Preparation of disulfide-linked peptides

Abstract

Methods and apparatus for producing disulfide-linked peptides are provided in which a solution of an oxidizing agent and a solution of a peptide containing at least two sulfhydryl groups are added simultaneously to a reaction vessel under conditions such that the average concentration of oxidizing agent in the reaction vessel is essentially zero during the simultaneous additions.

Description

[Technical Field]

[0001] The present invention generally relates to the field of industrial or laboratory-scale peptide synthesis. In particular, an improved method for producing peptides containing at least one intramolecular disulfide bond, particularly for the production of peptides containing one or more cystine residues, is disclosed. The present invention is directed to a method for effectively producing such peptides. Disulfide bonds are found in many peptides, and they are often important for peptide activity in that they stabilize the tertiary structure of the peptide. Therefore, the chemical synthesis of disulfide-bonded peptides is of considerable commercial importance. [Background technology]

[0002] Chemical peptide synthesis is generally well known in the art and typically proceeds from the C-terminus to the N-terminus of the peptide (see, e.g., "Chemical Synthesis of Peptides," vol. 1, pp. 111-114, 1999). During synthesis, peptide bond formation between the alpha amino group of a first amino acid and the alpha carboxyl group of a second amino acid should be favored over unintended side reactions. This is generally achieved by the use of suitable protecting groups.

[0003] Two standard approaches to chemical peptide synthesis are distinguished: liquid phase peptide synthesis (LPPS) and solid phase peptide synthesis (SPPS). In addition to LPPS and SPPS, hybrid approaches can be utilized in which fragments are first synthesized by one of the techniques mentioned above and then joined together using the other method.

[0004] The so-called Fmoc SPPS, which relies on the use of 9-fluorenylmethyloxycarbonyl (Fmoc) as a temporary amino-protecting group, is the most widespread form of SPPS. A growing peptide chain is anchored to an insoluble polymer resin via its C-terminal amino acid. The peptide is assembled by the sequential addition of the Fmoc-protected amino acids that make up its sequence.

[0005] Successive cycles of amino acid addition are performed, each consisting of a) cleavage of the Nα-protecting Fmoc group from the resin-bound peptide, b) a washing step, c) coupling of a protected amino acid, and d) a washing step. The peptide is then typically cleaved from the solid support, the protecting groups removed, and the peptide isolated.

[0006] The synthesis of disulfide-linked peptides typically involves the first synthesis of the full-length peptide sequence, followed by the formation of intramolecular disulfide bonds. In this latter step, intramolecular reactions compete with intermolecular disulfide bond formation, leading to disulfide-linked dimers and multimers as by-products. To suppress these undesired side reactions, several strategies have been applied.

[0007] For example, Patent Document 1 discloses a method involving oxidation on an SPPS resin with hydrogen peroxide prior to cleavage from the solid support. However, this pseudo-dilution approach can lead to side reactions of disulfide bonds during subsequent deprotection and cleavage from the solid support. Furthermore, monitoring oxidation on the resin is more demanding than monitoring oxidation reactions in solution, and the reproducibility of this approach is highly dependent on achieving regenerable resin loading and swelling. Alternatively, immobilizing oxidizing agents on a solid support has been reported (Postma and Albericio, 2014). However, this is cost-intensive and can lead to oxidation of sensitive amino acids.

[0008] The most common strategy is to perform the oxidation reaction in a very dilute peptide solution. An oxidizing reagent, such as iodine, is usually added to the peptide solution to initiate the reaction. Alternatively, Patent Document 2 teaches a method in which a peptide containing at least two cysteine ​​moieties is dissolved in an acidic aqueous solution at approximately 0.5-10 g / L and the peptide solution is slowly added to a buffer solution containing an oxidizing agent. This addition is carried out under conditions in which the concentration of sulfhydryls, i.e., linear peptides, in the reaction mixture is maintained essentially at zero throughout the reaction. Similarly, Non-Patent Document 2 discloses a method for disulfide bond formation by slowly adding a dilute peptide solution (free thiol form, 0.1-10 mM) under nitrogen to a 10 mM aqueous solution of KFe(CN) (20% excess oxidizing agent).

[0009] However, the above-mentioned methods require handling impractically large amounts of peptide solution, limiting the scalability of the method. When side reactions at the side chains of Trp or Met need to be avoided, Non-Patent Document 3 teaches adding the peptide solution and the oxidant solution to the reaction mixture simultaneously and at the same rate to produce a reaction solution containing a stable and optimal ratio of both educts. To reduce solvent usage without compromising product quality, Non-Patent Document 4, Patent Document 3, and Patent Document 4 teach the use of nanofiltration with recycling of the permeate after the completion of the oxidation reaction. Non-Patent Document 5 teaches the application of membrane filtration to the condensation reaction of peptide fractions. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 5,656,721 [Patent Document 2] U.S. Patent No. 4,216,141 [Patent Document 3] WO2016 / 042066 [Patent Document 4] WO2013 / 156600 [Non-patent literature]

[0011] [Non-Patent Document 1] brochure "Solid Phase Peptide Synthesis Bachem - Pioneering Partner for Peptides" published by Global Marketing, Bachem group, June 2014 [Non-patent document 2] Annis et al., Methods in Enzymology, Vol. 289, pp. 198-221, 1997 [Non-patent document 3] Misicka and Hruby, Polish J.Chem., vol. 68, pp. 893-899, 1994 [Non-patent document 4] Ormerod et al., Org. Process Res. Dev. 2015, vol. 19, pp. 841-848 [Non-patent document 5] Marchetti et al., Sustainable nanotechnology and the environment: Advances and achievements, 2013, vol. 1124, pp. 121-150. Summary of the Invention [Problem to be solved by the invention]

[0012] There remains a need in the art for simple, reliable, scalable and cost-effective methods for producing disulfide-linked peptides suitable for industrial-scale applications. [Means for solving the problem]

[0013] The present inventors have surprisingly discovered such a procedure in which concentrated solutions of peptide and oxidizing agent are added simultaneously to a reaction mixture. This addition is performed so that the oxidation reaction to form disulfide bonds occurs substantially instantly, maintaining the oxidizing agent concentration during the simultaneous addition at a substantially constant level, essentially equivalent to zero. This contrasts with the teachings of the prior art, in which the oxidizing agent is typically used in excess from the start of the reaction. In the procedure according to the present invention, the addition of the oxidizing agent is continued after the peptide addition is complete, which can result in excess oxidizing agent at the end of the addition. Unexpectedly, and contrary to the teachings of Misicka and Hruby, which avoid excess oxidizing agent, the present inventors have not observed over-oxidation of the Trp side chain. The method and apparatus of the present invention are surprisingly capable of producing high-quality disulfide-bonded peptides at high concentrations. Without being bound by theory, it is believed that this is achieved by precisely controlling the reaction conditions throughout the oxidation reaction.

[0014] In general, several abbreviations and definitions are used throughout this application: Acm Acetamidomethyl Boc tert.butyloxycarbonyl Bzl Benzyl Dpm Diphenylmethyl DTE Dithioerythritol DTT Dithiothreitol EDT 1,2-ethanedithiol Fmoc 9-Fluorenylmethyloxycarbonyl LPPS Liquid Phase Peptide Synthesis Mmt Methoxytrityl Mtt Methyltrityl Phacm phenylacetamidomethyl SPPS Solid Phase Peptide Synthesis StBu tert.butyl mercapto tBu tert.butyl TFA trifluoroacetic acid TIPS Triisopropylsilane Trt Trityl

[0015] Unless otherwise specified, pH values ​​are given for the temperature at which the particular aqueous solution will be used. Unless otherwise specified, the term "about" means a deviation of plus or minus 10% from the given numerical value.

[0016] Amino acids are referred to interchangeably by either their full name (e.g., alanine), their three-letter code (e.g., Ala) or their one-letter code (e.g., A) according to WIPO Standard ST.25. L-amino acids are generally referred to unless the enantiomer is explicitly specified. However, it should be noted that the present invention can be practiced using D-amino acids and other stereoisomers as well.

[0017] As used herein, the terms "peptide" and "polypeptide" are understood interchangeably. Unless otherwise indicated, peptide sequences are shown herein beginning at the N-terminus (left) and ending at the C-terminus (right). Peptides are characterized by the presence of at least one peptide bond (-CO-NH-) between at least two amino acids, i.e., moieties containing a carboxyl (-COOH) and a primary or secondary amino group (-NHR). Thus, it should be understood that the term "peptide" is not limited to peptide structures derived from natural amino acids. "Peptide" also includes, inter alia, peptide derivatives, peptides containing unnatural amino acids, peptides containing D-amino acids, and peptides containing covalently or non-covalently attached linkers, dyes, or other moieties.

[0018] The term "analog" or "analogs" as used herein refers to a peptide whose sequence is derived from a first peptide sequence by replacement of up to 50% of amino acid moieties, and / or by deletion of up to 10% of the amino acid moieties of said first peptide sequence, and / or by addition of up to 10 amino acid moieties.

[0019] Preferred analogs are derived from the first peptide sequence by replacement of up to 20% of the amino acid moieties, and / or by deletion of up to 10% of the amino acid moieties of said first peptide sequence, and / or by addition of up to 10 amino acid moieties.

[0020] The term "derivative" or "derivatives," as used herein, refers to a compound that can be obtained from a first compound by chemical reaction. As a result, a derivative can differ from the first compound by the presence or absence of a substituent. For example, an amino acid derivative for use in SPPS typically differs from the amino acid from which it is derived by the presence of at least an amino-protecting group.

[0021] The term "protecting group", as used herein, is understood in the broadest sense as a group introduced into a molecule by chemical modification of said group in order to block said functional group from reacting in a subsequent method step, for example to prevent side reactions of an amino acid side chain. Examples of amino protecting groups are the Boc group and the Fmoc group, and examples of carboxylic acid protecting groups are non-reactive esters such as methyl ester, benzyl ester or tert.butyl ester. Examples of sulfhydryl protecting groups include, for example, Acm, Phacm, Trt, Mtt, Mmt, Dpm, Bzl, tBu and StBu protecting groups.

[0022] For purposes of this application, the terms "unpurified" and "crude" are used interchangeably to designate peptide preparations that are essentially the direct product of the synthesis and isolation process and have not yet been subjected to specific purification steps. Chemical synthesis typically yields crude peptide preparations with a purity of about 40-80%.

[0023] In the context of the present invention, the term "purified" is used to designate a peptide composition that has been subjected to a particular purification step, e.g., preparative chromatography. Such compositions may be highly or partially purified and have a purity of up to 100%. However, it should be understood that the present invention applies advantageously to crude, partially purified, and purified peptide compositions.

[0024] Unless otherwise specified, peptide purity is referred to herein as "HPLC purity," i.e., the relative peak area observed by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) with UV detection at wavelengths between 205 and 230 nm, i.e., at the absorption maximum of the peptide bond. In other words, this value is determined as the area % of a given peak divided by the sum of the areas of all peaks observed in a chromatogram obtained by analytical RP-HPLC with UV detection at wavelengths between 205 and 230 nm. This measure is common practice in the art, and those skilled in the art routinely design product-specific RP-HPLC protocols and perform quantification in accordance with established guidelines, e.g., as set forth in the United States Pharmacopeia. The suitability of RP-HPLC protocols for detecting peptide contaminants is routinely assessed by determining peak purity by LC-MS. Under the assumption that all peptide components have the same absorption due to their similar structure, RP-HPLC purity can be used as a proxy for purity expressed as a mass percentage [% (w / w)].

[0025] The present invention is directed, inter alia, to a method for producing a peptide having at least one disulfide bond. As used herein, the phrase "disulfide bond" refers to a persulfide bond (-SS-), which is typically formed between two thiol (also known as sulfhydryl) groups by oxidation. Most commonly, peptides contain disulfide bonds formed between the side chains of two cysteine ​​moieties. Such peptides are sometimes referred to as cystine-containing peptides. However, it should be understood that disulfide bonds can be formed between any sulfhydryl-containing moiety within a peptide molecule. For example, sulfhydryl groups derived from the side chains of other amino acids, such as homocysteine ​​or penicillamine groups, or from any substituents or linkers introduced into a peptide molecule, are also involved in disulfide bond formation. The sulfhydryl group can have a protecting group, such as an Acm, Phacm, or Mmt protecting group. Preferably, the phrase "sulfhydryl group" as used herein refers to a "free," i.e., unprotected, sulfhydryl group (-SH).

[0026] As used herein, a peptide having at least one sulfhydryl group can be referred to as a "reduced peptide." Typically, a reduced peptide has two or more sulfhydryl groups. As used herein, a peptide having at least one disulfide bond can be referred to as an "oxidized peptide." A peptide having both at least one disulfide bond and at least one sulfhydryl group can be referred to as "oxidized," "partially oxidized," "reduced," or "partially reduced."

[0027] The present invention relates inter alia to a method for producing a peptide having at least one intramolecular disulfide bond, comprising the steps of: a) providing a liquid reaction medium in a reaction vessel, the vessel allowing for mixing of its contents; b) providing a solution of peptides, wherein the peptides contain at least two sulfhydryl groups; c) providing a solution of an oxidizing agent; d) simultaneously adding said solution of oxidizing agent and said solution of peptide to said reaction medium in the reaction vessel through spatially separated inlets while mixing the contents of the reaction vessel, said addition comprising: i) the average concentration of the peptide in its reduced state in the reaction vessel, C1, is less than the concentration C0, the final concentration of the peptide in the reaction vessel, at the end of the peptide addition, regardless of its oxidation state; ii) During the time of simultaneous addition of oxidizing agent and peptide, the average concentration of oxidizing agent within the reaction vessel is maintained at essentially zero. The process is as follows: and e) continuing the addition of the oxidizing agent beyond the end of the peptide addition The present invention provides a method comprising:

[0028] The present invention relates inter alia to a method for producing a peptide having at least one intramolecular disulfide bond, comprising the steps of: a) providing a liquid reaction medium in a reaction vessel, the vessel allowing for mixing of its contents; b) providing a solution of peptides, wherein the peptides contain at least two sulfhydryl groups; c) providing a solution of an oxidizing agent; d) simultaneously adding said solution of oxidizing agent and said solution of peptide to said reaction medium in the reaction vessel through spatially separated inlets while mixing the contents of the reaction vessel, said additions being performed such that the average concentration of oxidizing agent in the reaction vessel is maintained essentially zero during the time of simultaneous addition of oxidizing agent and peptide; and e) continuing the addition of the oxidizing agent beyond the end of the peptide addition The present invention provides a method comprising:

[0029] It should be understood that the present invention can be applied to, for example, a peptide containing one free sulfhydryl group and one protected sulfhydryl group, or a peptide containing two sulfhydryl groups protected by suitable protecting groups, by oxidation of the sulfhydryl group protected by a suitable protecting group. In such embodiments, the rate of removal of the protecting group is preferably faster than the rate of intermolecular disulfide bond formation. In some embodiments of the present invention, the sulfhydryl group contained in the peptide is not protected by a protecting group, i.e., the sulfhydryl group is a free sulfhydryl group. In some embodiments of the present invention, the peptide contains at least two free sulfhydryl groups. In other embodiments of the present invention, the peptide contains at least one free sulfhydryl group.

[0030] In one embodiment, the concentration C1 is decreased during the time of simultaneous addition of the oxidizing agent and the peptide in step d).

[0031] As used herein, the phrase "concentration within the reaction vessel" may be synonymous with the phrase "concentration within the liquid contained within the reaction vessel."

[0032] As used herein, the expression "average concentration" is used to refer to the concentration of a substance in a volume that results, or is likely to result, from the instantaneous and uniform distribution of said substance within that volume. For example, the average concentration of reduced peptide in a reaction vessel is calculated by dividing the molar amount of reduced peptide in the reaction vessel by the total amount of liquid in the reaction vessel.

[0033] Those skilled in the art will understand that the composition of the reaction medium in step a) is influenced by the properties of the peptide to be produced. Preferably, the reaction medium is capable of dissolving the reduced peptide at a concentration C1 or greater. Those skilled in the art will understand that the average concentration C1 of the reduced peptide in the reaction vessel can be calculated by assuming instantaneous and uniform distribution of the added peptide solution in the liquid contained in the reaction vessel. The reduced peptide is expected to be consumed by the oxidation reaction, and its concentration may vary over time. The average concentration C1 is preferably averaged over the time period of step d). Most preferably, the reaction medium is capable of dissolving the peptide at a concentration C0 or greater. C0 is the final concentration of the peptide in the reaction vessel at the end of peptide addition, regardless of its oxidation state. In other words, C0 is defined as the maximum average concentration of all peptides in the liquid contained in the reaction vessel during the oxidation reaction. Those skilled in the art will understand that C0 is typically calculated based on the concentration of the peptide solution, the amount of peptide solution added, the amount of oxidant solution added until the end of peptide addition, and the initial amount of reaction medium. If the contents of the reaction vessel are subjected to simultaneous membrane filtration, the calculation may be based on the concentration of the peptide solution added, the amount of peptide solution added, and the final amount of reaction mixture contained in the reaction vessel and, if appropriate, in the holding loop.

[0034] It is preferred that the reaction medium does not react with either the oxidizing agent or the peptide. In one embodiment, the reaction medium is essentially inert to the oxidizing agent, reduced peptide, and oxidized peptide. The pH of the reaction medium is preferably selected to support disulfide bond formation and stabilize disulfide bonds. In one embodiment, the pH of the reaction medium is less than 7.0, preferably less than 5.0. In one embodiment, the pH of the reaction medium is less than 7.0, less than 6.5, less than 6.0, less than 5.5, less than 5.0, less than 4.5, less than 4.0, less than 3.5, or less than 3.0. However, depending on the oxidizing agent selected, it may be preferable to use a reaction medium with a (slightly) basic pH, for example, pH 7.0-8.0. Those skilled in the art will understand that the reaction medium is preferably highly polar.

[0035] In one embodiment of the present invention, the reaction medium is an aqueous solution. In a preferred embodiment of the present invention, the reaction medium is selected from the group consisting of water, aqueous acetic acid, aqueous trifluoroacetic acid, or aqueous formic acid. In a particularly preferred embodiment, the reaction medium is selected from the group consisting of the following: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (v / v) aqueous trifluoroacetic acid, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95%, or 100% (v / v) aqueous acetic acid, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (v / v) aqueous formic acid. In other embodiments of the present invention, the reaction medium can include the above components. In other embodiments of the present invention, the reaction medium can further include one or more organic solvents such as DMF, DMSO, methanol, ethanol, isopropyl alcohol, acetic acid, dioxane, trifluoroethanol, hexafluoroisopropanol, acetonitrile, tetrahydrofuran, etc. In still other embodiments, the reaction medium can comprise or consist essentially of one or more organic solvents such as DMF, methanol, ethanol, isopropyl alcohol, acetonitrile, and the like.

[0036] The term "preparing a solution of a peptide containing at least two sulfhydryl groups" is understood in the broadest sense to mean obtaining any liquid composition containing a peptide containing at least two sulfhydryl groups. The peptide can be obtained by any means known in the art. For example, the peptide can be obtained by solid-phase peptide synthesis (SPPS) or liquid-phase peptide synthesis (LPPS), or a combination thereof. It is well known to those skilled in the art that oxidation of sulfhydryl groups, such as cysteine ​​residues, can lead to the formation of sulfonic acids, such as cysteic acid, and must be avoided during peptide synthesis. This can be achieved by using suitable sulfhydryl protecting groups, such as diphenylmethyl, acetamidomethyl, or phenylacetamidomethyl protecting groups, and / or by working under protective gas, preferably under nitrogen. After SPPS, the peptide is typically cleaved from the resin using a cleavage cocktail containing TFA and one or more scavengers such as water, triisopropylsilane (TIPS), dithiothreitol (DTT), dithioerythritol (DTE), anisole, thioanisole, or 1,2-ethanedithiol (EDT). For example, the cleavage cocktail may contain 80-90% TFA and 5-10% each of water, TIPS, and DTE or EDT. Other examples of cleavage cocktails are TFA / thioanisole / anisole / EDT (90:5:3:2), TFA / water / TIPS (90:5:5) v / v / v, TFA / water / phenol (90:5:5) v / v / v, TFA / EDT / HO / TIPS (85:10:2.5:2.5) v / v / v / v, TFA / water / EDT / TIPS (90:5:2.5:2.5) v / v / v / v, TFA / water / DTE / TIPS (90:5:2.5:2.5) v / v / v / v, TFA / water / EDT / TIPS (90:4:3:3) v / v / v / v, TFA / water / EDT (90:5:5) v / v / v, TFA / DTE / HO / TIPS 85:10:2.5:2.5 (v / w / v / v) and TFA / water / DTE (90:5:5) v / v / v.The peptides are then typically precipitated from the cleavage cocktail using an organic antisolvent, such as diethyl ether, diisopropyl ether, methyl tert-butyl ether (MTBE), or a mixture of the above ethers with, for example, acetonitrile or hexane. Alternatively, simple polypeptide chains can be obtained from biotechnological methods, and optionally subsequently modified by chemical / synthetic means. Preferably, the peptides are obtained from SPPS, LPPS, or a combination thereof. More preferably, the peptides are obtained from a method comprising or consisting of Fmoc-SPPS.

[0037] In some embodiments of the invention, the peptide contained in the solution of step b) comprises exactly two sulfhydryl groups. In further embodiments of the invention, the sulfhydryl groups of the peptide are moieties of a cysteine ​​side chain. In further embodiments, the peptide comprises exactly two cysteine ​​moieties. Some embodiments of the invention include peptides such as somatostatin (CAS Registry Number 38916-34-6), lanreotide (CAS Registry Number 108736-35-2), octreotide (CAS Registry Number 83150-76-9), dotatate (CAS Registry Number 177943-88-3), edotreotide (CAS Registry Number 204318-14-9), aprotinin (CAS Registry Number 9087-70-1), oxytocin (CAS Registry Number 50-56-6), (Arg8)-vasopressin (CAS Registry Number 113-79-1), ... The present invention relates to the production of peptides selected from the group consisting of benzodiazepine (CAS Registry Number 11000-17-2), pramlintide (CAS Registry Number 151126-32-8), linaclotide (CAS Registry Number 851199-59-2), ziconotide (CAS Registry Number 107452-89-1), eptifibatide (CAS Registry Number 188627-80-7), desmopressin (CAS Registry Number 16679-58-6), calcitonin (CAS Registry Number 90779-69-4), and atosiban (CAS Registry Number 90779-69-4). The above names are well known to those skilled in the art, and the corresponding chemical structures / amino acid sequences can be easily and unambiguously identified. For example, INNs (International Nonproprietary Names) can be found in the list published by the World Health Organization on its website (https: / / www.who.int / ), among others. Additionally, CAS Registry Numbers, which are widely used and unambiguous identifiers, can be used to retrieve the chemical structures of the above compounds. However, it should be understood that the above peptide names do not imply a limitation to any particular salt form of the peptide.

[0038] Additionally, the present invention is applicable to the production of disulfide-linked derivatives and analogs of the above peptides.

[0039] It will be understood by those skilled in the art that the peptide contained in the solution of step b) can optionally have any counterion known in the art, such as an anion or cation, for example, chloride, acetate, carbonate, hydrocarbon, sodium, potassium, magnesium, bromide, perchlorate, ammonium, phosphate, or sulfate, any ion of the cleavage solution (e.g., TFA ion) and / or the cation or anion of a protecting group residue. Furthermore, the peptide can optionally be covalently or noncovalently bound to traces of one or more scavengers, such as, for example, triisopropylsilane (TIPS), dithiothreitol (DTT), dithioerythritol (DTE), anisole, thioanisole, or 1,2-ethanedithiol (EDT), or may contain traces of one or more antisolvents, such as diisopropyl ether, diethyl ether, acetonitrile, hexane, or methyl tert.butyl ether.

[0040] The solution of step b) can contain one or more of the above ions and compounds, as well as any adducts, polymers, or other products formed by chemical reactions of the above compounds. In one embodiment, the solution of step b) can contain crude or unpurified peptides, which can have a purity of 30-90%, e.g., greater than 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, unpurified peptides with a purity of 40-80% are used. In one embodiment, the crude peptide composition is obtained by precipitating the peptide from the cleavage cocktail after SPPS. In a preferred embodiment, the solution of step b) can contain partially or highly purified peptides, i.e., peptide compositions that have been subjected to specific purification steps, such as preparative chromatography.

[0041] The solution of step b) may preferably be an aqueous solution and may contain further additives to dissolve the peptide in question. For example, the solution of step b) may be obtained by dissolving or diluting the crude or purified peptide product in an aqueous liquid. The aqueous liquid may be, for example, pure water, an aqueous trifluoroacetic acid solution [e.g., 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1% (v / v)], an aqueous acetic acid solution [e.g., 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (v / v)], an aqueous formic acid solution [e.g., 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% (v / v)], or an aqueous formic acid solution [e.g., 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% (v / v)]. %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (v / v)], an aqueous potassium acetate solution [e.g., 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, or 300 mM], or an aqueous sodium acetate solution [e.g., 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, or 300 mM].

[0042] Preferably, the pH value of the solution in step b) is in the same range as the pH value of the liquid reaction medium provided in step a). In another embodiment, the solution in step b) can comprise one or more organic solvents such as DMF, methanol, isopropyl alcohol, acetonitrile, etc. In another embodiment, the solution in step b) can be obtained by dissolving or diluting the crude or purified peptide product in an organic solvent or a mixture of organic solvents such as DMF, methanol, isopropyl alcohol, acetonitrile, etc. Preferably, the solution in step b) is prepared to minimize the content of components other than the peptide, and this solution can be reacted with either the liquid reaction medium provided in step a) or the oxidizing agent provided in step c). Preferably, the solution in step b) is miscible with the reaction medium provided in step a).

[0043] Those skilled in the art will understand that the optimal concentration of peptide in the solution of step b) depends on the molecular properties of the peptide, and those skilled in the art will routinely optimize this concentration. Generally, the peptide concentration can be selected to avoid peptide aggregation and / or precipitation while keeping the volume of the solution of step b) small. In some embodiments, the concentration of peptide in the solution of step b) is selected to be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mmol / L or more. In other embodiments, the concentration of peptide in the solution of step b) is selected to be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / l or more.

[0044] The solution in step c) may preferably be miscible with the solutions in steps a) and b). In principle, it will be understood by those skilled in the art that any oxidizing agent capable of oxidizing sulfhydryl chains can be used in step c). Preferably, the oxidizing agent does not have a high tendency for side reactions, such as oxidation of the side chains of methionine, tyrosine, or tryptophan. Preferably, the oxidizing agent is selected from iodine, hydrogen peroxide, 2,2'-dipyridyl disulfide, dimethyl sulfoxide (DMSO), or an aqueous solution of potassium hexacyanoferrate [K3Fe(CN)6].

[0045] In one embodiment, for example, a solution of iodine in aqueous potassium iodide is used in a molar ratio of 1 part iodine to 3 parts potassium iodide, or 1 part iodine to 3 parts sodium iodide. In other embodiments, the solution in step c) is selected from dimethyl sulfoxide in water, 2,2'-dipyridyl disulfide in isopropyl alcohol, or hydrogen peroxide in water. As used herein, the expression "oxidizing agent" refers to a chemical moiety capable of oxidizing sulfhydryl groups to form disulfide bonds. Thus, when referring to the concentration of an oxidizing agent, this concentration refers to the concentration of the oxidizing agent in its oxidized state. Those skilled in the art will understand that the concentration of the oxidizing agent in the solution in step c) depends on the desired rate of addition of the oxidizing agent to the reaction vessel, the desired flow rate into the reaction vessel, and the molecular properties of the oxidizing agent. Thus, as a matter of routine work, those skilled in the art will optimize the concentration of the oxidizing agent for the particular task at hand.

[0046] In some embodiments, the concentration of the oxidizing agent can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, or 500 mmol / L or more. Preferably, the solution of step c) is prepared to minimize the content of components other than the oxidizing agent, and this solution can be reacted with either the liquid reaction medium provided in step a) or the peptide solution provided in step b). Preferably, the solution of step c) is miscible with the reaction medium provided in step a). In one embodiment, the solution of step c) is an aqueous solution. Preferably, the pH value of the solution is selected to favor favorable reaction conditions in the reaction vessel.

[0047] Those skilled in the art will understand that the volume of the reaction medium in step a) and the solutions in steps b) and c) will depend on the total amount of peptide to be oxidized and the desired reaction conditions, in particular the desired final concentration C0 of the peptide in the reaction vessel at the end of peptide addition. Those skilled in the art will routinely optimize the concentration C0 of the peptide specific to the production. In general, it may be advantageous to maintain the peptide concentration C0 as high as possible while avoiding peptide aggregation and multimer formation due to disulfide bond exchange.

[0048] In some embodiments of the invention, the concentration C0 is greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 g / l. In some embodiments of the invention, the concentration C0 is greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 mM. In some embodiments of the invention, the concentration C0 is not as high as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 g / l. In some embodiments of the invention, the concentration C0 is not as high as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 mM. Those skilled in the art will understand that the ratio of concentrations C0 / C1 will depend on peptide characteristics and desired reaction conditions, and can optimize said ratio according to the specific task at hand. For example, the ratio C0 / C1 can be at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500. In one embodiment, the ratio C0 / C1 is at least 10, more preferably at least 100, and most preferably at least 1000. To calculate this ratio, C1 is averaged in time over the period of step d).

[0049] Step d) of the method according to the present invention involves the simultaneous addition of a peptide solution and an oxidizing agent solution to the reaction medium in the reaction vessel. This addition is carried out through spatially separated inlets while mixing the contents of the vessel. Those skilled in the art will appreciate that mixing of the contents of the reaction vessel can be achieved by any suitable means. For example, an agitator including a rotating impeller can be used. Such an impeller can be a turbulent mixer that induces axial, mixing, or radial flow of the liquid in the reaction vessel. Known impellers include marine-type propellers, pitch-blade turbines, flat-blade turbines, and flat-blade paddles. The use of baffle blades can help improve mixing. Alternatively, or additionally, mixing can be achieved by bubbling a gas through the liquid. Alternatively, or additionally, mixing can be achieved by liquid circulation, for example, by a pumping circuit. Those skilled in the art will typically select a mixing means to achieve efficient distribution of the substances in the reaction medium while avoiding foaming. The positioning of the inlets for the peptide solution and the oxidizing agent solution can vary depending on the design of the reaction vessel and the mixing means. Figure 1 shows various designs. Generally, each solution can be added either from the top of the reaction vessel (i.e., above the surface of the reaction medium), in the center of the reaction vessel, or from the bottom of the reaction vessel.

[0050] For example, the peptide solution and the oxidant solution can be added from the top; the peptide solution can be added from the top while adding the oxidant solution in the middle; the peptide solution can be added from the top while adding the oxidant solution from the bottom; the peptide solution and the oxidant solution can be added in the middle; the peptide solution can be added in the middle while adding the oxidant solution from the top; the peptide solution can be added in the middle while adding the oxidant solution from the bottom; the peptide solution can be added from the bottom while adding the oxidant solution in the middle; the peptide solution can be added from the bottom while adding the oxidant solution in the middle; the peptide solution can be added from the bottom while adding the oxidant solution in the middle; the peptide solution can be added from the bottom while adding the oxidant solution from the top; or the peptide solution and the oxidant solution can be added from the bottom.

[0051] In one embodiment, only the peptide solution is added from the top. In another embodiment, only the oxidizing agent solution is added from the top. In another embodiment, both solutions are added below the surface of the reaction medium. Furthermore, the inlets can be located on the same side of the reaction vessel, at opposite locations, or between both locations. In other words, as shown in panel j) of Figure 1, when planning the location of the inlets from the top of the reaction vessel, the angle between both inlets can be any angle between 0° and 360°, provided they are spatially separated. Addition of the reagent solutions to the reaction vessel is generally performed by flowing the liquids through the respective inlets. Suitable mechanical means for achieving this can be useful. For example, the liquid flow can be driven by vacuum suction, nitrogen pressure, or a pump. In some embodiments of the present invention, step d) can include starting the flow of the solution prepared in step b) into the reaction vessel followed by starting the flow of the solution prepared in step c) into the reaction vessel. Control of the flow can be achieved by any means, preferably by using a dosing pump.

[0052] The introduction of the flow of the solutions of steps b) and c) into the reaction vessel can be continuous and / or controlled by a feedback loop. For example, the flow rate of the solution of step b) and / or the solution of step c) can be controlled based on the optical properties of the liquid contained in the reaction vessel. In some embodiments, the flow rate of the solution of step c) is controlled by a feedback signal generated by a monitoring system in the reaction vessel. It is noted that this monitoring system may take the form of a dip probe or flow cell and can be located within the reaction vessel itself, or, in one embodiment, within the recirculation loop or in a bypass to such a loop if the reaction vessel is part of a pumping circuit. For example, photometric or redox potential measurements can provide a feedback signal that regulates the flow rate of reagents into the reaction vessel. Such measurements and corresponding control signals can be generated, for example, by a transmission dip probe or redox electrode. The redox electrode can be used a) to determine the redox potential in the reaction vessel (and, if necessary, within the recirculation loop) during the simultaneous addition of the peptide solution and the oxidizing solution, and b) to provide a feedback signal to the introduction pump. Even when using complex mixtures, such as those obtained when using crude peptide products in oxidation reactions, this setup surprisingly makes it possible to control the redox potential in the reaction vessel so that it is at or slightly below the equivalence point, where the molar amount of reducing chemical groups in the reaction vessel corresponds to the molar amount of added oxidation equivalents. This therefore makes it possible to automatically control the flow rate of the solution of step c) based on a feedback signal obtained from a redox electrode in the reaction mixture. In one embodiment of the present invention, the flow rate of the solution of step c) is controlled based on a feedback signal generated by the redox electrode in the reaction vessel, and during step d) of the method, the redox potential in the reaction vessel is adjusted to be slightly below the equivalence point. In some embodiments of the present invention, the inflow of the solutions of steps b) and c) into the reaction vessel can be independently controlled and continuously adjusted.

[0053] In the method according to the present invention, the peptide solution and the oxidizing agent solution are added in step d) so that the average concentration of the oxidizing agent is essentially zero, i.e., so that the amount of oxidizing agent is limited to disulfide bond formation. Under these conditions, any oxidizing agent added is essentially immediately consumed by the oxidation reaction. In some embodiments, the average oxidizing agent concentration is considered to be essentially zero as long as it is below the detection limit of the monitoring system used to monitor the oxidizing agent concentration. In some embodiments, this can be achieved by controlling the color of the reaction mixture, for example, with a dip probe. In some embodiments, when iodine is used as the oxidizing reagent, the concentration of the oxidizing agent in the reaction mixture is essentially zero as long as the bulk color of the reaction mixture is clear or yellow. In some embodiments, a redox electrode can be used to determine the equivalence point and control the flow rate to maintain the determined potential at or below the equivalence point on average.

[0054] The average concentration of oxidizing agent in the reaction vessel can be considered essentially zero if the average concentration is less than 0.0005 equivalents / L, less than 0.0004 equivalents / L, less than 0.0003 equivalents / L, less than 0.0002 equivalents / L, or less than 0.0001 equivalents / L. It should be understood that one equivalent of oxidizing agent is defined as the stoichiometric amount of oxidizing agent required to convert the amount of peptide at concentration C0 in the reaction vessel from a fully reduced state to a fully oxidized state. In some embodiments of the present invention, the flow rates of the peptide solution and the oxidizing agent solution can be adjusted so that concentration C1, i.e., the average concentration of reduced peptide in the liquid contained in the reaction vessel and the average concentration of oxidizing agent in the liquid contained in the reaction vessel, reaches a steady state during the simultaneous addition period. In some embodiments of the present invention, concentration C1 may decrease during the simultaneous addition period due to dilution by the amount of added solution.

[0055] In other embodiments, the flow rates of the peptide solution and the oxidant solution can be adjusted so that the average concentration C1 increases over time.

[0056] After step d), the addition of oxidizing agent continues beyond the end of peptide addition to the reaction vessel (step e). This addition can be performed while maintaining the average oxidizing agent concentration at essentially zero. In other embodiments, the addition of oxidizing agent can cause the average oxidizing agent concentration in the reaction vessel to increase above zero. Figure 2 shows a schematic representation of the evolution of the average concentrations of reduced peptide, total peptide and oxidizing agent over time in a reaction vessel according to one embodiment of the invention. In further embodiments of the invention, steps d) and e) are alternately repeated at least once. In some embodiments of the invention, steps d) and e) are alternately repeated at least 2, 4, 6, 8, 10 or more than 10 times.

[0057] In some embodiments of the invention, the total amount of oxidizing agent added to the reaction vessel in steps d) and e) is selected so that the amount of excess (i.e., unreacted) oxidizing agent contained in the reaction vessel at the end of step e) corresponds to at least 0.01 equivalents, e.g., at least 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.04, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1.00 equivalents of the total amount of disulfide (i.e., -SS-) bonds present in the fully oxidized peptide. In some embodiments of the invention, the total amount of oxidizing agent is selected so that the amount of excess (i.e., unreacted) oxidizing agent contained in the reaction vessel at the end of the last iteration of step e) corresponds to at least 0.01 equivalents, e.g., 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.04, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1.00 equivalents of the total amount of disulfide (i.e., -SS-) bonds present in the fully oxidized peptide. In one embodiment of the present invention, the total amount of oxidizing agent added to the reaction vessel corresponds to 1.0 to 2.0 equivalents of the total number of disulfide (i.e., -SS-) bonds present in the fully oxidized peptide, e.g., about 0.95, 1.00, 1.01, 1.02, 1.03, 1.03, 1.04, 1.05, 1.1, 1.15, 1.20, 1.25, 1.3, 1.35, 1.4, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00, or 2.04 equivalents.

[0058] In one embodiment, the total amount of oxidizing agent added to the reaction vessel corresponds to 1.0 to 1.5 equivalents of the total amount of disulfide bonds present in the fully oxidized peptide. As used herein, the expression "1 equivalent of disulfide bonds" is used to define the stoichiometric amount of oxidizing agent required to generate a given amount of disulfide bonds. For example, 1 mole of iodine (I2) is required to generate 1 mole of disulfide bonds.

[0059] In a further embodiment of the present invention, any excess oxidizing reagent that may accumulate during step e) is subsequently removed. In one embodiment of the present invention, after completion of step e), any excess oxidizing agent contained in the reaction vessel is removed by adding a suitable reducing agent. The reducing agent is added to the reaction vessel as a solid or a solution. It will be understood by those skilled in the art that a reducing agent suitable for the above purpose will reduce the oxidizing agent but will not essentially affect the disulfide bonds of the peptide. In some embodiments of the present invention, ascorbic acid or thiosulfate is used to remove the excess oxidizing agent. It will be understood by those skilled in the art that the amount of reducing agent can preferably be adjusted to the amount of excess oxidizing reagent present. This can be achieved, for example, by titrating the amount of reducing agent or by calculating the stoichiometric amount of reducing agent required to reduce the predicted excess oxidizing agent.

[0060] In another embodiment of the present invention, low molecular weight species are removed from the contents of the reaction vessel. This can be preferably achieved by subjecting the contents of the reaction vessel to membrane filtration. In one embodiment of the present invention, membrane filtration is performed continuously while steps d) and e) of the method of the present invention are performed. In one embodiment, membrane filtration is performed simultaneously with (also performed in the same way as) step d). In this embodiment, membrane filtration can be initiated before or after the start of step d) and before or after the end of step d). Preferably, membrane filtration is performed during the entire duration of step d). In one embodiment, membrane filtration is performed during the entire duration of step d) and is continued thereafter. If membrane filtration is continued after step d), membrane filtration can be used to concentrate the product solution and / or for diafiltration (i.e., salt and / or solvent exchange by adding an alternative buffer or solvent during filtration). In another embodiment, membrane filtration is performed on the contents of the reaction vessel after step e) is performed. In a preferred embodiment, membrane filtration is used to concentrate the oxidized peptide in the reaction vessel while simultaneously removing low molecular weight species. As used herein, the term "low molecular weight species" can refer to substances contained in the reaction vessel that have a lower molecular weight than the peptide to be produced.

[0061] Membrane filtration is a pressure-driven separation process that relies on the use of a semipermeable membrane that allows small molecules, such as buffer and solvent molecules, to pass through while retaining the peptide of interest. For purposes of the present invention, it is preferred to use a membrane with a molecular weight cutoff of 3 kDa or less, e.g., 3 kDa, 2 kDa, 1 kDa, 0.5 kDa, 0.2 kDa, or less. The liquid that passes through the membrane is referred to as the "permeate" or "filtrate," while the sample retained by the membrane is referred to as the "retentate." To avoid clogging of the membrane pores, a tangential flow filtration format (also known as cross-flow filtration) is advantageously used. For example, cross-flow ultrafiltration is preferentially used in embodiments in which the contents of the reaction vessel are mixed by liquid circulation.

[0062] For the purposes of the present invention, it is preferable to use a membrane that is compatible with acids, bases, and organic solvents. In a particularly preferred embodiment, a ceramic or polymeric membrane with a molecular weight cutoff of less than 0.5 kDa is used. However, it should be understood that the filter can be made of any material known in the context of filtration, such as plastic (e.g., nylon, polystyrene), metal, alloy, glass, ceramic, metal oxide, cellophane, cellulose, or composite materials, as long as the membrane achieves a suitable molecular weight cutoff. The filter can be hydrophobic or hydrophilic. The surface of the filter can be neutral, positively charged, or negatively charged.

[0063] In a further embodiment according to the invention, the method further comprises the step of purifying the peptide in its oxidized state.

[0064] Preferably, the peptide is subjected to purification after completion of step e) and optional removal of any excess oxidizing agent. Purification and isolation means optionally used in the present context include, for example, crystallization, lyophilization, one or more electrophoretic methods (e.g., gel electrophoresis or capillary (CE) electrophoresis), one or more additional precipitation-based methods (e.g., salting-in or salting-out), one or more dialysis methods (dialysis), and / or one or more chromatographic methods (e.g., gel permeation chromatography (GPC), size exclusion chromatography, ion exchange chromatography (IEC), high performance liquid chromatography (HPLC), reversed-phase HPLC (RP-HPLC), fast protein liquid chromatography (FPLC), flash chromatography (flash), rapid refluid liquid chromatography (RRLC), rapid separation liquid chromatography (RSLC), ultra fast flow liquid chromatography (UFLC), reversed-phase UFLC (RP-UFLC), ultra performance liquid chromatography (UPLC) or reversed-phase UPLC (RP-UPLC), preferably, the oxidized peptide is subjected to at least one dimension of reversed-phase HPLC.

[0065] A further aspect of the invention is an apparatus suitable for carrying out the method according to the invention, comprising: a) a reaction vessel equipped with means for mixing the contents of the reaction vessel, preferably the means for mixing the contents of the vessel is an agitator, a pumping circuit, or an agitator and a pumping circuit; b) two spaced apart inlets for liquid entry into said reaction vessel; c) two storage vessels, each connected to one of the inlets, one containing a liquid comprising an oxidizing agent and the other containing a liquid comprising a peptide to be oxidized; and d) an automated pump that allows for the flow of liquid from the storage vessel into the reaction vessel via an inlet and for this to be controlled; The present invention relates to an apparatus comprising:

[0066] Preferably, all materials from which the device is made are (substantially) inert to the reagents to which they are exposed. Preferably, the materials comply with applicable regulations for pharmaceutical products, cosmetics, and / or food and beverages, i.e., the materials preferably comply with Good Manufacturing Practice (GMP) for drugs and quasi-drugs. Furthermore, conductive materials can be used to minimize the risk of electrostatic ignition.

[0067] As used herein, the terms "reaction vessel" or "reactor" refer to a vessel suitable for collecting reagents of interest and preferably having at least one liquid outlet. The reaction vessel is shaped to allow efficient mixing of its contents and rinsing of its walls. The reaction vessel is preferably closed, i.e., suitable for avoiding contamination or unintentional release of its contents. The reaction vessel may be equipped with additional inlets and outlets for substances, preferably liquids. Such inlets are preferably equipped with valves that are closed unless substances are intentionally introduced into or removed from the reaction vessel. Preferably, the transfer of substances via the additional inlets and outlets is driven and controlled by automated equipment (e.g., pumps and valves) managed by a central or local control unit. The reaction vessel of the device of the present invention may be equipped with at least two spatially separated inlets for liquids, as described above. In one embodiment, the reaction vessel is equipped with at least one additional inlet that allows the introduction of a reducing agent into the reaction vessel to remove any excess oxidizing agent. In one embodiment, the reaction vessel comprises at least one further liquid inlet through which a buffer / wash liquid can be introduced into the reaction vessel, the latter liquid inlet being connected to a spray head within the reactor.

[0068] The reaction vessel can be made from any suitable material, such as metal, glass, enamel, or a polymer such as polypropylene, polyethylene, polyvinyl chloride, polystyrene, and polyetheretherketone. Preferably, a material is selected that is essentially inert to the reagents to which it is exposed. Furthermore, conductive materials can be used to minimize the risk of electrostatic ignition. In some embodiments, the storage vessel is made from stainless steel, Hastelloy alloy, or glass. The size and dimensions of the reaction vessel can be selected according to the intended scale. For example, reactors with internal volumes of (approximately) 10 to (approximately) 300 liters, (approximately) 20 to (approximately) 250 liters, (approximately) 30 to (approximately) 200 liters, (approximately) 40 to (approximately) 150 liters, (approximately) 50 to (approximately) 100 liters, or (approximately) 60 to (approximately) 75 liters can be used. For example, reactors with internal volumes of 10, 20, 30, 40, 50, 75, 100, 150, 200, or 250 liters can be used. In some embodiments, the reaction vessel has an internal volume of (about) 10 to (about) 30 liters.

[0069] The reaction vessel is adapted to operate under a protective atmosphere. For example, the reaction vessel can include a first controllable valve that can be connected to a vacuum source, a second controllable valve that can be connected to a source of inert gas such as nitrogen, and an electronic or mechanical pressure controller. Preferably, the valves are operated automatically, for example, by a central control unit. In other embodiments, the reaction vessel can include only a controllable valve connected to a source of inert gas such as nitrogen, allowing the protective gas to blanket the liquid contents of the vessel.

[0070] To allow for temperature control, the reaction vessel may be a jacketed reactor. Preferably, a temperature sensor within the reactor provides a feedback signal that controls the circulation of cooling or heating fluid within the reactor jacket.

[0071] As used herein, the term "storage container" refers to a container in which a substance of interest can be stored under suitable conditions. Those skilled in the art can define suitable conditions depending on the circumstances, for example, to maintain the integrity of the substance or process safety according to the specifications of a given process. The storage container can be made of any suitable material, for example, metal, glass, enamel, or a polymer such as polypropylene, polyethylene, polyvinyl chloride, polystyrene, and polyetheretherketone. In some embodiments, the storage container is made of stainless steel or a Hastelloy alloy. The size of the storage container can be selected according to the intended scale of synthesis. In some embodiments, the storage container has an internal volume of (about) 1 to (about) 100 liters, for example, (about) 1, 2, 5, 10, 15, 20, 25, or 30 liters. In some embodiments, the storage container has an internal volume of (about) 5 to (about) 20 liters.

[0072] To allow for temperature control, the storage vessel may be jacketed. Preferably, a temperature sensor within the reactor provides a feedback signal that controls the circulation of cooling or heating fluid within the vessel jacket.

[0073] In some embodiments, the storage container is further adapted to contain a protective gas. For example, the container can include a pressure controller, a controllable valve that can be connected to a vacuum source, and a second controllable valve that can be connected to a source of an inert gas such as nitrogen.

[0074] Each storage vessel is connected to an inlet of the reaction vessel via a liquid conduit. As detailed above with respect to the method of the present invention, the inlets are spatially separated and positioned so that individual reagents are added from above the reactor to the surface of the liquid contained therein, or agents are added below the surface of the liquid. The liquid flow rate from the storage vessel to the reaction vessel is preferably controlled by a pump, sometimes referred to herein as a dosing pump. Those skilled in the art will routinely determine the specifications of the dosing pump depending on the dimensions of the particular apparatus at hand and the expected concentrations of the reagents used.

[0075] In one embodiment, the apparatus can further comprise a monitoring system within the reaction vessel, the system providing a feedback signal to an input pump that regulates the flow rate of the oxidant solution into the reaction vessel. In one embodiment, the apparatus can further comprise a monitoring system within the reaction vessel, the system providing a feedback signal to a pump that regulates the flow rate of the peptide solution into the reaction vessel. In one embodiment, the apparatus can further comprise a monitoring system within the reaction vessel, the system providing a feedback signal to a pump that regulates the flow rate of the peptide solution into the reaction vessel and to a pump that regulates the flow rate of the oxidant solution into the reaction vessel. In one embodiment, the apparatus can further comprise a monitoring system within the reaction vessel, the system providing a feedback signal to control at least one automated pump that provides and controls the flow of liquid from a storage vessel into the reaction vessel via an inlet.

[0076] In a preferred embodiment, the flow rate of said solution of oxidizing agent is controlled by a monitoring system, preferably by a feedback signal generated by a redox potential probe in the reaction vessel, which monitoring system may preferably be located in or at the reaction vessel, the holding loop and / or the bypass loop.

[0077] In a preferred embodiment, the apparatus further comprises a monitoring system in or attached to the reaction vessel, or in or attached to the holding loop, or in or attached to a bypass to the holding loop, which system provides a feedback signal to control at least one of said automated pumps (6), preferably said monitoring system comprising an oxidation-reduction potential probe.

[0078] The monitoring system may comprise an optical probe having a suitable spectral range, such as a dip probe for UV, Vis, NIR, or IR (e.g., a Raman probe), or a redox potential probe, e.g., a suitable Pt or Au electrode. Alternatively to the use of a dip probe, the sensor may be in the form of a flow cell, which in such embodiments is integrated into a holding loop or bypass loop, in which case the reaction vessel is part of a recirculation circuit. In each of the foregoing embodiments, the monitoring system can be used to monitor any parameter suitable for determining the concentration of reduced peptide, the concentration of total peptide, the concentration of oxidation reagent, the concentration of any other product or educt of the oxidation reaction, and / or the ratio between any of the above. In particular, specific optical properties or redox potentials of the solution in the reaction vessel can be determined by the monitoring system. Using one or more monitoring systems, e.g., optical probes and redox probes, several parameters can be monitored in parallel.

[0079] As used herein, the phrase "redox potential" or "oxidation-reduction potential" or "ORP" reflects the tendency of a solution to release or take up electrons. Redox potential is expressed as a single voltage in millivolts (mV). Redox potential can be determined by measuring the voltage between a supporting electrode and a reference electrode. As used herein, the phrases "redox probe," "orp electrode," "redox potential probe," and "redox electrode" are used interchangeably and include configurations in which the supporting electrode and the reference electrode are constructed in a single component. Such a setup can be referred to as a redox combination electrode.

[0080] In one embodiment, the monitoring system is capable of monitoring the concentration of oxidant in the reaction vessel and providing feedback to a pump that regulates the flow of the oxidant solution to the reaction vessel. In one embodiment, the monitoring system is capable of monitoring the ratio of oxidant to reduced peptide in the reaction vessel and providing feedback to a pump that regulates the flow of the oxidant solution to the reaction vessel. In one embodiment, the monitoring system is capable of monitoring the ratio of oxidant to reduced peptide in the reaction vessel and providing feedback to a pump that regulates the flow of the reduced peptide solution to the reaction vessel. Suitable monitoring systems include, among others, a transmission dip probe having a suitable spectral range, e.g., a UV-Vis transmission dip probe, and a redox electrode, e.g., a suitable Pt or Au electrode.

[0081] Therefore, a further aspect of the present invention is an apparatus suitable for carrying out the method according to the present invention, comprising: a) a reaction vessel equipped with means for mixing the contents of the reaction vessel, preferably the means for mixing the contents of the vessel is an agitator or a pumping circuit; b) two spaced apart inlets for liquid entry into said reaction vessel; c) two storage vessels, each connected to one of said inlets, one containing a liquid comprising an oxidizing agent and the other containing a liquid comprising the peptide to be oxidized; d) an automated pump that allows for the flow of liquid from the storage vessel into the reaction vessel via an inlet and to be controlled; and e) a monitoring system capable of monitoring the concentration of oxidant in the reaction vessel and providing a feedback signal to control at least one of said automated pumps. The present invention relates to an apparatus comprising:

[0082] As used herein, the phrase "monitoring the concentration of a compound" is used to describe measuring any parameter directly or indirectly related to the absolute or relative concentration of the compound of interest. The monitoring system may include a sensor, e.g., a transmission dip probe having a suitable spectral range, such as a UV-Vis or NIR (near-infrared) transmission dip probe, or a redox electrode, such as a suitable Pt or Au electrode. The monitoring system may further include a controller unit that receives the sensor input (actual value) and the user input (target value), calculates any adjustments required for the operation of the dosing pump, and provides appropriate control signals to one or more dosing pumps.

[0083] In one embodiment, the monitoring system is capable of monitoring the redox potential of the liquid in the reaction vessel and provides feedback to a pump that regulates the flow rate of the reduced peptide solution to the reaction vessel and to a pump that regulates the flow rate of the oxidizing agent solution to the reaction vessel. In one embodiment, the monitoring system is capable of monitoring the redox potential of the liquid in the reaction vessel and provides feedback to a pump that regulates the flow rate of the reduced peptide solution to the reaction vessel. In one embodiment, the monitoring system is capable of monitoring the redox potential of the liquid in the reaction vessel and provides feedback to a pump that regulates the flow rate of the oxidizing agent solution to the reaction vessel. In some embodiments, the monitoring system comprises a platinum electrode, preferably a composite platinum electrode, such as a composite platinum ring electrode with a ceramic diaphragm.

[0084] Therefore, a further aspect of the present invention is an apparatus suitable for carrying out the method according to the present invention, comprising: a) a reaction vessel equipped with means for mixing the contents of the reaction vessel, preferably the means for mixing the contents of the vessel is an agitator or a pumping circuit; b) two spaced apart inlets for liquid entry into said reaction vessel; c) two storage vessels, each connected to one of said inlets, one containing a liquid comprising an oxidizing agent and the other containing a liquid comprising the peptide to be oxidized; d) an automated pump that allows for the flow of liquid from the storage vessel into the reaction vessel via an inlet and to be controlled; and e) a monitoring system capable of monitoring the redox potential within the reaction vessel and providing a feedback signal to control at least one of said automated pumps. The present invention relates to an apparatus comprising:

[0085] In some embodiments, the monitoring system comprises a redox potential probe, preferably a metal combination electrode, most preferably a composite platinum electrode.

[0086] In one embodiment, the apparatus may further comprise means for subjecting the contents of the reaction vessel to membrane filtration, e.g., by ultrafiltration or nanofiltration. Preferably, this is achieved by integrating the reaction vessel into a pumping circuit, such that liquid / retentate is circulated from the reaction vessel back to the reaction vessel via a cross-flow filtration unit. Exemplary embodiments of this aspect of the invention are shown in Figures 3 and 5. The permeate removed from the filtration unit preferably contains buffer, scavenger, and any small molecule compounds such as reduced oxidants.

[0087] Therefore, a further aspect of the present invention is an apparatus suitable for carrying out the method according to the present invention, comprising: a) a reaction vessel equipped with means for mixing the contents of the reaction vessel, preferably the means for mixing the contents of the vessel is an agitator or a pumping circuit; b) two spaced apart inlets for liquid entry into said reaction vessel; c) two storage vessels, each connected to one of said inlets, one containing a liquid comprising an oxidizing agent and the other containing a liquid comprising the peptide to be oxidized; d) an automated pump that allows for the flow of liquid from the storage vessel into the reaction vessel via an inlet and to be controlled; and e) means for subjecting the contents of the reaction vessel to membrane filtration; The present invention relates to an apparatus comprising:

[0088] As used herein, the expression "subjecting the contents of the reaction vessel to membrane filtration" describes a setup in which the retentate, i.e., the peptide-containing solution, is contained in or recycled to the reaction vessel. In contrast to the teachings of the prior art, the inventors have found that such a setup is advantageous in that it can achieve completion of peptide oxidation and improve the purity of the oxidized product. Without being bound by this theory, it is believed that this may be because such a setup allows i) the amount of fluid in the reaction vessel to be maintained, and / or ii) the concentration of low molecular weight contaminants to be kept (essentially) constant over time.

[0089] In one embodiment, the means for subjecting the contents of the reaction vessel to membrane filtration may include a membrane integrated into the reaction vessel (dead-end configuration), with flow through the membrane being driven by applying nitrogen pressure to the solution within the reaction vessel.

[0090] In one embodiment, the means for subjecting the contents of the reaction vessel to membrane filtration can include a membrane filtration unit and a liquid conduit capable of circulating the liquid / retentate from the reaction vessel through the membrane filtration unit and back to the reaction vessel (cross-flow configuration). This liquid conduit can be referred to as a holding loop. In one embodiment, the holding loop emerges from the outlet of the reaction vessel and leads, via a membrane filtration unit, to a dedicated inlet of the reaction vessel. In another embodiment, the holding loop emerges from the outlet of the reaction vessel and leads, via a membrane filtration unit, to an inlet of the same reaction vessel as one of the storage vessels. In this case, the storage vessel is connected to the inlet via the holding loop, i.e., the liquid conduit emerging from the storage vessel drains into the holding loop. In this latter embodiment, the flow of the holding loop is used to pre-dilute the reagents before the flow enters the reaction vessel.

[0091] Flux of the liquid through the retention loop can be achieved by a recirculation pump integrated into the retention loop. The pump can preferably be dimensioned to ensure a linear velocity of 1-5 m / s at the membrane surface. The pressure in this loop is regulated by the pump performance and by a pressure control valve integrated into the retention loop. The formation of permeate is driven by pressure applied to the liquid. The permeate is removed from the membrane filtration unit. Alternatively, the flux of the liquid from the retention loop can be achieved by a recirculation pump, and pressure-driven permeate formation can be achieved by applying nitrogen pressure to the reactor.

[0092] The membrane filtration unit may preferably be configured in a cross-flow configuration. The cross-flow membrane filtration unit may comprise a membrane housing, which may have one feed inlet and one outlet for the permeate and retentate, respectively. The membrane housing is sealed and may hold a membrane module. Those skilled in the art can preferably routinely select a suitable membrane module depending on the details of the oxidation reaction (e.g., product retention and filtration) and the equipment at hand. Common examples include cylindrical modules made from multi-channel or single-channel ceramic membranes, spiral-wound modules made from polymer membranes, membrane cassettes, or hollow fibers. Metal or composite membranes can also be used. The membrane surface area can be sized to achieve the desired operating time.

[0093] For purposes of the present invention, it is preferred to use membranes for nanofiltration and ultrafiltration, as described above with respect to the method of the present invention. Membranes with a molecular weight cutoff of 10 kDa or less, for example, 9 kDa, 5 kDa, 3 kDa, 2 kDa, 1 kDa, 0.5 kDa, 0.2 kDa, or less, can be used. In a preferred embodiment, ceramic or polymeric membranes with a molecular weight cutoff of less than 0.5 kDa are used. However, it should be understood that the membrane can be made of any material known in the context of filtration, such as polymers (e.g., nylon, polystyrene), metals, alloys, glass, ceramics, metal oxides, cellophane, cellulose, or composite materials, so long as the membrane achieves a suitable molecular weight cutoff. The membrane may be hydrophobic or hydrophilic. The membrane surface may be neutral, positively charged, or negatively charged. It is preferred to use membranes that are compatible with acids, bases, and organic solvents and stable at the pressures and temperatures used. Similarly, the housing and seals of the membrane filtration unit are preferably stable against these influences.

[0094] To enable temperature control within the retentate loop, the retentate loop can be equipped with a heat exchanger, such as a shell-and-tube heat exchanger, a plate heat exchanger, a plate-and-frame heat exchanger, or a spiral plate heat exchanger. The flow of the heating / cooling medium within the heat exchanger can be in the same direction as the flow of retentate within the retentate loop (co-current), opposite the flow of the retentate loop (counter-current), or crossing the flow of the retentate loop (cross-current). Hybrid configurations such as cross-counter-current and multi-pass flow are also possible. Those skilled in the art will routinely select a heat exchanger of the appropriate type and dimensions to achieve the required heating / cooling.

[0095] Temperature control within the apparatus of the present invention can be achieved by the combination of one or two jacketed storage vessels, a jacketed reaction vessel, and a heat exchanger integrated into the holding loop. In another embodiment, temperature control within the apparatus of the present invention can be achieved by the combination of one or two jacketed storage vessels and a jacketed reaction vessel. In another embodiment, temperature control within the apparatus of the present invention can be achieved by the combination of one or two jacketed storage vessels and a heat exchanger integrated into the holding loop. In another embodiment, temperature control within the apparatus of the present invention can be achieved by the combination of a jacketed reaction vessel and a heat exchanger integrated into the holding loop. These temperature control means can be selected to control the temperature of the liquid within the apparatus to any desired temperature selected from the range of -10°C to 50°C, e.g., 5°C to 50°C or 10°C to 30°C, preferably with an accuracy of + / -1°C. In another embodiment, these temperature control means can be selected to control the temperature of the liquid within the vessel to any desired temperature selected from the range of 5°C to 50°C.

[0096] The apparatus of the present invention, as detailed above, can be used in a semi-continuous format, where continuous feeds of peptide solution and oxidation reagent are pumped into the reaction vessel for a specified period of time.

[0097] In addition to the batchwise oxidation of a defined amount of peptide, the apparatus of the present invention can also be used in a truly continuous manner, in which case the storage containers for the peptide solution and the oxidizing agent solution may have inlets that allow them to be permanently refilled during operation, and the product may be continuously removed from the holding loop. Preferably, in such a configuration, the amount of oxidation reagent is introduced so that the redox potential of the solution in the reaction vessel is at or slightly above the equimolar point, i.e., so that the reducible groups and the oxidizing reagent in the reaction vessel are present in equimolar amounts, or so that there is a slight excess of the oxidizing reagent over the reducible groups. Preferably, the removal of the product from the holding loop can be achieved by a liquid outlet located in the holding loop between the pressure control valve and the inlet to the reaction vessel. Preferably, the liquid outlet is used to continuously remove a certain volumetric flow rate of retentate from the holding loop, which volumetric flow rate is smaller than the volumetric flow rate of the total recirculation stream in the reaction vessel. For example, the volumetric flow rate withdrawn per minute can be less than 10%, e.g., 8%, 6%, 4%, or 2%, of the total recirculation flow rate of the liquid contained in the reaction vessel and holding loop. Those skilled in the art routinely optimize the flow withdrawal for the task at hand, depending, for example, on the flow rate and reaction rate in the holding loop. This mode of operation is preferred for rapid reactions and system configurations where reagents have sufficient residence time in the holding loop to complete the reaction before product withdrawal. This was the case in Experiment 5 shown below.

[0098] In a preferred embodiment, the peptide solution feed removed from the holding loop can be loaded directly onto a chromatography column, e.g., an RP-HPLC column, preferably by a loading pump. In another embodiment, the peptide solution feed removed from the holding loop can be collected in a storage tank. Optionally, the peptide solution removed from the holding loop is mixed with a solution of a reducing agent to remove / quench any excess oxidizing agent. The peptide collected in the storage tank can be subjected to further purification, e.g., a chromatographic purification step such as RP-HPLC, size exclusion chromatography, or ion exchange chromatography.

[0099] The apparatus of the present invention provides multiple options for controlling the reaction conditions of the oxidation reaction. As described above, the apparatus of the present invention can control the concentrations of reduced peptide, oxidation reagent, and oxidized peptide in the reaction vessel (e.g., by adjusting the reagent supply rate to the reaction vessel and / or the membrane filtration rate, i.e., the permeate formation rate), temperature (e.g., by adjusting the flux of the heating / cooling medium in the vessel jacket and the heat exchanger), and pressure (e.g., by controlling the recirculation pump, pressure control valve, and applied nitrogen flow), as well as the concentration of small molecules (e.g., by adjusting the membrane filtration rate, i.e., the permeate formation rate). Preferably, the adjustment of these parameters is performed by one or more automated devices that receive inputs from appropriate sensors in the reaction vessel or recirculation loop. Preferably, the sensors include one or more sensors selected from a pressure sensor, a temperature sensor, and a conductivity probe, a redox probe, and an optical probe (e.g., a UV-Vis probe or an NIR probe).

[0100] Automation control devices may be local control units or part of a central control unit. The central control unit may be organized to form various hierarchical levels of control, as in the case of a supervisory control and data acquisition (SCADA) control system architecture. For example, a control unit may include one or more remote monitoring computers that collect data from and send control commands to peripheral devices and one or more peripheral devices, such as remote terminal units (RTUs), programmable logic controllers (PLCs), and user interfaces, such as GUI panels. The PLCs and monitoring SCADA software may receive inputs from field sensors, such as temperature, pressure, redox potential, or conductivity sensors, among others. One or more SCADA monitoring computing platforms may further interact with a manufacturing execution system (MES), which in turn interacts with an enterprise resource planning (ERP) system. Furthermore, one or more SCADA monitoring computing platforms may perform logging tasks by sending specific process parameters to a dedicated database. In some embodiments, the control unit comprises at least one PLC equipped with sensors and actors that control the operation of at least one SCADA system, dosing pumps, recirculation pumps, and pressure control valves. In some embodiments, the control unit further controls the outflow of product from the holding loop, the outflow of liquid from the reaction vessel, the inflow of additional reagents, such as reducing agents or buffer / wash liquids, into the reaction vessel, and the flow of heating / cooling fluids through the vessel jacket and / or heat exchangers of the holding loop. In some embodiments, the control unit can further control the flow of protective gas (e.g., nitrogen) to the reaction vessel and the reaction vessel's mixing equipment.

[0101] In some embodiments, the control device is adjusted to maintain the amount of liquid in the reaction vessel and in the recirculation loop at a constant value within a certain range. Those skilled in the art will readily recognize that under these conditions, the feed flow rate, filtration rate, and retentate removal rate (if applicable) are interdependent. For example, an increase in the amount of oxidizing equivalents in the reduction vessel is adjusted by reducing the inflow of oxidizing agent while offsetting the increase in oxidizing equivalents by either decreasing the filtration rate, and / or increasing the inflow of peptide solution, and / or adding buffer solution to the reaction vessel. Similarly, an increase in the concentration of small molecule contaminants contained in the peptide solution is offset by adding a certain amount of buffer solution to the reaction vessel while increasing the filtration rate. Those skilled in the art will determine which parameters, in particular, affect the specific oxidation reaction at hand and configure the system to control those parameters.

[0102] The following aspects relate to particularly preferred embodiments of the device of the present invention: Reference signs shown in parentheses are for illustration only and should not be construed as limiting.

[0103] In a first aspect, the apparatus comprises: a) a reaction vessel (1) equipped with means for mixing the contents of the vessel, preferably the means for mixing the contents of the vessel is a stirrer or a pumping circuit; b) two spatially separated inlets (4) for liquid entry into said reaction vessel; c) two storage vessels (5), each connected to one of said inlets, one containing a liquid comprising an oxidizing agent and the other containing a liquid comprising the peptide to be oxidized; d) an automated pump (6) that allows for the liquid to flow from the storage vessel to the reaction vessel via an inlet and to be controlled; and e) means for subjecting the contents of the reaction vessel to membrane filtration; Equipped with.

[0104] In a second embodiment, the means for subjecting the contents of the reaction vessel to membrane filtration comprises or corresponds to a membrane filtration unit (7) and a retention loop (15) for circulating liquid from the reaction vessel through the membrane filtration unit and back to the reaction vessel.

[0105] In a third embodiment, the membrane filtration unit (7) according to the second embodiment is a cross-flow filtration unit, preferably the membrane filtration unit comprises a membrane housing with a nanofiltration membrane module having a molecular weight cut-off of less than 0.5 kDa.

[0106] In a fourth embodiment, the holding loop (15) of the apparatus according to the second embodiment further comprises a recirculation pump (11) and a pressure control valve (10).

[0107] In a fifth aspect, the apparatus according to any of the above aspects further comprises at least one temperature control means, which is selected from the group consisting of a jacketed storage vessel, a jacketed reaction vessel, or a heat exchanger integrated into the holding loop (15).

[0108] In a sixth aspect, the reaction vessel (1) of the device according to any of the above aspects further comprises an additional liquid inlet, preferably the liquid inlet making it possible to supply a buffer or a washing medium to the reaction vessel.

[0109] In a seventh aspect, the apparatus according to any of the above aspects further comprises a sensor (13) in the reaction vessel, which sensor provides a feedback signal to control at least one of said automated pumps.

[0110] In an eighth aspect, the sensor according to the seventh aspect is selected from an optical sensor and a redox potential probe, preferably the sensor is a combined metal electrode, most preferably a composite platinum electrode.

[0111] In a ninth aspect, the apparatus according to the seventh or eighth aspect further comprises a controller unit which receives inputs from said sensors in the reaction vessel and provides control signals to one or more dosing pumps.

[0112] In a tenth aspect, the apparatus according to any of the above aspects further comprises at least one sensor selected from a temperature sensor, a pressure sensor or an optical sensor in the reaction vessel (1), in the holding loop (15) or in a bypass to the holding loop.

[0113] In an eleventh aspect, the device according to any of the above aspects further comprises a central control unit, which receives inputs from the sensors and controls the operation of the pump, the pressure within the device, and optionally the pressure within the device.

[0114] It should be noted that the above explanations and descriptions of embodiments of the method according to the invention are likewise applicable to the device according to the invention and vice versa.

[0115] The following figures and examples, including the experiments performed and results achieved, are presented for illustrative purposes only and should not be construed as limiting the scope of the claims. [Brief explanation of the drawings]

[0116] [Figure 1] 1 shows various positions of the inlet for the solution containing the reduced peptide and the oxidizing agent in the reaction vessel. Panels a) to i) show the reaction vessel from a side view, and panel j) shows a top view illustrating the (imaginary) angle α enclosed between both inlets. Panels a) to i) illustrate possible positions of the inlet (4) relative to the surface of the reaction medium (2), which is mixed by the mixing means (3). [Figure 2]1 shows a simulation of the change in concentration of reduced peptide (open circles), total peptide (closed triangles) and oxidant (closed diamonds) in the reaction vessel over time during steps d) and e) in one embodiment of the method of the invention. The dashed line indicates the start of step e). [Figure 3] 1 shows an embodiment of the apparatus according to the present invention, in which a reaction vessel (1) is connected to a pumping circuit including a membrane filtration unit. A solution of reduced peptide and oxidizing agents (A, B) is fed to the reaction vessel (1) through spatially separated inlets (4). The reaction vessel (1) may be equipped with a mixer (not shown). A holding loop (15) circulates the liquid from the outlet of the reaction vessel (1) through a cross-flow filtration unit (7) and back to the reaction vessel (1), thereby subjecting the contents of the reaction vessel (1) to membrane filtration. The liquid backflow rate and pressure in the holding loop are controlled by a recirculation pump (11) and a pressure control valve (10). The formed permeate (P) is discharged from the membrane filtration unit (7) to a permeate tank (8). [Figure 4] Figure 1 shows analytical HPLC traces of a peptide oxidized according to Comparative Example 2 (trace i) and the same peptide oxidized according to Example 3 (trace ii). It can be seen that the peptide oxidized according to the invention (trace ii) contains significantly fewer disulfide-linked oligomers (see area highlighted by arrow). [Figure 5] FIG. 3 shows another embodiment of the apparatus of the present invention, in which the apparatus of FIG. 3 is further complemented by a monitoring system (13) that provides a feedback signal to a dosing pump that controls the flow rate of reagents from storage vessel B to the reaction vessel. The holding loop contains two pressure sensors (9) that allow monitoring of the pressure before and after the nanofiltration unit. A liquid drain (12) allows the system to be emptied. A line (14) connected to a liquid outlet in the holding loop allows the product to be continuously withdrawn from the holding loop. The flux in this line is driven by a pump (not shown) integrated into this line. [Figure 6]Figure 1 shows analytical HPLC traces of a peptide oxidized according to Comparative Example 3 (trace a), and the same peptide oxidized according to Example 5.1 (trace b) and the long-term run of Example 5.2 (trace c). It can be seen that the peptide oxidized according to inventive Example 5 contains significantly fewer high molecular weight contaminants, which are expected to be oligomers (see areas highlighted by oval shapes). [Explanation of symbols]

[0117] 1 reaction vessel 2. Reaction medium 3 Mixing means 4 Liquid inlet 5. Storage containers 6. Pump for adding liquid 7. Membrane filtration unit 8 Permeate container 9. Pressure Sensor 10 Pressure control valve 11 Pump for recirculating liquids 12 Liquid discharge 13 Feedback Sensors and Control Devices 14 Continuous take-out line 15 Retention Loop [Example]

[0118] General method: Peptide 1 [HD-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-L-threoninol] and peptide 2 [(deamino-Cys 1 ,D-Tyr(Et) 2 ,Thr 4 ,Orn 8 )-oxytocin] was synthesized by standard methods and SPPS using Fmoc-amino acid derivatives. Peptide cleavage and simultaneous cleavage of protecting groups were performed using a cleavage cocktail containing at least 80% TFA, water, and a scavenger. The crude peptide was precipitated from the cleavage cocktail using diisopropyl ether as an antisolvent.

[0119] Comparative Example 1: Oxidation of Peptide 1 Crude peptide 1, produced by the general procedure, was dissolved in 30% acetic acid and diluted with 5% aqueous acetonitrile to a final peptide concentration of 1.3 g / L. 0.01 M aqueous iodine was slowly added to the peptide solution. After completion of the reaction, unreacted iodine was reduced by the addition of ascorbic acid. The resulting final peptide concentration was 0.95 g / L, as determined by analytical RP-HPLC, and the purity of the oxidized peptide was 90%.

[0120] Comparative Example 2: Oxidation of Peptide 1 A 0.069 M solution of iodine in 0.207 M aqueous potassium iodide was added continuously to 110 ml of stirred 30% aqueous acetic acid. A solution of crude peptide 1 (50 g / L) in 30% aqueous acetic acid was added continuously via a separate inlet. During the simultaneous addition, the reaction mixture turned reddish-brown, demonstrating an average iodine concentration greater than zero. After completion of the oxidation reaction, excess iodine was quenched by the addition of ascorbic acid, and the reaction product was analyzed by analytical RP-HPLC. The content of disulfide-linked oligomers was found to be 12.03%. The resulting final peptide concentration was 17.5 g / L.

[0121] Example 3 Oxidation of peptide 1 Crude peptide 1, produced by the general procedure, was dissolved in 30% aqueous acetic acid to a concentration of 50 g / L. This solution was added continuously to 110 ml of stirred 30% aqueous acetic acid. A 0.069 M iodine solution in 0.207 M potassium iodide was added continuously via a separate inlet. During the simultaneous additions, the reaction mixture turned slightly yellow, demonstrating that the average iodine concentration was essentially zero. When the addition of the iodine solution continued beyond the peptide addition, the color of the reaction mixture turned reddish-brown. After completion of the oxidation reaction, excess iodine was quenched by the addition of ascorbic acid, and the reaction product was analyzed by analytical RP-HPLC. The disulfide-linked oligomer content was found to be 7.63%. The resulting final peptide concentration was 17.5 g / L. Thus, as illustrated in Figure 4, the method according to the present invention is able to reduce the amount of disulfide-linked oligomers by 63% compared to the prior art method of Example 2. Furthermore, the concentration of oxidized peptide obtained is significantly higher than that obtained by the prior art method of Example 1.

[0122] Example 4 Oxidation of peptide 2 The crude peptide produced by the general procedure was dissolved in 60% aqueous acetic acid to a concentration of 100 g / L. This solution was added continuously to the stirred solution of acetic acid. Simultaneously, a solution of 0.075 M iodine in 0.227 M aqueous potassium iodide was added via a separate inlet. During the simultaneous additions, the reaction mixture turned slightly yellow, demonstrating that the average iodine concentration was essentially zero. When the addition of the iodine solution continued beyond the peptide addition, the color of the reaction mixture turned brown. Excess iodine was quenched by the addition of ascorbic acid, and the reaction products were analyzed by analytical RP-HPLC. The resulting final peptide concentration was 27.8 g / L.

[0123] Example 5 Oxidation of peptide 1 by nanofiltration Crude peptide 1, produced by the general procedure, was dissolved in 30% aqueous acetic acid at a concentration of 50 g / L. A solution of 0.052 M iodine in 0.159 M aqueous potassium iodide was prepared. The two solutions were filled into first and second storage vessels, respectively. These storage vessels were connected to a stirred reaction vessel via remote inlets. The reaction vessel served as the feed vessel for the nanofiltration system, which consisted of a holding loop with a pressure sensor and pressure control valve, a recirculation pump, and a ceramic monochannel membrane (filtration area: 0.0104 m). 2 The reactor vessel and the holding loop of the nanofiltration system were filled with 2 L of 30% aqueous acetic acid.

[0124] The oxidation reaction was initiated by simultaneously adding the crude peptide solution and the iodine solution to the reaction vessel. During the simultaneous additions, the reaction mixture turned slightly yellow, demonstrating that the average iodine concentration was essentially zero. The addition of the iodine solution continued beyond the peptide addition. Liquid circulation within the nanofiltration system was initiated prior to the start of reagent flow into the reaction vessel and maintained throughout the reagent addition period. The pressure in the retention loop was set to 23 bar. Permeate and retentate samples were taken at regular time points, quenched by the addition of ascorbic acid (0.5 mL sample + 50 μL of 0.5 M ascorbic acid), and then analyzed by analytical RP-HPLC. After completion of the oxidation reaction, the nanofiltration system was depressurized and drained, and the retentate was collected. The retentate was quenched by the addition of ascorbic acid, and the reaction products were analyzed by analytical RP-HPLC.

[0125] 5.1 Short-term operation The total run time was approximately 2.2 hours. The final peptide concentration obtained was 5 g / L with a purity of 92.59%. The content of disulfide-linked oligomers was found to be less than 5%.

[0126] 5.2 Long-term operation The total run time was approximately 9 hours. The final peptide concentration obtained was 20 g / L with a purity of 90.44%. The content of disulfide-linked oligomers was found to be less than 5%.

[0127] No linear peptides were detected in the permeate.

[0128] Thus, as illustrated in Figure 6, the method according to the present invention is able to significantly further reduce the amount of disulfide-linked oligomers compared to the method of Example 3. Furthermore, the concentration of the resulting oxidized peptides is higher than in the prior art methods of Examples 1-4 and can be further increased by adjusting the operation of the nanofiltration system.

Claims

1. 1. A method for producing a peptide having at least one intramolecular disulfide bond, comprising: a) providing a liquid reaction medium in a reaction vessel having a pH below 5.0, wherein the vessel allows for mixing of its contents; b) providing a solution of peptides, wherein the peptides contain at least two sulfhydryl groups; c) providing a solution of an oxidizing agent; d) simultaneously adding said solution of oxidizing agent and said solution of peptide to said reaction medium in the reaction vessel through spatially separated inlets while mixing the contents of the reaction vessel, said addition comprising: i) the average concentration of the peptide in its reduced state in the reaction vessel, C1, is less than the concentration C0, the final concentration of the peptide in the reaction vessel, at the end of the peptide addition, regardless of its oxidation state; ii) during the time of simultaneous addition of oxidizing agent and peptide, the average concentration of oxidizing agent within the reaction vessel is maintained at essentially zero, where essentially zero means that the amount of oxidizing agent is limited to disulfide bond formation; The process is carried out as follows: e) The addition of the oxidizing agent is continued beyond the end of the peptide addition. The method comprising:

2. 2. The method of claim 1, wherein the total amount of oxidizing agent added to the reaction vessel is equivalent to 1.0 to 1.5 equivalents of the total amount of disulfide bonds present in the fully oxidized peptide.

3. The method according to any one of claims 1 to 2, wherein steps d) and e) are alternately repeated at least once.

4. 4. The method of any one of claims 1 to 3, further comprising the step of removing any excess oxidizing agent contained in the reaction vessel after completion of step e).

5. 5. The method of any one of claims 1 to 4, wherein mixing of the contents of the reaction vessel is achieved by an agitator, by gas aeration, by liquid circulation, or any combination thereof.

6. The method of any one of claims 1 to 5, wherein low molecular weight species are removed from the contents of the reaction vessel.

7. 7. The method of claim 6, wherein membrane filtration is carried out simultaneously with step d).

8. The method of any one of claims 1 to 7, wherein the flow rate of the solution of oxidizing agent is controlled by a feedback signal generated by a monitoring system.

9. The method of any one of claims 1 to 8, further comprising the step of purifying the peptide in its oxidized state.

10. The method according to any one of claims 1 to 9, wherein the oxidizing agent is selected from the group consisting of iodine, hydrogen peroxide, dimethyl sulfoxide, 2,2'-dipyridyl disulfide and aqueous potassium hexacyanoferrate(III).

11. 11. The method of any one of claims 1 to 10, wherein the sulfhydryl group is part of a cysteine ​​side chain and / or the peptide contains exactly two sulfhydryl groups and / or the peptide is selected from the group consisting of somatostatin, lanreotide, octreotide, dotatate, edotreotide, aprotinin, oxytocin, (Arg8)-vasopressin, vasopressin, linaclotide, ziconotide, eptifibatide, desmopressin, pramlintide, calcitonin and atosiban.

12. 12. The method according to any one of claims 1 to 11, wherein during step d) the ratio C0 / C1 is at least 10.

13. 5. The method of claim 4, wherein the removal is carried out by addition of a suitable reducing agent.

14. A method according to any one of claims 1 to 13, wherein low molecular weight species are removed from the contents of the reaction vessel by membrane filtration.

15. 15. The method of any one of claims 1 to 14, wherein the flow rate of said solution of oxidizing agent is controlled by a monitoring system via a feedback signal generated by a redox potential probe in the reaction vessel.

16. 16. The method of any one of claims 1 to 15, wherein the flow rate of said solution of oxidizing agent is controlled by a monitoring system via a feedback signal generated by an oxidation-reduction potential probe in the reaction vessel, the monitoring system being located in or at the reaction vessel, the holding loop and / or the bypass loop.

17. 17. The method of any one of claims 1 to 16, further comprising purifying the peptide in its oxidized state by preparative chromatography.

18. 18. The method according to any one of claims 1 to 17, wherein during step d) the ratio C0 / C1 is at least 100.

19. 19. The method according to any one of claims 1 to 18, wherein during step d) the ratio C0 / C1 is at least 1000. The method according to any one of claims 1 to 10.

20. 17. An apparatus suitable for carrying out the method according to claim 16, comprising: a) a reaction vessel (1) equipped with means (3) for mixing the contents of the vessel; b) two spatially separated inlets (4) for liquid entry into said reaction vessel; c) two storage vessels (5), each connected to one of said inlets, one containing a liquid comprising an oxidizing agent and the other containing a liquid comprising the peptide to be oxidized; d) an automated pump (6) that allows for the liquid to flow from the storage vessel to the reaction vessel via an inlet and to be controlled; and e) a monitoring system within or attached to the reaction vessel, or within or attached to the holding loop, or within or attached to a bypass to the holding loop, that provides a feedback signal for controlling at least one of the automated pumps (6), the monitoring system including an oxidation-reduction potential probe; and f) means for subjecting the contents of the reaction vessel to membrane filtration, said means comprising a membrane filtration unit (7) and a holding loop (15) capable of circulating liquid from the reaction vessel (1) to the membrane filtration unit (7) and back to the reaction vessel (1); The device comprising:

21. 21. Apparatus according to claim 20, wherein the reaction vessel (1) equipped with means (3) for mixing the contents of the vessel is characterized in that the means for mixing the contents of the vessel is an agitator or a pumping circuit.

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