Purification of peptide coupling reagents

Abstract

The present invention provides a method for purifying a crude product of a peptide coupling reagent by removing foreign particles using an optical sorting machine. The method includes steps for detecting and ejecting foreign particles to obtain a purified peptide coupling reagent, specifically phosphonium or uronium salts. Additionally, the invention encompasses the solid purified product obtainable by this method and its application in peptide coupling reactions, enhancing the efficiency and quality of pharmaceutical synthesis processes.

Description

Attorney Docket No. P25-123-SEC-WO01PURIFICATION OF PEPTIDE COUPLING REAGENTSCross-Reference to Related Application

[0001] The present application claims the benefit of priority of U.S. provisional patent application no. 63 / 772,149, filed on March 14, 2025, the content of which is hereby incorporated in its entirety by reference.Field of Invention

[0002] The synthesis of amides from amines and carboxylic acids is a critical step in the preparation of active pharmaceutical ingredients (APIs), with, for example, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) and related peptide coupling reagents playing a vital role in this process. The performance of these peptide coupling reagents can significantly impact the efficiency and quality of API synthesis.

[0003] The present application provides an innovative and efficient method for purifying a crude product in particle form of a peptide coupling reagent such as, for example, PyBOP, by removing foreign particles, thereby enhancing its efficacy in amide formation. The purification method employs an advanced optical sorting machine, which utilizes detection signals from an optical detector to detect foreign particles based on their size, shape, and / or color. This system is designed to eject foreign particles from the material stream along a predetermined trajectory, effectively removing impurities from the crude product. Specifically, it is capable of detecting and removing orange-colored particles with a minimum size in three dimensions (3D) of 100 to 300 pm, preferably 150 to 250 pm, more preferably 200 pm, as well as dark orange, non-transparent, or black particles of a minimum size in three dimensions (3D) of 50 to 150 pm, preferably 75 to 125 pm, more preferably 100 pm.

[0004] The optical sorting system machine is equipped with an advanced camera inspection system that enhances its ability to monitor and analyze powder materials in real-time. This system comprises three high-resolution, high-speed cameras strategically positioned to inspect the crude productAttorney Docket No. P25-123-SEC-WO01particles as they flow through the sorting process. Employing the waterfall principle, the crude product is allowed to trickle down an edge, which facilitates meticulous control over the sorting process on a particle-by-particle basis. This method ensures that each individual particle is thoroughly examined, enhancing the accuracy of the sorting operation. During the inspection, any foreign particles that are identified as impurities are directed towards a specialized flap mechanism designed for their removal. This targeted approach ensures that only the acceptable particles continue through the sorting process. The remaining particles, classified as acceptable, are then collected and systematically distributed into separate packaging containers, each designated for further processing based on specific criteria. The removal of foreign particles is accomplished through a precisely controlled air stream that is selectively applied to the crude product. This air stream is directed with accuracy to ensure that only the identified impurities are displaced, minimizing disruption to the flow of acceptable particles.

[0005] Additionally, the sorting machine preferably incorporates a dosing mechanism that utilizes three scales, each associated with a packaging container. The scales automatically halt when the target net weight is achieved, prompting a request for manual packaging container replacement.

[0006] To our knowledge, this is the first instance of a peptide coupling reagent such as, for example, PyBOP being purified through this method for use in the synthesis of APIs, including, for example, semaglutide and related pharmaceutical compounds. This innovative purification method represents a significant advancement in the preparation of high-quality amides, with the potential to improve the overall production process for various APIs.

[0007] As the demand for high-purity reagents continues to grow in the pharmaceutical industry, this purification method according to the present invention plays a crucial role in streamlining production workflows, reducing costs, and ultimately enhancing the quality of therapeutic agents. This innovation not only represents a technical achievement but also underscores the ongoing commitment to improving pharmaceutical manufacturingAttorney Docket No. P25-123-SEC-WO01processes and ensuring the delivery of safe, effective medications to patients.

[0008] Provided is a method for purifying a crude product in particle form of a peptide coupling reagent by removing foreign particles from said crude product to obtain a purified peptide coupling reagent utilizing an optical sorting machine, wherein the peptide coupling reagent is a phosphonium salt or an uranium salt. There is further provided a purified peptide coupling reagent obtainable by said method. Said purified peptide coupling reagent can be used in a peptide coupling reaction.Background

[0009] WO 90 / 10009 A1 relates to phosphonium salts as peptide coupling reagents, particularly focusing on a new reagent based on the general formula' R '\l— P+— YR' / AL -l3where R and R' together form a tetramethylene group, Y represents chlorine, bromine, or the benzotriazoyloxy group (OBt), and A represents the hexafluorophosphate group (PFe-). This invention aims to improve peptide synthesis by offering a coupling reagent that avoids the drawbacks of existing reagents like BOP, particularly in terms of efficiency and the formation of guanidinated by-products. The document outlines various synthesis methods and applications of these reagents in peptide synthesis.

[0010] WO 2009 / 138985 A2 relates to the field of peptide synthesis, specifically the development of novel coupling agents designed to enhance the efficiency and yield of peptide bond formation. It emphasizes the use of coupling agents containing unique leaving groups, such as HBTLI (2-(1H-benzotriazol-1-yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate) and DCC (N,N'-dicyclohexylcarbodiimide), to facilitate the coupling of amino acids, including those with challenging structures like secondary and tertiary alpha amines. The synthesis methods described include solid-phase and Solution-Attorney Docket No. P25-123-SEC-WO01phase techniques, highlighting the importance of minimizing racemization during peptide formation. The document outlines various chemical structures and the effectiveness of these coupling agents in producing high-purity peptides.

[0011] WO 2005 / 027978 A2 pertains to the field of peptide chemistry, specifically the development of novel glucagon-like peptide-1 (GLP-1) derivatives. It emphasizes the use of peptide coupling reactions to synthesize these analogs, utilizing reagents such as HBTII (2-(1 H-benzotriazol-1 -yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and DCC (N,N'-dicyclohexylcarbodiimide). The synthesis involves solid-phase peptide synthesis techniques, employing protecting groups like Fmoc and Boc, and specific removal methods for Mtt and Dde groups. These methods aim to enhance the stability and bioavailability of GLP-1 analogs for therapeutic applications.

[0012] WO 2006 / 097537 A2 relates to novel acylated GLP-1 analogs aimed at providing prolonged therapeutic effects for type 2 diabetes and obesity. These analogs improve the stability and bioavailability of GLP-1 through structural modifications, including lipophilic groups for enhanced albumin binding. Described are synthesis methods focusing on peptide coupling reactions using reagents like HBTU (2-(1 H-Benzotriazol-1 -yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate) and HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) in solid-phase peptide synthesis, along with the use of protecting groups such as Mtt and Dde.

[0013] US 2023 / 033325 A1 describes an optical sorter integrating advanced features for detecting and sorting materials based on optical signals. The machine comprises a light source that irradiates sorting targets on a conveyance route with light. It employs optical sensors to detect light emitted from the light source and reflected from the sorting targets, enabling the identification of foreign objects and defective products.

[0014] The present invention addresses the technical problem of lower yields in peptide synthesis reactions attributed to impurities in couplingAttorney Docket No. P25-123-SEC-WO01reagents, specifically PyBOP. The presence of these impurities adversely affects the efficiency and effectiveness of peptide coupling processes, leading to suboptimal yields in the final peptide products.

[0015] Impure PyBOP may contain various contaminants, including approximately 0.02 wt% triethylamine hydrochloride (NEts HCI), 0.07 wt% PyBOP oxide, and around 0.1 wt% of other impurities such as 1-chloro-2-nitrobenzene and benzotriazole, each present at levels under 100 ppm. These impurities not only interfere with the coupling reaction but also introduce variability in the quality of the synthesized peptides, resulting in lower overall yields and more complicated purification schemes. This issue is particularly critical in the production of peptide APIs such as, for example, semaglutide, a polypeptide drug where high purity and yield are essential for ensuring the efficacy and safety of the final product.

[0016] The presence of impurities in peptide coupling reagents such as, for example, PyBOP significantly hampers the scalability and reliability of peptide API production, necessitating the development of a method to purify peptide coupling reagents and enhance their yield in peptide coupling reactions.Object of Invention

[0017] The object of the present invention is to provide a method for purifying a peptide coupling reagent that is present as a crude product in particle form. The method aims to be efficient and continuous, making it suitable for large-scale production. By ensuring the removal of impurities from the peptide coupling reagent, the invention seeks to enhance the yield and quality of peptide synthesis reactions, thereby addressing the technical problems associated with the use of impure coupling reagents.

[0018] A further object of the present invention is to provide a method for purifying a peptide coupling reactant that is designed to be scalable and adaptable, facilitating its integration into existing manufacturing workflows for the preparation of peptide coupling reagents to be used for the synthesis ofAttorney Docket No. P25-123-SEC-WO01peptide-based therapeutics.

[0019] A further object of the present invention is to provide a purified peptide coupling reagent that is obtainable by the aforementioned method and that can be used in a peptide coupling reaction.Summary

[0020] In a first embodiment, the present invention provides a method for purifying a crude product in particle form of a peptide coupling reagent by removing foreign particles from said crude product to obtain a purified peptide coupling reagent utilizing an optical sorting machine, wherein the peptide coupling reagent is a phosphonium salt or an uranium salt, wherein the optical sorting machine comprises (i) a first container to accommodate a crude product in particle form, (ii) an optical detector to detect foreign particles in said crude product based on a detection signal, (iii) an ejector to remove said foreign particles along a predetermined trajectory from the crude product to provide a purified product, and (iv) a second container to collect said purified product, and wherein the method comprises the following steps:(A) providing a crude product in particle form of a peptide coupling reagent in the first container of the optical sorting machine;(B) detecting foreign particles in said crude product based on a detection signal from the optical detector;(C) removing said foreign particles along a predetermined trajectory from the crude product to obtain a purified peptide coupling reagent; and(D) collecting said purified peptide coupling reagent in the second container of the optical sorting machine.

[0021] In a second embodiment, the present invention further provides a purified peptide coupling reagent that is obtainable or obtained by the method according to the present invention.

[0022] In a third embodiment, the present invention further provides for theAttorney Docket No. P25-123-SEC-WO01use of the purified peptide coupling reagent, as described in the second embodiment of the present invention, in peptide coupling reactions.Brief Description of the Figures

[0023] FIG 1 : Representation of the chemical structures of the amide coupling reagents: PyBOP (benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphon-ium hexafluorophosphate), PyBroP (bromo-tris-pyrrolidino-phosphonium hexafluorophosphate), BOP (benzotriazole-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), HATLI (1-[bis(dimethylamin)methylene]-1 H-1 ,2,3-triazol[4,5-b]pyridinium-3-oxid-hexafluorophosphate), HBTII (2-(1 H-benzotriazole-1 -y l)-1 , 1 ,3,3-tetramethyluronium hexafluorophosphate), and HCTLI (O-(1 H-6-chlorobenzotriazole-1 -y l)-1 , 1 ,3,3-tetramethyluronium hexafluorophosphate).

[0024] FIG 2: Infrared (IR) spectrum obtained from a white reference PyBOP crystalline material. The spectrum illustrates the characteristic absorption peaks associated with the functional groups present in the purified PyBOP.

[0025] FIG 3: Infrared (IR) spectrum obtained from an orange PyBOP crystalline material (crude product). The spectrum illustrates the characteristic absorption peaks associated with the functional groups present in the impure PyBOP.

[0026] FIG 4: High-performance liquid chromatography (HPLC) spectrum of a white reference PyBOP crystalline material, acquired in electrospray ionization (ESI) positive mode. The chromatogram illustrates the base peak chromatogram (BPC), highlighting the retention times and relative intensities of the purified PyBOP.

[0027] FIG 5: High-performance liquid chromatography (HPLC) spectrum of an orange PyBOP crystalline material (crude product), acquired in electrospray ionization (ESI) positive mode. The chromatogram illustrates the base peak chromatogram (BPC), highlighting the retention times and relativeAttorney Docket No. P25-123-SEC-WO01intensities of the impure PyBOP.

[0028] FIG 6a: Microscopic image of sorted PyBOP.

[0029] FIG 6b: Microscopic image of unsorted PyBOP.

[0030] FIG 6c: Microscopic image of rejected PyBOP.

[0031] FIG 7a: Microscopic image of sorted PyBOP with scale bar.

[0032] FIG 7b: Microscopic image of unsorted PyBOP with scale bar.

[0033] FIG 7c: Microscopic image of rejected PyBOP with scale bar.

[0034] FIG 8: Bench top images of sorted PyBOP, unsorted PyBOP, and rejected PyBOP.Detailed Description

[0035] In a first embodiment, the present invention relates to a method for purifying a crude product in particle form of a peptide coupling reagent, specifically through the removal of foreign particles to yield a purified peptide coupling reagent. This purification process is executed utilizing an optical sorting machine designed to enhance the efficiency and effectiveness of the purification. Said optical sorting machine comprises (i) a first container to accommodate a crude product in particle form, (ii) an optical detector to detect foreign particles in said crude product based on a detection signal, (iii) an ejector to remove said foreign particles along a predetermined trajectory from the crude product to provide a purified product, and (iv) a second container to collect said purified product.

[0036] In said method for purifying, the peptide coupling reagent is a phosphonium salt or an uronium salt. The method comprises the steps of providing a crude product in particle form of a peptide coupling reagent, detecting foreign particles in said crude product, removing said foreign particles along a predetermined trajectory from the crude product to obtain a purified peptide coupling reagent, and collecting said purified peptide coupling reagent. The method for purifying enhances the yield and quality of peptideAttorney Docket No. P25-123-SEC-WO01synthesis by ensuring the removal of visible impurities from peptide coupling reagents that could negatively impact the reaction outcomes.

[0037] Thus, in a first embodiment of the present invention, there is provided a method for purifying a crude product in particle form of a peptide coupling reagent by removing by removing foreign particles from said crude product to obtain a purified peptide coupling reagent utilizing an optical sorting machine, wherein the peptide coupling reagent is a phosphonium salt or an uronium salt, wherein the optical sorting machine comprises (i) a first container to accommodate a crude product in particle form, (ii) an optical detector to detect foreign particles in said crude product based on a detection signal, (iii) an ejector to remove said foreign particles along a predetermined trajectory from the crude product to provide a purified product, and (iv) a second container to collect said purified product, and wherein the method comprises the following steps:(A) providing a crude product in particle form of a peptide coupling reagent in the first container of the optical sorting machine;(B) detecting foreign particles in said crude product based on a detection signal from the optical detector;(C) removing said foreign particles along a predetermined trajectory from the crude product to obtain a purified peptide coupling reagent; and(D) collecting said purified peptide coupling reagent in the second container of the optical sorting machine.

[0038] It is preferred that the peptide coupling reagent is selected from the list consisting of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), benzotriazole-1 -yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), 1-[bis(dimethylamin)methylene]-1 H-1 ,2,3-triazol[4,5-b]pyridinium-3-oxid-hexafluorophosphate (HATLI), 2-(1 H-benzotriazole-1 -yl)-1 , 1 ,3,3-tetramethyluronium hexafluorophosphate (HBTII), O-(1 H-6-chlorobenzotriazole-1 -yl)-1 , 1 ,3,3-tetramethyluroniumAttorney Docket No. P25-123-SEC-WO01hexafluorophosphate (HCTLI), 1 , 1 ,3,3-tetramethyl-2-(4,5,6,7-tetrachloro-1 ,3-dioxoisoindolin-2-yl)isouronium hexafluorophosphate(V) (CITU), (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholinocarbenium hexafluorophosphate (COMII), O-(benzotriazol-1 -yl)oxybis-(pyrrolidino)-uronium hexafluorophosphate (HAPyll), O-(benzotriazol-1-yl)-N,N,N',N'-bis(tetra-methylene)uronium hexafluorophosphate (HBPyll), N,N,N',N'-tetramethyl-S-(1-oxido-2-pyridyl)thiuronium hexafluorophosphate (HOTT), 0-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTII), 1 , 1 ,3,3-tetramethyl-2-(2-oxopyridin-1 (2H)-yl)isouronium hexafluorophosphate (HPTLI), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate (HSTLI), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), 6-chloro-benzotriazole-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyClock), 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyOxim), 1-[bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxide (TATLI), N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTLI), O-(6-chlorobenzotriazole-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TCTLI), O-(3,4-dihydro-4-oxo-1 ,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), 2-(endo-5-norbornene-2.3-dicarboxylimide)-1 , 1 ,3,3-tetramethyluroniumtetrafluoroborate (TNTLI), O-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTII), O-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTLI), and N,N,N',N-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTLI). This highlights the versatility of the method for purifying according to the present invention across different classes of peptide coupling reagents.

[0039] It is more preferred that the peptide coupling reagent is selected from the list consisting of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), and benzotriazole-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP). In particular, the method may focus on specific reagents such as PyBOP, PyBroP, and BOP for enhancedAttorney Docket No. P25-123-SEC-WO01purification outcomes. The method for purifying according to the present invention is particularly effective for purifying these specific peptide coupling reagents.

[0040] It is most preferred that the peptide coupling reagent is benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP). The method for purifying according to the present invention is particularly effective for purifying PyBOP.

[0041] FIG 1 illustrates various exemplary peptide coupling reagents, highlighting their chemical structures and properties.

[0042] The method for purifying focuses on the physical properties of the crude product in particle form of the peptide coupling reagent, which preferably has a crystalline, transparent-broken structure (sugar-like characteristics) to ensure optimal purification. Regarding the sugar-like characteristics, the particles are characterized by elongated cube shapes that slant to one side, giving them a distinctive appearance reminiscent of sugar crystals. This unique morphology contributes to their functional properties, such as flowability and dissolution rates, making them suitable for various applications. If the crude product does not possess a crystalline, transparent-broken structure and instead exhibits powdery or flour-like characteristics, the separation process may be compromised, resulting in suboptimal purification.

[0043] The preferred minimum grain size of the peptide coupling reagent is approximately 100 pm in three dimensions (3D), while the preferred maximum grain size is approximately 3 mm in three dimensions (3D). More preferably, the minimum grain size of the peptide coupling reagent is approximately 200 pm and the maximum grain size is approximately 2.5 mm in three dimensions (3D). Most preferably, the minimum grain size of the peptide coupling reagent is approximately 200 pm and the maximum grain size is approximately 2 mm in three dimensions (3D).

[0044] It is preferred in the method for purifying according to the present invention that the foreign particles in the crude product are detected and removed based on at least one specific characteristics selected from size,Attorney Docket No. P25-123-SEC-WO01shape, and color. This emphasizes the importance of these properties in achieving effective detection and removal of foreign particle impurities and purification of the peptide coupling reagent. For instance, foreign particles may include orange-colored particles of a minimum size in three dimensions (3D) of 100 to 300 pm, preferably 150 to 250 pm, more preferably 200 pm and / or dark orange, non-transparent, or black particles of a minimum size in three dimensions (3D) of 50 to 150 pm, preferably 75 to 125 pm, more preferably 100 pm. This highlights the precision of the optical sorting machine in detecting and removing foreign particles as impurities.

[0045] It is preferred in the method for purifying according to the present invention that the foreign particles in the crude product are detected and removed based on specific characteristics, including:a) orange-colored particles of a minimum size in three dimensions (3D) of 100 to 300 pm, preferably 150 to 250 pm, more preferably 200 pm; and / orb) dark orange, non-transparent, or black particles of a minimum size in three dimensions (3D) of 50 to 150 pm, preferably 75 to 125 pm, more preferably 100 pm.

[0046] It is preferred that the optical detector of the optical sorting machine is a camera inspection system comprising three or more high-resolution cameras. It is more preferred that the optical detector is a camera inspection system comprising three high-resolution cameras. This enhances the accuracy and efficiency of the method for purifying according to the present invention.

[0047] It is preferred that the optical sorting machine further comprises (v) a conveyor system that transports the crude product from the first container, past the optical detector to the ejector. This ensures an efficient and streamlined workflow, thereby optimizing the purification process.

[0048] It is preferred that the optical sorting machine further comprises (vi) a dosing mechanism that automatically and continuously dispenses the purified product collected in the second container into individual packaging. ThisAttorney Docket No. P25-123-SEC-WO01enables automated and efficient handling and packaging of the purified product, minimizing manual intervention and enhancing throughput.

[0049] It is preferred that the ejector of the optical sorting machine operates based on a waterfall principle, allowing particle-by-particle control as the crude product trickles down over an edge. This ensures precise removal of foreign particle impurities while minimizing the loss of the desired product.

[0050] It is preferred that the optical sorting machine further comprises (vii) a flap mechanism to transport the removed foreign particles into a designated removal area. This ensures that the purification process is both effective and clean.

[0051] Preferably, the second container of the optical sorting machine is attached to three packaging containers, each equipped with a scale that stops the dosing process upon reaching a predetermined net weight of the purified product. This provides a systematic approach to packaging and ensures accurate weight measurements of the purified product.

[0052] Preferably, the delivery rate of the purified peptide coupling reagent is in the range from 20 to 80 kg / h, preferably from 40 to 60 kg / h, more preferably 50 kg / h, allowing for the manual handling of packaging operations, including:a) filling the first container from a 10 to 40 kg, preferably 20 to 30 kg, more preferably 25 kg primary packaging container of the crude product of the peptide coupling reagent;b) removing a filled 0.1 to 10 kg, preferably 0.5 to 5 kg, more preferably 1 kg final packaging container of the purified peptide coupling reagent; and c) sealing the final packaging container with a lid and filling it with a protective gas atmosphere.

[0053] Suitable optical sorting machines are commercially available from Meliscout, a leader in advanced sorting technology. A particularly preferred embodiment of the present invention involves the use of an automated opticalAttorney Docket No. P25-123-SEC-WO01sorting system, such as those developed by Meliscout, which significantly enhances both the precision and efficiency of the purification process for peptide coupling reagents.

[0054] The integration of this advanced technology into the purification workflow allows for real-time detection and removal of foreign particles, ensuring that only high-purity peptide coupling reagents are collected. The automated optical sorting system operates with a high degree of accuracy, utilizing sophisticated optical detection methods to identify and classify particles based on specific characteristics such as size, shape, and color. This capability is crucial for maintaining the integrity of the purified product, as even minor impurities can adversely affect the efficacy and safety of peptide-based pharmaceuticals.

[0055] Additionally, the use of Meliscout's optical sorting technology allows for scalability in production. As the demand for peptide coupling reagents grows, the automated system can be easily adapted to handle larger volumes without compromising the quality of the purification process. This scalability ensures that manufacturers can meet market demands while maintaining optimal product quality.

[0056] In a second embodiment, the present invention relates to a purified peptide coupling reagent obtainable or obtained by the method for purifying according to the first embodiment of the present invention. The method for purifying according to the present invention results in a purified peptide coupling reagent that is obtainable through the described method, which can be utilized in various peptide coupling reactions, enhancing the efficiency and quality of such reactions in practical applications. This embodiment emphasizes the end product of the purification process.

[0057] In a third embodiment, the present invention relates to the use of the purified peptide coupling reagent according to the second embodiment of the present invention in a peptide coupling reaction. This embodiment highlights the practical application of the purified product in synthesizing peptidesAttorney Docket No. P25-123-SEC-WO01effectively.

[0058] It is preferred that the peptide coupling reaction is carried out in the presence of a carbodiimide or a carbonyl compound. Preferred carbodiimides are N,N’-dicyclohexylcarbodiimide (DCC), N,N’-diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). A preferred carbonyl compound is 1 ,1’-carbonyldiimidazole (CDI).

[0059] The purified peptide coupling reagent obtained through the method described in this invention demonstrates significant utility in peptide coupling reactions, which are essential for the synthesis of various active pharmaceutical ingredients (APIs). The enhanced purity and quality of the peptide coupling reagent not only improve the yield of the desired peptide products but also ensure the overall efficiency of the synthesis process.

[0060] The practical applications are exemplified by several peptide-based APIs that can benefit from the use of the purified peptide coupling reagent. Notable examples include:

[0061] Semaglutide: A glucagon-like peptide-1 (GLP-1) analogue used in the treatment of type 2 diabetes and obesity. The synthesis of semaglutide involves complex peptide coupling reactions, where the purity of the reagents is critical for achieving the desired therapeutic efficacy.

[0062] Enfuvirtide: An HIV fusion inhibitor that is composed of a 36-amino-acid peptide. The production of enfuvirtide requires precise peptide coupling techniques to ensure the correct sequence and structure, making the use of a high-purity coupling reagent indispensable.

[0063] Liraglutide: Another GLP-1 analogue used for glycemic control in type 2 diabetes. The synthesis of liraglutide benefits from the use of purified coupling reagents, which help minimize side reactions and improve the overall yield of the final product.

[0064] Ziconotide: A peptide derived from the cone snail venom, utilized for the treatment of severe chronic pain. The synthesis of ziconotide involvesAttorney Docket No. P25-123-SEC-WO01intricate peptide coupling reactions, where the quality of the coupling reagents directly influences the potency and safety of the drug.

[0065] Teduglutide: A GLP-2 analogue used in the treatment of short bowel syndrome. Similar to semaglutide and liraglutide, the synthesis of teduglutide relies on efficient peptide couplings, where the use of purified reagents can significantly enhance the production process.

[0066] These examples illustrate the critical role that the purified peptide coupling reagent plays in the pharmaceutical industry, particularly in the synthesis of peptide-based therapeutics. By utilizing the purified reagent, manufacturers can achieve higher yields, reduce impurities, and enhance the overall quality of the APIs produced. Furthermore, the improved efficiency in peptide coupling reactions can lead to more cost-effective production processes, ultimately benefiting patients through the availability of high-quality medications. The implications of this invention extend beyond individual drug synthesis, potentially impacting the broader field of peptide chemistry and its applications in drug development.

[0067] As used herein, the term “about” or “approximately”, when used in connection with a measurable numerical variable, refers to the indicated value of the variable and to all values of the variable that tare within the experimental error of the indicated value (e.g., within 95% confidence limit for the mean) or within ± 10%, preferably ± 5%, of the indicated value, whichever is greater.

[0068] The present invention is further illustrated by the examples following hereinafter which shall in no way be construed as limiting. The skilled person will acknowledge that various modifications, additions and alternations may be made to the invention without departing from the spirit and scope of the invention as defined in the appended claims.Attorney Docket No. P25-123-SEC-WO01ExamplesMaterials Used

[0069] All chemicals utilized in the following examples were sourced from Sigma-Aldrich. The materials included N-protected amino acid, such as N-(tert-butoxycarbonyl)-L-phenylalanine (Boc-Phe-OH), C-protected amino acid hydrochloride, such as glycine ethyl ester hydrochloride, diisopropylethylamine (DIPEA), dichloromethane (DCM), and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (98%, Sigma-Aldrich, Product No. 377848), which was used as PyBOP crude product in particle form. Additional equipment included the optical sorting machine “PowderSecure” (Meliscout), HPLC for analysis, and round-bottom flask. Working Example 1 - Purification of PyBOP Crude Product

[0070] The PyBOP crude product particles were subjected to purification using a “PowderSecure” optical sorting machine from Meliscout. The sorting machine employs optical detection to identify and remove impurities based on their absorption characteristics. These impurities are present in particle form and exhibit an orange, dark orange, non-transparent, or black appearance. The primary components of these impurities include triethylamine hydrochloride (NEts HCI), PyBOP oxide, 1-chloro-2-nitrobenzene, and benzotriazole.

[0071] Using the optical sorting machine, the impurity particles were detected and mechanically separated from the purified PyBOP crystals. The machine's optical detection system triggers the removal of impurities based on their optical / spectral characteristics including orange particles (with a minimum size of 100 to 300 pm in three dimensions (3D)), dark orange, nontransparent particles, and black particles (both with a minimum size of 50 to 150 pm in three dimensions (3D)). Following the purification process, the purified PyBOP crystals were analyzed using IR and HPLC.

[0072] IR spectra were obtained for both the purified PyBOP crystals (see FIG 2) and the PyBOP crude product (see FIG 3). It was noted that the crudeAttorney Docket No. P25-123-SEC-WO01product lacks an absorption band near 1724 cm-1in the IR spectra, while impurities exhibit additional signals near 1671 cm-1, 1531 cm-1, 1428 cm-1, and 1423 cm-1.

[0073] HPLC spectra were obtained for both the purified PyBOP crystals (see FIG 4) and the PyBOP crude product (see FIG 5).

[0074] Microscopic images of purified (sorted) PyBOP are shown in FIGs 6a and 7a, microscopic images of crude (unsorted) PyBOP are shown in FIGs 6b and 7b, and microscopic images of PyBOP impurities (rejected PyBOP) are shown in FIGs 6c and 7c. FIG 8 shows bench top images of purified (sorted) PyBOP, crude (unsorted) PyBOP, and PyBOP impurities (rejected PyBOP).

[0075] The removal of the above-mentioned impurities resulted in higher conversion rates in subsequent peptide synthesis reactions as shown in the following Working Example 2.Working Example 2 - Peptide Coupling Reaction

[0076] In a round-bottom flask, N-(tert-butoxycarbonyl)-L-phenylalanine (0.250 g, 0.94 mmol) was dissolved in 6 mL dichloromethane (DCM). To this solution, 1 equivalent of PyBOP (0.490 g, 0.94 mmol) and 1.1 equivalents of glycine ethyl ester hydrochloride (0.145 g, 1.04 mmol) were added. Finally, 2.75 equivalents of diisopropylethylamine (DIPEA) (0.335 g, 2.59 mmol) were introduced into the flask. The reaction mixture was stirred at 25 °C for 30 min. to allow for peptide coupling. The reaction mixture was then analyzed using High-Performance Liquid Chromatography (HPLC) to assess the conversion rates. During the analysis, it was noted that the starting materials constituted 0.75% of the mixture when 1-chloro-2-nitrobenzene was present. In contrast, when pure PyBOP was used, the percentage of starting materials decreased to 0.48%. This indicates that the presence of 1-chloro-2-nitrobenzene may hinder the reaction's efficiency, resulting in a higher retention of starting materials in the mixture. The comparison suggests that pure PyBOP facilitates a more effective reaction, leading to a lower concentration of unreactedAttorney Docket No. P25-123-SEC-WO01starting materials.Working Example 3 - Peptide Coupling Reaction

[0077] In a round-bottom flask, N-(tert-butoxycarbonyl)-L-phenylalanine (0.100 g, 0.38 mmol) was dissolved in 6 mL dichloromethane (DCM). To this solution, 1 equivalent of PyBOP (0.196 g, 0.38 mmol) and 1.1 equivalents of glycine methyl ester hydrochloride (0.52 g, 0.42 mmol) were added. Finally, 2.75 equivalents of diisopropylethylamine (DIPEA) (0.133 g, 1.03 mmol) were introduced into the flask. The reaction mixture was stirred at 25 °C for 30 min. to allow for peptide coupling. The reaction mixture was then analyzed using High-Performance Liquid Chromatography (HPLC) to assess the conversion rates. In separate experiments under the above reaction conditions, the PyBOP used was spiked with 5%, 10% and 15% rejected PyBOP. The concentration of product observed by HPLC decreased with the addition of more rejected PyBOP material which supports that the material removed by the optical sorter hinders the coupling reaction compared to the reaction using purified PyBOP (see Table 1).

[0078] Table 1 : Comparison of peptide coupling performance between sorted PyBOP and PyBOP spiked with 5%, 10%, and 15% rejected PyBOP.PyBOP Absorbance Product Absorbance (mAU) Concentration (mAU)*5(mg / mL)Sorted PyBOP 333.377 0.242916 1666.885 PyBOP (5% rejected) 270.903 0.189147 1354.515 PyBOP (10% rejected) 227.987 0.152211 1139.935 PyBOP (15% rejected) 216.591 0.142403 1082.955Working Example 4 - Peptide Coupling Reaction

[0079] In a round bottom flask, N-(tert-butoxycarbonyl)-L-phenylalanine (0.250 g, 0.94 mmol) was dissolved in 12 mL dichloromethane (DCM). To this solution, 1.1 equivalent of PyBOP (0.539 g, 1.03 mmol) and 1.1 equivalents ofAttorney Docket No. P25-123-SEC-WO01glycine methyl ester hydrochloride (0.130 g, 1.03 mmol) were added. Finally, 3.0 equivalents of diisopropylethylamine (DIPEA) (0.121 g, 2.82 mmol) were introduced into the flask. The reaction mixture was stirred at 25 °C for 3 hours, to allow for peptide coupling. The reaction mixture was then analyzed using High-Performance Liquid Chromatography (HPLC) to assess the conversion rates. In separate experiments under the above reaction conditions, the PyBOP used was sorted, unsorted and rejected PyBOP. The concentration of product observed by HPLC increased with the purity of the PyBOP used which supports that the material removed by the optical sorter hinders the coupling reaction compared to the reaction using sorted PyBOP (see Table 2).

[0080] Table 2: Comparison of peptide coupling performance between sorted PyBOP, unsorted PyBOP and rejected PyBOP.PyBOP Product Absorbance (mAU)Concentration(mg / mL)Sorted PyBOP 1.8495077 2200.076Unsorted PyBOP 1.6011421 1911.500Rejected PyBOP 0.8739349 1066.558Conclusion

[0081] The purification method effectively enhances the quality of PyBOP crystals by utilizing optical detection and mechanical separation, leading to improved yields in peptide synthesis applications. The absence of the specified absorption bands in the IR spectra of the purified product confirms the successful removal of impurities.

[0082] Furthermore, the analyses presented in Working Examples 3 and 4 demonstrate that purified (sorted) PyBOP exhibits significantly enhanced catalytic activity compared to PyBOP containing a defined proportion of impurities (referred to as rejected PyBOP) or to crude (unsorted) PyBOP. This indicates that the purification process effectively removes detrimentalAttorney Docket No. P25-123-SEC-WO01impurities, thereby optimizing the catalytic performance of PyBOP in various applications.

[0083] The examples presented herein are intended solely for illustrative purposes and should not be interpreted as limiting the scope of the invention as defined in the accompanying claims. It is important to recognize that the claims encompass a broader range of applications and variations that may not be explicitly detailed in the examples. Thus, the invention's scope should be understood in its entirety, allowing for modifications and alternative embodiments that fall within the appended claims.

Claims

Attorney Docket No. P25-123-SEC-WO01Claims1. A method for purifying a crude product in particle form of a peptide coupling reagent by removing foreign particles from said crude product to obtain a purified peptide coupling reagent utilizing an optical sorting machine, wherein the peptide coupling reagent is a phosphonium salt or an uronium salt, wherein the optical sorting machine comprises (i) a first container to accommodate a crude product in particle form, (ii) an optical detector to detect foreign particles in said crude product based on a detection signal, (iii) an ejector to remove said foreign particles along a predetermined trajectory from the crude product to provide a purified product, and (iv) a second container to collect said purified product, and wherein the method comprises the following steps:(A) providing a crude product in particle form of a peptide coupling reagent in the first container of the optical sorting machine;(B) detecting foreign particles in said crude product based on a detection signal from the optical detector;(C) removing said foreign particles along a predetermined trajectory from the crude product to obtain a purified peptide coupling reagent; and(D) collecting said purified peptide coupling reagent in the second container of the optical sorting machine.

2. The method according to claim 1 , wherein the peptide coupling reagent is selected from the list consisting of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), benzotriazole-1 -yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), 1-[bis(dimethylamin)methylene]-1H-1 ,2,3-triazol[4,5-b]pyridinium-3-oxid-hexafluorophosphate (HATLI), 2-(1 H-benzotriazole-1-yl)-1 , 1,3,3-tetramethyluronium hexafluorophosphate (HBTII), and O-(1H-6-chlorobenzotriazole-1 -y l)-1 , 1 ,3,3-tetramethyluronium hexafluorophosphate (HCTU).Attorney Docket No. P25-123-SEC-WO013. The method according to claim 1 or 2, wherein the peptide coupling reagent is selected from the list consisting of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), and benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP).

4. The method according to any one of claims 1 to 3, wherein the peptide coupling reagent is benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP).

5. The method according to any one of claims 1 to 4, wherein the foreign particles in the crude product are detected and removed based on at least one specific characteristics selected from size, shape, and color.

6. The method according to any one of claims 1 to 5, wherein the foreign particles in the crude product are detected and removed based on specific characteristics, including:a) orange-colored particles of a minimum size in three dimensions (3D) of 100 to 300 pm, preferably 150 to 250 pm, more preferably 200 pm; and / or b) dark orange, non-transparent, or black particles of a minimum size in three dimensions (3D) of 50 to 150 pm, preferably 75 to 125 pm, more preferably 100 pm.

7. The method according to any one of claims 1 to 6, wherein the optical detector is a camera inspection system comprising three or more high-resolution cameras.

8. The method according to any one of claims 1 to 7, wherein the optical sorting machine further comprises (v) a conveyor system that transports the crude product from the first container, past the optical detector to the ejector.

9. The method according to any one of claims 1 to 8, wherein the optical sorting machine further comprises (vi) a dosing mechanism that automatically and continuously dispenses the purified product collected in the second container into individual packaging.Attorney Docket No. P25-123-SEC-WO0110. The method according to any one of claims 1 to 9, wherein the ejector operates based on a waterfall principle, allowing particle-by-particle control as the crude product trickles down over an edge.

11. The method according to any one of claims 1 to 10, wherein the optical sorting machine further comprises (vii) a flap mechanism to transport the removed foreign particles into a designated removal area.

12. The method according to any one of claims 1 to 11 , wherein the second container is attached to three packaging containers, each equipped with a scale that stops the dosing process upon reaching a predetermined net weight of the purified product.

13. The method according to claim 12, wherein the delivery rate of the purified peptide coupling reagent is in the range from 20 to 80 kg / h, preferably from 40 to 60 kg / h, more preferably 50 kg / h, allowing for the manual handling of packaging operations, including:a) filling the first container from a 10 to 40 kg, preferably 20 to 30 kg, more preferably 25 kg primary packaging container of the crude product of the peptide coupling reagent;b) removing a filled 0.1 to 10 kg, preferably 0.5 to 5 kg, more preferably 1 kg final packaging container of the purified peptide coupling reagent; andc) sealing the final packaging container with a lid and filling it with a protective gas atmosphere.

14. Purified peptide coupling reagent obtainable by the method according to any one of claims 1 to 13.

15. Use of the purified peptide coupling reagent according to claim 14 in a peptide coupling reaction.

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