40%) pramlintide with HPLC purity 97.3% was obtained. The PMI for the oxidation plus purification step was calculated as 2878 kg / kg, a reduction of about 81 % relative to the conventional high dilution iodine oxidation protocol for pramlintide.

[0226] 18. Example 17: Oxidation of human calcitonin with DHA on column

[0227] Because reduced calcitonin is poorly soluble with a maximal solubility of around 5 mg / ml in most aqueous buffers, on-column oxidation is attractive. Experiments were performed on a C8 column (10x250 mm) column on a UHPLC-System equipped with an automated fraction collector. Eluents A: 3% ACN 1 % AcOH and B: 80% ACN 1 % AcOH were used. The column temperature during the whole procedure was 40°C. At first, the column was equilibrated with 1.5 CV of eluent A. Then, the solution of linear calcitonin precursor (5 g / l; 40.7 ml) was loaded on the column and washed with 1 .3 CV Eluent A. The column load was 0.26 g / cm2. Next, an isocratic step 1.3 CV of 20% B was applied. The loaded linear peptide was oxidized by pumping a solution of DHA (0.7 g / l) in 50 mM NH4OAc 15% ACN pH 4.9 for 50 min (3 CV). The molar ratio of total DHA divided by calcitonin peptide was estimated to be at least 4.5. Afterwards, the column was washed with eluent A (1 CV) and the product was eluted using a gradient program 0%B - T - 20%B - 5T - 52%B. Collected fractions were analyzed with analytical HPLC (see general methods) and all fractions with calcitonin purity >94% were pooled together and lyophilized. Using this protocol, 60 mg (gross yield 203.5 Z60 *100 = 34%) calcitonin with HPLC purity 98.0% was obtained and the PMI was calculated as 4681 kg / kg. A representative HPLC chromatogram is given in Figure 10.

[0228] If 0.02% TFA was used as additive in eluents instead of 1 % AcOH, 59 mg (gross yield 29%) calcitonin with HPLC purity 97.5% was obtained.

[0229] By comparison, a traditional high-dilution protocol using cystine / air as oxidation agent required a two-dimensional purification to achieve similar purity to yield 97.4% purity (gross yield 28%); the PMI was 6357 kg / kg. Hence, compared to the high dilution protocol using cystine / air, the on-column protocol with DHA resulted in improved product purity, a better yield, and overall PMI reduction by around 26%.

[0230] 19. Example 18: Oxidation of human calcitonin on column with DHA pulse protocol

[0231] The same equipment and eluents as in Example 17 were used. The column temperature during the whole procedure was 40°C. At first, the column was equilibrated with 1 .5 CV of eluent A. Then, the solution of linear calcitonin precursor (5 g / l; 40.7 ml) was loaded on the column and washed with 50 mM NH4Ac 5% ACN pH 8. The column load was 0.26 g / cm2. The loaded linear peptide was oxidized using a pulse protocol, where the DHA solution (1.3 g / L, pH 5.1 , for 3 min) was pumped over the column in alternation with 50 mM NH4OAc 5 % ACN (pH 8, for 5 min) at the same flow. Altogether, 15 pulses were applied. Afterwards, the column was washed with eluent A (1 CV) and the product was eluted using a gradient program 0%B - T - 20%B - 5T - 52%B. The collected fractions were analyzed with analytical HPLC (see general methods) and all fractions with calcitonin purity >94% were pooled together and lyophilized. Using this protocol, 26 mg (gross yield 13%, recovery 36% calcitonin with HPLC purity 98.4% was obtained. However, had less stringent pooling criteria (end purity 95%) been applied, the recovery would have been 76% and the expected yield can be calculated as 26%.

[0232] A representative HPLC chromatogram for the pulse oxidation is given in Figure 11 .

[0233] 20. Example 19: Oxidation of octreotide in solution with temporary excess of DHA

[0234] Dehydroascorbic acid was prepared in situ by adding 391 mg iodine (1 .54 mmol) to a solution of 311 mg ascorbic acid (1 .77 mmol) in 17 ml 1 M NH4OAC 10% ACN pH 5.4. The solution was colorless, indicating complete conversion of iodine to iodide. A solution (8ml) of octreotide linear crude peptide in 30 % NMP (50 g / l) was added, via a syringe pump (8 ml / h) to the reaction vessel (total amount added: <0.39 mmol, 3.9 fold excess of DHA over total amount of linear peptide on average; the excess within the first minute of addition is estimated as 1.77 mmol I (8ml / 60min *1 min *50mg / ml / 1021 mg / mmol) = 271 fold). After addition, the reaction mixture was stirred for 1 h at room temperature. The product purity in the reaction mixture was 88.2%.

[0235] Conclusion: When using DHA as oxidizing agent, it was not necessary to control its amount in the reaction mixture so as to avoid a temporary molar excess of oxidizing agent vs. peptide. Higher product purity (88.2% vs. 84.2% in comparative Example 10) could be obtained as compared to the standard procedure of Example 10, where both the peptide and iodine as the oxidizing agent were subjected to pseudodilution.

Claims

Claims1 ) A method for the manufacture of a peptide (Pox) comprising an intramolecular disulfide bond, comprising the following steps: a. Providing a composition comprising a chemically synthesized peptide (Pred), which has at least two free sulfhydryl groups; b. Combining the composition of step a with at least one equivalent of dehydroascorbic acid, relative to the total amount of free sulfhydryl groups contained in the composition of step a, so as to obtain an aqueous reaction composition (C1 ) having a pH of 3.0 to 8.0 and comprising dehydroascorbic acid and the peptide (Pred), wherein the peptide (Pred) is either present in this aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column at an initial amount of at least 0.05 g net peptide per cm2column diameter or is dissolved in the aqueous reaction composition (C1 ) at an initial concentration of at least 1 mg net peptide per ml, or wherein the initial amount of dehydroascorbic acid in the aqueous reaction composition (C1 ) is at least one equivalent, relative to the total amount of free sulfhydryl groups contained in the composition of step a; and c. Maintaining the aqueous reaction composition (C1 ) for a time sufficient to allow conversion of the peptide (Pred) to the disulfide bonded peptide (Pox), thereby obtaining the disulfide bonded peptide (Pox).2) The method according claim 1 , wherein the initial amount of dehydroascorbic acid in the aqueous reaction composition (C1 ) is at least 1 equivalent relative to the total amount of free sulfhydryl groups contained in the composition of step a, and the final concentration of the net disulfide bonded peptide (Pox) in the aqueous reaction composition (C1 ) is at least 1 mg / ml after step c.3) The method according to any one of claims 1 ) and 2), wherein the peptide (Pred) is either present in this aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column at an initial amount of at least 0.1 g net peptide per cm2column diameter or isdissolved in the aqueous reaction composition (C1 ) at an initial concentration of at least 3 mg net peptide per ml.4) The method according to any one of claims 1 ) to 3), wherein step b comprises combining the composition of step a with a total amount of 1.3 to 10 equivalents of dehydroascorbic acid, relative to the total amount of free sulfhydryl groups contained in the composition of step a, preferably wherein the initial amount of dehydroascorbic acid in the aqueous reaction composition (C1 ) is 1.3 to 10 equivalents, relative to the total amount of free sulfhydryl groups contained in the composition of step a.5) The method according to any one of claims 1 ) to 4), where the reaction composition (C1 ) comprises an aqueous buffer, preferably wherein the buffer comprises components selected from the group consisting of acetic acid, citric acid, formic acid, phosphoric acid, carbonic acid, and ammonium.6) The method according to any one of claims 1 ) to 5), wherein step a comprises the following: a.a1 . Conducting solid phase peptide synthesis; a.b1 . Cleaving the peptide from the solid support and removing at least the protecting groups from two sulfhydryl moieties so as to obtain a cleaved and at least partially deprotected peptide with at least two free sulfhydryl groups (Pred); a.c. Isolating the peptide (Pred); a.d. Dissolving the isolated peptide (Pred) to obtain a solution thereof; a.e. Optionally, flowing the solution of step a.d. through a reversed phase liquid chromatography column, so as to allow adsorption of the peptide (Pred) to the stationary phase of the column; and a.f. Optionally, eluting the peptide (Pred) from the column so as to obtain an eluted solution comprising the peptide (Pred).7) The method according to any one of claims 1 ) to 6), comprising the following steps: a.

1. Providing an aqueous solution (S1 ) comprising a chemically synthesized peptide (Pred), which has at least two free sulfhydryl groups; a.2 Flowing the solution (S1 ) through a reversed phase liquid chromatography column, so as to allow adsorption of the peptide (Pred) to the stationary phase of the column;b.1 Contacting the adsorbed peptide on the stationary phase with a solution (S3) having a pH between 3.0 and 8.0, and comprising at least one equivalent of dehydroascorbic acid, relative to the amount of free sulfhydryl groups on the stationary phase; c.1 Maintaining the adsorbed peptide (Pred) on the stationary phase in contact with the solution (S3) for a time sufficient to allow conversion of the adsorbed peptide (Pred) to an adsorbed disulfide bonded peptide (Pox); and d.1 Eluting the adsorbed disulfide bonded peptide (Pox) from the stationary phase with a solution (S5) containing an organic modifier.8) The method according to claim 7), wherein the elution is performed such that the disulfide bonded peptide (Pox) is separated from at least one unwanted peptidic component.9) The method according to any one of claims 7) and 8), further comprising one or both of the following steps: a.3 washing the chromatography column after step a.2 with at least one aqueous liquid (S2); and c.2 washing the chromatography column after step c.1 with at least one aqueous liquid (S4).10) The method according to any one of claims 1 ) to 9), wherein the aqueous reaction composition (C1 ) has a pH of 4.0 to 7.0.11 ) The method according to any one of claims 7) to 10), wherein the solution (S1 ), the solution (S3), and, if used, the liquid (S2) and the liquid (S4), comprise between 1 % and 40% of an organic modifier.12) The method according to any one of claims 1 ) to 11 ), wherein the reversed phase chromatography column contains a stationary phase selected from the group consisting of C4 silica, C8 silica, C18 silica, phenyl silica, biphenyl silica, phenyl-hexyl silica, and polar-embedded solid phases.13) The method according to any one of claims 7) to 12), wherein the organic modifier is selected from the group consisting of acetonitrile, methanol, ethanol, isopropanol, and mixtures thereof.14) The method according to any one of claims 1 ) to 13), wherein the disulfide bonded peptide (Pox) obtained is conjugated to another moiety, thereby yielding a disulfide bonded and conjugated peptide.15) The method according to any one of claims 1 ) to 14), further comprising subjecting the disulfide bonded peptide (Pox) or the disulfide bonded and conjugated peptide to one or more additional purification steps; or further comprising a step of drying, preferably freeze-drying, the disulfide bonded peptide (Pox) or the disulfide bonded and conjugated peptide; or further comprising subjecting the disulfide bonded peptide (Pox) or the disulfide bonded and conjugated peptide to one or more additional purification steps followed by a step of drying, preferably freeze-drying, the disulfide bonded peptide (Pox) or the disulfide bonded and conjugated peptide.16) The method according to any one of claims 1 ) to 15), wherein in step c., the disulfide bonded peptide (Pox) is obtained at a concentration of at least 1 mg net peptide per ml or in an amount of at least 0.05 g net peptide per cm2column diameter.17) The method according to any one of claims 1 ) to 16), wherein the peptide (Pred) is either present in this aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column at an initial amount of 0.05 to 1.5 g net peptide per cm2column diameter or is dissolved in the aqueous reaction composition (C1 ) at an initial concentration of 1 to 30 mg net peptide per ml; or wherein the initial amount of dehydroascorbic acid in the aqueous reaction composition (C1 ) is 1 to 50 equivalents relative to the total amount of free sulfhydryl groups contained in the composition of step a, and the final concentration of the net disulfide bonded peptide (Pox) in the aqueous reaction composition (C1 ) is 1 to 30 mg / ml.18) The method according to any one of claims 9 to 17), wherein the solution (S3) has a pH between 3.0 and 5.0, and the aqueous liquid (S4) has a pH of 7.0 to 9.0, and steps b.1 , c.1 and c.2 are repeated multiple times.19) The method according to any one of claims 1 to 18), wherein the total amount of net peptide (Pred), which is provided in step a. is least 1 g, preferably 100 to 50’000 g.20) The method according to any one of claims 1 ) to 19)16), wherein the total amount of net disulfide bonded peptide (Pox) obtained in step c. is at least 1 g, preferably at least 10 g.21 ) The method according to any one of claims 7) to 20), wherein the total amount of net disulfide bonded peptide (Pox) obtained in step d.1 . is at least 1 g, preferably at least 10 g.22) An aqueous reaction composition (C1 ) having a pH of 3.0 to 8.0 comprising dehydroascorbic acid and a chemically synthesized peptide (Pred), which has at least two free sulfhydryl groups, wherein the peptide (Pred) is either present in this aqueous reaction composition (C1 ) adsorbed to a stationary phase of a reversed phase liquid chromatography column at an initial amount of at least 0.05 g net peptide per cm2column diameter or is dissolved in the aqueous reaction composition (C1 ) at an initial concentration of at least 1 mg net peptide per ml, and wherein the initial amount of dehydroascorbic acid in the reaction composition (C1 ) is at least one equivalent, relative to the total amount of disulfide bonds to be formed.

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