Trichloroacetic acid based cleavage reagent for peptide synthesis

Abstract

The present invention refers to a method of preparing a peptide, wherein a peptide precursor is incubated with a cleavage cocktail comprising trichloroacetic acid and one or more solvents and / or scavengers at elevated temperatures. Furthermore, the invention relates to a cleavage cocktail comprising trichloroacetic acid and the use for cleaving an acid-cleavable solid support or tag off a peptide precursor.

Description

[0001] Bachem Holding AG, 1 BAC75006PC1

[0002] Novo Nordisk A / S September 29, 2025

[0003] Cleavage reagent for peptide synthesis

[0004] The present invention refers to a method of preparing a peptide, wherein a peptide precursor is incubated with a cleavage cocktail comprising trichloroacetic acid and one or more solvents and / or scavengers at various temperatures. Furthermore, the invention relates to a cleavage cocktail comprising trichloroacetic acid and the use for cleaving an acid-cleavable non-peptidic moiety off a peptide precursor.

[0005] There are two standard approaches used in the context of chemical peptide synthesis, namely solid phase peptide synthesis (SPPS) and liquid phase peptide synthesis (LPPS). In addition, hybrid approaches may be utilized, wherein typically, fragments may first be synthesized by one of the above techniques, preferably SPPS, and then joined together using the other approach, preferably LPPS. This strategy may be employed for large (i.e. , long) peptides with challenging sequences. A variation of the above synthetic approaches are forms of synthesis where a (typically hydrophobic) tag (also referred to as solubility-modifying support) is used to support isolation of the growing peptide chain from other components of the reaction mixture by means of extraction, (nano)filtration, and / or precipitation steps (support-assisted peptide synthesis), which may be designated as tag-assisted peptide synthesis (TAPS). TAPS may also be considered as a variation of LPPS because TAPS allows carrying out the coupling reaction in a liquid phase. Hence, support-assisted peptide synthesis may be considered as a modified form subsumable under LPPS.

[0006] SPPS as well as LPPS and hybrid forms thereof are routinely based on sequentially coupling of building blocks (typically amino acid moieties optionally having protected side chains) using orthogonal protecting groups. In a typical approach, functional groups of the side chains of the peptide moieties are protected by one or more side chain protecting groups, while the site to which the next building block is to be conjugated (most typically the alpha-amino group) is temporarily protected by a temporary protecting group. Often, side chain protecting groups on the one hand and the temporary protecting group on the other hand are cleavable under different Bachem Holding AG, 2 BAC75006PC1

[0007] Novo Nordisk A / S September 29, 2025 conditions. A typical synthesis cycle includes cleaving off the temporary protecting group (most typically off the alpha-amino group) and coupling of next building block. This is repeated for necessary number of cycles until the optionally modified peptide strand is obtained. In this immediately obtained peptide strand, functional groups of side chains are typically still protected by the side chain protecting groups.

[0008] In a branched peptide (such as, e.g., a dendrimer), additionally or alternatively one or more side chains may be protected by temporary protecting groups allowing coupling of one or more building blocks to this site.

[0009] Inter alia, there are protecting groups that are cleavable under acidic conditions (acid-cleavable protecting groups) and protecting groups that are cleavable under other conditions such as basic conditions (base-cleavable protecting group).

[0010] Typical acid-cleavable protecting groups may include, for instance, tertbutyloxycarbonyl (Boc), tert-butyl (tBu), trityl (Trt), 2,2,4,6,7-pentamethyl-2,3- dihydrobenzofuran-5-sulfonyl (Pbf), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 4-Methoxy-2,3,6-trimethylphenylsulfonyl (Mtr), 9-Xanthydryl (Xan) 3- methylpent-3-yl ester (OMpe), 4-methyltrityl (Mtt), monomethoxytrityl (Mmt), 2,4- dimethoxybenzyl (Dmb), and diphenylmethyl (Dpm). Acid-cleavable protecting groups may be used to protect, e.g., amino acid side chains, or the peptide backbone. An example of the latter use is protection of a backbone amide group with 2,4-dimethoxybenzyl (Dmb). Furthermore, pseudoprolines may serve as an acid-cleavable protecting group. The term “pseudoproline” as used herein refers to temporary proline mimics, which can be readily obtained from Ser and Thr by oxazolidine formation and from Cys by thiazolidine formation. The person skilled in the art is well-aware of such pseudoproline dipeptides, in particular of 2,2- dimethyloxazolidines.

[0011] Typical base-cleavable protecting groups include, for instance, fluorenylmethyloxycarbonyl (Fmoc).

[0012] A common pattern of protecting groups, which may be referred to as Fmoc / tBu reaction scheme, may be based on: Bachem Holding AG, 3 BAC75006PC1

[0013] Novo Nordisk A / S September 29, 2025 base-cleavable protecting groups (e.g., Fmoc) as temporary protecting group (most typically at the alpha-amino group of building blocks), which is cleaved off at each reaction cycle releasing an amino group for further reaction (most typically at the alpha-amino group after conjugation of the building block), and acid-cleavable protecting groups as side chain protecting groups.

[0014] In case of SPPS, the cleaving conditions of side chain protecting groups and the linkage to the solid support are often essentially the same. This means that a single cleavage cocktail can facilitate the deprotection of side chains of the peptide and, concomitantly, releasing the peptide from the solid support. Most typically, the solid support is conjugated via a linker to the C-terminal end of the peptide. At end of synthesis, cleavage from support and removal of permanent protecting groups may be achieved concomitantly. Often, the peptide is fully deprotected in such step.

[0015] Various solid support materials are known in the art. For instance, such solid support may be swellable polystyrene beads crosslinked with 1 -2% divinyl benzene or swellable polystyrene beads with grafted hydrophilic compounds (e.g., TentaGel®). Various linkers are known for covalently binding a peptide to a carrier, which linkers are often cleavable under acidic conditions, cf. Brochure “Solid phase peptide synthesis”, Bachem May 2020, published by Global Marketing, Bachem AG.

[0016] In the case of Fmoc / tBu SPPS, the linker between the peptide strand and the solid support is typically also acid-cleavable. This allows suitable cleavage cocktails to cleave the side chain protecting groups and the solid support from the optionally modified peptide.

[0017] Such orthogonal protection scheme may also be used in LPPS, including the example of Fmoc / tBu reaction scheme. Optionally, this may also include cleaving an optionally acid-cleavable tag off the peptide such as, e.g., from its C-terminal end.

[0018] The cleavage of acid-cleavable groups as used in peptide synthesis for acid- cleavable non-peptidic moieties such as, e.g., solid support and / or tag and / or protecting groups typically requires harsh acidic conditions. These harsh conditions Bachem Holding AG, 4 BAC75006PC1

[0019] Novo Nordisk A / S September 29, 2025 should, however, be also able to maintain the peptide bonds within the peptide strand, should minimize side reactions of the peptide strand and / or its side chains and should allow solubilizing a large variety of peptides of different sequences.

[0020] In former times, hydrofluoric acid (HF) was considered for cleavage. Today, HF is still used for special peptide synthesis schemes. Typically, it is tried to avoid HF due to its danger when used, its toxicity and the complex means that are required when handling HF.

[0021] Thus, for decades, concentrated trifluoroacetic acid (TFA) has been the standard reagent for cleavage from solid support and / or tag and / or for cleaving of acid- cleavable protecting groups. In the frequently used Fmoc / tBu scheme, TFA may be used for cleaving the permanent protecting groups (most typically of the side chain functional groups) and optionally concomitantly cleaving the peptide from a solid support. There are, however, environmental concerns associated with per- and polyfluorinated substances (PFAS) such as TFA.

[0022] Therefore, alternatives to TFA are urgently needed. To reduce environmental concerns, such reagent should be a PFAS-free reagent.

[0023] A severe challenge in finding alternatives to TFA for cleaving acid-cleavable non- peptidic moieties such as solid support and / or tag and / or protecting groups is to strike a fine balance in that complete cleavage from the one or more acid-cleavable non-peptidic moieties of interest is needed, but the peptide should concomitantly not undergo degradation. In particular, hydrolysis of peptide bonds, deamidation reactions (e.g. Asn => Asp), and aspartimide formation should be essentially avoided. As far as possible, it is additionally desirable that other side reactions should be minimized such as the formation of tert-butyl adducts, the formation of SO3 adducts, the oxidation of methionine and tryptophane side chains, and the demethylation of methionine. Moreover, it is particularly desirable that an alternative cleavage cocktail should be able to swell the (often gel-like) solid support to which the peptide is bound in SPPS. Additionally, the cleavage cocktail should be able to solubilize a large variety of peptides of various sequences, including peptides with large hydrophobic side chains. In other words, an alternative cleavage cocktail Bachem Holding AG, 5 BAC75006PC1

[0024] Novo Nordisk A / S September 29, 2025 should be widely, if possible universally usable in peptide synthesis. It should allow replacing TFA.

[0025] Due to the challenges as noted above, there is still an unmet need for TFA-free cleavage cocktails that could replace TFA. Palladino and Stetsenko (Organic Letters, 2012, 14(24):6346-6349) reported a novel TFA-free cleavage and global deprotection in Fmoc-solid-phase peptide synthesis by dilute (0.1 to 1 M) HCI in fluoro alcohols like 2,2,2-trifluoro ethanol (TFE) or 1 ,1 , 1 ,3,3, 3-hexafluoroiso- propanol (HFIP) as solvents. However, just as TFA, TFE and HFIP fall in the category of Per- and Polyfluorinated Substances, and other protic and aprotic solvents tested in their place were found to be ineffective.

[0026] In the past, efforts have been made to replace TFA, used for the cleavage of the Boc protecting group at an alpha-amino group of the growing peptide in Boc-based SPPS. Less hazardous reagents including diluted non-fluorinated acids like methanesulfonic acid (MSA), benzenesulfonic acid, trichloroacetic acid (TCA), sulfuric acid, hydrochloric acid have been tested by Houghten et al. (Int. J. Peptide Protein Res., 1986, 27:653-658) to cleave the N-alpha-Boc group of small test peptides.

[0027] It is found that alternative cleavage cocktails based on trifluoromethanesulfonic acid (TFMSA) and methanesulfonic acid (MSA) do not bear an optimal performance for cleavage reactions. Deprotecting agents such as liquid HF, TFMSA or MSA are not considered as mild as TFA (Palladino and Stetsenko, Organic Letters, 2012, 14(24):6346-6349).

[0028] Trichloroacetic acid (TCA) has been described occasionally as a component of cleavage cocktails in specific SPPS protocols at room temperature. As TCA has a melting / thawing point of approximately 59 °C, it is solid at room temperature. US 5,371 ,186 states that in one example a short-length peptide was cleaved from the resin overnight at room temperature by 95% “trichloroacetic acid” containing 5% phenol as a scavenger. This experiment cannot be reproduced, because TCA is a solid material at room temperature. It was found that the crystallization temperature of TCA in 5% phenol (approximately 9.3 M) is in a range of approximately 35 °C. Bachem Holding AG, 6 BAC75006PC1

[0029] Novo Nordisk A / S September 29, 2025

[0030] Indeed, the solid form of TCA renders the use of TCA rather challenging. TCA was typically dissolved at low concentrations such as 2.5% to 10% weight per volume in combination with other agents such as MSA in various solvents such as taught by Houghten et al. (Int. J. Peptide Protein Res., 1986, 27:653-658).

[0031] There is still an unmet need for a method of preparing a peptide that includes a step of effectively cleaving off an acid-cleavable non-peptidic moiety such as an acid- cleavable support (e.g. a solid support or tag) from a peptide precursor that allows omittance of TFA. Such method should be based on a cleavage cocktail that is not based on a per- and polyfluorinated substances (PFAS) such as TFA. It should be feasible for different peptide sequences and for cleaving different acid-cleavable moieties such as protecting groups and solid support or tag concomitantly.

[0032] Surprisingly, it has been found that a cleavage cocktail containing trichloroacetic acid and one or more solvents and / or scavengers allows such method of preparing a peptide that includes a step of effectively cleaving off the acid cleavable support, in particular the acid-cleavable solid support or the acid-cleavable tag from a peptide precursor. Protecting groups can be cleaved off concomitantly, thereby yielding a globally deprotected peptide. As experimentally shown, such method is effective at room temperature and at elevated temperatures. Per- and polyfluorinated substances (PFAS) such as TFA and or PFAS-containing solvents and / or scavenges as well as harsh agents such as HF and MSA could surprisingly be entirely omitted. The cleavage efficacy is high and peptide structure remains intact. The method is comparably rapid. Short peptides and also rather long peptides of more than 20 amino acid moieties in lengths could be effectively prepared.

[0033] A first aspect of the present invention relates to a method of preparing a peptide, wherein the method comprises the steps of:

[0034] (i) contacting

[0035] (A) a peptide precursor that is covalently bound to at least one acid- cleavable support; with

[0036] (B) a cleavage cocktail comprising:

[0037] (b1 ) trichloroacetic acid, Bachem Holding AG, 7 BAC75006PC1

[0038] Novo Nordisk A / S September 29, 2025

[0039] (b2) one or more solvents selected from the group consisting of diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole, and

[0040] (b3) optionally a salt; and

[0041] (ii) incubating the components of step (i), thereby cleaving off the at least one acid-cleavable support from the peptide precursor resulting in the peptide, wherein the amount of trichloroacetic acid in the cleavage cocktail is in the range from 2 to 12 g per 1 mL of said one or more solvents (b2) (when the volume of the solvent is the volume at 25 °C), and the amount of trichloroacetic acid in the cleavage cocktail, based on the total mass of the cleavage cocktail, is at least 60% by weight.

[0042] Surprisingly, it has been found that cleavage cocktails according to the first aspect of the invention remain liquid at mild temperatures, despite the rather high melting point of trichloroacetic acid, while exhibiting good reactivity in cleaving acid- cleavable supports off from peptide precursors. Preferably, the amount of the amount of trichloroacetic acid in the cleavage cocktail is in the range from 3 to 12 g per 1 mL, most preferably 4 to 8 g per 1 mL of said one or more solvents (b2) (when the volume of the solvent is the volume at 25 °C). When the amount of trichloroacetic acid in the cleavage cocktail is high, the cleavage cocktails tend to solidify at mild temperatures such as below 40 °C. Moreover, when the amount of trichloroacetic acid in the cleavage cocktail too low, the reactivity of the cleavage cocktails tends to be reduced, and the reaction time tends to increase. It was found that cleavage cocktails according to the first aspect of the invention, where the amount of trichloroacetic acid is in the range from 4 to 8 g per 1 mL of said one or more solvents (b2) (when the volume of the solvent is the volume at 25 °C) work particularly well at room temperature and at slightly increased temperatures, e.g. at 25-45°C.

[0043] Moreover, it has been surprisingly found that the selection of the solvents (b2) allows conducting the cleaving of acid cleavable supports off from peptide precursors to occur with substantially reduced formation of by-products such as aspartimides compared with other solvents. Bachem Holding AG, 8 BAC75006PC1

[0044] Novo Nordisk A / S September 29, 2025

[0045] A second aspect of the present invention relates to a method of preparing a peptide, wherein the method comprises the steps of:

[0046] (i) contacting

[0047] (A) a peptide precursor that is covalently bound to an acid-cleavable support; with

[0048] (B) a cleavage cocktail comprising:

[0049] (b1 ) trichloroacetic acid,

[0050] (b2) one or more solvents selected from the group consisting of diphenylsulfide, monohalogenated thiophenols, and monohalogenated thioanisoles, and

[0051] (b3) optionally a salt; and

[0052] (ii) incubating the components of step (i), thereby cleaving off the acid-cleavable support from the peptide precursor resulting in the peptide.

[0053] It has been found that cleavage cocktails according to the second aspect of the invention perform even better than cleavage cocktails containing thioanisol in some cases.

[0054] Unless explicitly stated otherwise, the definitions and embodiments described herein are applicable to both the first and the second aspects of the invention.

[0055] As used herein, the term “and / or” is used to express what is called an “inclusive or”. In other words: the condition “A and / or B” is met if at least one of A or B is true (that is: either A is true, or B is true, or both are true). It is noted that some solvents such as phenol can be considered both a solvent and a scavenger, as phenol may react with carbocations, thereby removing this species from the reaction mixture. Commonly, the cleavage cocktail of the invention will comprise at least one solvent and at least one scavenger.

[0056] As used herein, unless specified otherwise, rounding conventions are applied for numbers. This means that, e.g., the number 95% is understood to comprise any number from 94.50% to 95.49%.

[0057] The method of the present invention allows the omittance of per- and polyfluorinated substances (PFAS) such as trifluoroacetic acid (TFA) in the cleavage step and Bachem Holding AG, 9 BAC75006PC1

[0058] Novo Nordisk A / S September 29, 2025 optionally in the whole method of preparing the peptide. This may have environmental benefits.

[0059] Per- and polyfluorinated substances (PFAS) may be defined “as fluorinated substances that contain at least one fully fluorinated methyl or methylene carbon atom (without any H / CI / Br / l atom attached to it), i.e. , with a few noted exceptions, any chemical with at least a perfluorinated methyl group (-CF3) or a perfluorinated methylene group (-CF2-) is a PFAS” (OECD (2021 ), Reconciling Terminology of the Universe of Per- and Polyfluoroalkyl Substances: Recommendations and Practical Guidance, OECD Series on Risk Management, No. 61 , OECD, Publishing, Paris, as published by the Organisation for Economic Co-operation and Development (OECD)).

[0060] As used herein, the term “peptide” and “polypeptide” may be understood interchangeably in the broadest sense as generally understood in the art. Typically, a peptide is characterized by the presence of at least one peptide bond (-CO-NH-) between at least two amino acid moieties. More preferably, a peptide comprises more than one peptide bond interconnecting amino acid moieties.

[0061] As used herein, the term “amino acid moiety” may be understood in the broadest sense as generally understood in the art. An amino acid moiety is typically derivable from a chemical entity comprising at least one primary or secondary amino group (- NH2 or -NH-) or a salt thereof and at least one carboxyl (-COOH) group or a salt thereof.

[0062] Typically, an amino acid moiety embedded in a peptide has the formula

[0063] -HmN-CR1R2-(R3)n-CO-, wherein

[0064] R1and R2may be independently from each other any residue, preferably a residue selected from the group consisting of: hydrogen; deuterium; Bachem Holding AG, 10 BAC75006PC1

[0065] Novo Nordisk A / S September 29, 2025 halogen such as, e.g., Cl, F, Br, or I; a linear or branched and / or cyclic Ci-Cio-(hetero)alkyl, optionally substituted by one or more functional groups such as, e.g., a primary (-NH2), secondary (-NH-), tertiary (-N\), or quaternary ( / N+\) amino group or a salt thereof, -COOH or a salt thereof, a guanidinium group or a salt thereof, OH, -SO3H or a salt thereof, =0, =S, -SO4H or a salt thereof, -SO-, -S-, -SO2-, -SO3-, -SO4-, -SOsN^ or a salt thereof, -SH, - PO4H2 or a salt thereof, -CO-NH-, -CO-O-, -CS-NH-, -CS-O-, -CO-S-, -O-alkyl, -O- aryl, -NH-CO-NH2 and / or -CS-S-; a C4-C3o-(hetero)alkyl-(hetero)aryl, optionally substituted by one or more functional groups such as, e.g., -NH2 or a salt thereof, -NH- or a salt thereof, -COOH or a salt thereof, a guanidinium group or a salt thereof, -OH, -SO3H or a salt thereof, -SO4H or a salt thereof, =0, =S, -SO-, -S-, -SO2-, -SO3-, -SO4-, -SOsN^ or a salt thereof, -SH, -PO4H2 or a salt thereof, -CO-NH-, -CO-O-, -CS-NH-, -CS-O-, -CO-S-, and / or -CS-S-; m is = 0 or 1 , with the proviso that, where m is 0, one of R1, R2, or R3is bonded to the nitrogen atom, thereby forming a heterocyclic structure;

[0066] R3is a linear or branched and / or cyclic Ci-Cio-(hetero)alkylene, optionally substituted by one or more functional groups such as, e.g., a primary, secondary, tertiary, or quaternary amino group or a salt thereof, -COOH or a salt thereof, a guanidinium group or a salt thereof, -OH, -SO3H or a salt thereof, -SO4H or a salt thereof, =0, =S, -SO-, -S-, -SO2-, -SO3-, -SO4-, -SO3NH2 or a salt thereof, -SH, - PO4H2 or a salt thereof, -CO-NH-, -CO-O-, -CS-NH-, -CS-O-, -CO-S-, O-alkyl, -0- aryl, -NH-CO-NH2 and / or -CS-S-; and a C3-C3o-(hetero)arylene, optionally substituted by one or more functional groups such as, e.g., a primary, secondary, tertiary, or quaternary amino group or a salt thereof, -COOH or a salt thereof, a guanidinium group or a salt thereof, -OH, -SO3H or a salt thereof, -SO4H or a salt thereof, =0, =S, -SO-, -S-, -SO2-, -SO3-, -SO4-, - SO3NH2 or a salt thereof, -SH, -PO4H2 or a salt thereof, -CO-NH-, -CO-O-, -CS-NH- , -CS-O-, -CO-S-, and / or -CS-S-; a C4-C3o-(hetero)alkylene-(hetero)arylene, optionally substituted by one or more functional groups such as, e.g., -NH2 or a salt thereof, -NH- or a salt thereof, -COOH or a salt thereof, a guanidinium group or a salt thereof, -OH, -SO3H or a salt thereof, -SO4H or a salt thereof, =0, =S, -SO-, -S-, -SO2-, -SO3-, -SO4-, -SOsN^ or a salt Bachem Holding AG, 11 BAC75006PC1

[0067] Novo Nordisk A / S September 29, 2025 thereof, -SH, -PO4H2or a salt thereof, -CO-NH-, -CO-O-, -CS-NH-, -CS-O-, -CO-S- , and / or -CS-S-; and n is 0, 1 or 2,

[0068] For the avoidance of doubt, expressions like -SO4H, and -PO4H2 refer to sulfate groups and phosphate groups, i.e. the connection to the carbon atom is via an oxygen atom.

[0069] A peptide may optionally also be crosslinked such as, e.g., via a disulfide bond (-S- S-) or via a synthetic linker such as, e.g., a linkage resulting from divalent epoxy linkers or the intermediate formation of a Schiff base (-N=).

[0070] In a preferred embodiment, at least one of the amino acid moieties embedded in a peptide has the formula

[0071] -HmNCHR1CO-, wherein m and R1are defined as above.

[0072] In a preferred embodiment, an amino acid moiety in the sense of the present invention has a molecular weight of not more than 300 Da or not more than 250 Da.

[0073] As amino acid moiety may comprise proteinogenic amino acid moieties (alanine (Ala), cysteine (Cys), aspartic acid / aspartate (Asp), glutamic acid / glutamate (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (lie), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), pyrrolysine (Pyl), proline (Pro), glutamine (Gin), arginine (Arg), serine (Ser), threonine (Thr), Selenocysteine (Sec), valine (Vai), tryptophan (Trp) and tyrosine (Tyr)) and / or non-proteinogenic amino acid moieties. Typically, the proteinogenic amino acid moieties are each L-amino acid moieties (while glycine is non-chiral).

[0074] In a preferred embodiment, a peptide comprises at least one proteinogenic amino acid moiety or more than one proteinogenic amino acid moieties. In a preferred Bachem Holding AG, 12 BAC75006PC1

[0075] Novo Nordisk A / S September 29, 2025 embodiment, most amino acid moieties in a peptide by molar number are proteinogenic amino acid moieties.

[0076] A non-proteinogenic amino acid moiety may be a non-proteinogenic natural amino acid moiety or a non-natural amino acid moiety. A non-proteinogenic amino acid moiety may be any non-proteinogenic amino acid moiety known in the art, in particular such having a molecular weight of not more than 300 Da or not more than 250 Da. For instance but not limited, a non-proteinogenic amino acid moiety may be a D-amino acid moieties, beta- amino acid moieties, gamma- amino acid moieties, nipecotic acid, isonipecotic acid, pipecolic acid, Aib (a-aminoisobutyric acid), epsilon-aminohexanoic acid (epsilon-aminocaproic acid), AEEAc (8-amino-3,6- dioxaoctanoic acid) etc.

[0077] It will be understood that the term “peptide” is not limited to peptides consisting of amino acid moieties. A peptide may also comprise one or more modifications of amino acid moieties.

[0078] For instance, the peptide may be conjugated with one or more residues. In a preferred embodiment, such a conjugating residue, if present, does not comprise more than 50 carbon atoms or does not comprise more than 30 carbon atoms, or does not comprise more than 20 carbon atoms. In a preferred embodiment, such a conjugating residue, if present, has a molecular mass below 500 Da, or below 350 Da.

[0079] Such conjugating residue may be understood in the broadest sense. For instance, an amino group of the peptide (i.e., alpha-amino group and / or side chain amino group) may be conjugated with an organic acid group of a conjugating residue thereby forming a further amide bond (-NH-CO-). Such conjugation to an amino group of the peptide may be an acylation by conjugating an organic acid (e.g., acetylation, forming -NH-COCH3), or an acid derivative such as, e.g., 16-(tert- butoxy)-16-oxohexadecanoyl moiety, gamma-L-glutamoyl(N-alpha-hexadecanoyl). For instance, a carboxy group of the peptide (i.e., C-terminal group and / or side chain carboxy group) may be conjugated with an amino group of a conjugating residue thereby forming a further amide bond (-CO-NH- or -CO-NH2 or salt thereof). There Bachem Holding AG, 13 BAC75006PC1

[0080] Novo Nordisk A / S September 29, 2025 is a plethora of further conjugating residues available. Alternatively, peptide synthesis may also be conducted in a reverse order starting from the N-terminal end.

[0081] As used herein, the term “salt thereof” may be understood in the broadest sense as a charged (i.e. , ionic) form of the aforementioned component and the one or more counterions thereof. As used herein, the term “salt” may be understood in the broadest sense as generally understood in art. A salt may contain one or more cations and one or more anions. Depending on the charged groups, a peptide and / or a peptide precursor of the present invention may serve as a cation and / or as an anion. Preferably, a cation of a salt is not a proton (H+). Preferably, an anion of a salt is not a hydroxyl ion (OH-). Preferably, a cation of a salt is not a proton (H+) and an anion of a salt is not a hydroxyl ion (OH-).

[0082] Non-limiting examples of a cation may be Na+, K+, Ca2+, Mg2+, NH4+, choline.

[0083] Non-limiting examples of an anion may be Cl SO42; SO32; NO3; NO2; PO43; HPO42; H2PO4; CO32; HCO3; acetate (Ac, CH3-COO ), pamoate, citrate, trifluoroacetate, trichloroacetate, salicylate, or formate (Fm, HCOO ).

[0084] Non-limiting examples for salts are selected from the list consisting of NaCI, KCI, CaCI2, MgCI2, NH4CI, NaNO3, KNO3, Ca(NO3)2, Mg(NO3)2, NH4NO3, NaNO2, KNO2, Ca(NO2)2, Mg(NO2)2, NH4NO2, Na2SO4, K2SO4, CaSO4, MgSO4, (NH4)2SO4, Na2SO3, K2SO3, CaSO3, MgSO3, (NH4)2SO3, Na3PO4, K3PO4, Ca3(PO4)2, Mg3(PO4)2, (NH4)3PO4, Na2HPO4, K2HPO4, CaHPO4, MgHPO4, (NH4)2HPO4, NaH2PO4, KH2PO4, Ca(H2PO4)2, Mg(H2PO4)2, NH4H2PO4, Na2CO3, K2CO3, CaCO3, MgCO3, (NH4)2CO3, NaHCO3, KHCO3, Ca(HCO3)2, Mg(HCO3)2, NH4HCO3, NaAc, KAc, CaAc2, MgAc2, NH4AC, Na(HCOO), K(HCOO), Ca(HCOO)2, Mg(HCOO)2, NH4(HCOO), and combinations of two or more thereof.

[0085] As used herein, a “peptide precursor” is a peptide that is conjugated to least one cleavable non-peptidic moiety. Typically, it is intended that such one or more cleavable non-peptidic moieties are cleaved off the peptide precursor in the course of the method of the present invention to result in the peptide of interest. Bachem Holding AG, 14 BAC75006PC1

[0086] Novo Nordisk A / S September 29, 2025

[0087] In a preferred embodiment, a “peptide precursor” is a peptide that is conjugated to least one acid-cleavable non-peptidic moiety. Typically, it is intended that such one or more acid-cleavable non-peptidic moieties are cleaved off the peptide precursor in the course of the method of the present invention to result in the peptide of interest.

[0088] As used herein, an acid-cleavable protecting group is characterized in that at least 80% by mole of the acid-cleavable group bound to the group it protects are cleaved off upon incubation with a cleavage reagent comprising 94% (v / v) trifluoroacetic acid (TFA), 2% (v / v) triisopropylsilane (TIS), 2% (v / v) water, and 2% (w / v) dithiothreitol (DTT) for 2 hours at room temperature. Preferably such acid-cleavable protecting group bound to the group it protects is essentially not cleaved off upon incubation in dimethylformamid (DMF) or water for 2 hours at room temperature, i.e. , less than 10%, typically less than 1 % by mole is cleaved off.

[0089] According to the present invention the acid-cleavable non-peptidic moiety is a support, preferably is a solid support or a tag.

[0090] As used herein, the term “solid support” refers to a solid or gel-like structure, which may be macroscopic. The solid support enables separation of the peptide chain being synthesized from other components of the reaction mixture by filtration. The solid support may be any solid support known in the art. As generally understood in the art, the expressions “support”, “carrier” and “resin” may be understood as being interchangeable with the term “solid support”. In a preferred embodiment, the solid support is composed of beads, preferably spherical beads.

[0091] As further described below, the solid support may be made up from, e.g., polystyrene, polystyrene-PEG composites, PEG (polyethylene glycol), PEGA (acrylamide-polyethylene glycol co-polymers), cross-linked ethoxylate acrylate (CLEAR), polyamides, polydimethylacrylamide, polyepsilonlysine (e.g. under the trademark SpheriTide), or any other support with the desired physical and chemical properties. Swellable resins based on beaded polystyrene with 1 % divinylbenzene are among the routinely used supports, typically having a size distribution of 200- 400 mesh or 100-200 mesh. Bachem Holding AG, 15 BAC75006PC1

[0092] Novo Nordisk A / S September 29, 2025

[0093] As used herein, the term “tag” refers to a molecular moiety, which is covalently bound to the peptide chain being synthesized and allows shifting solubility to a desired range. In other words: When bound, the tag modifies the solubility of the peptide precursor as compared to the same molecule without the tag. The peptide precursor with the tag bound to it is usually soluble in the reaction mixtures used for peptide synthesis. However, the tag facilitates separation of the peptide precursor from other components of the reaction mixture by extraction, precipitation and / or (nano) filtration. The terms “tag”, “pseudo-solid phase protecting group”, “soluble tag”, “soluble support”, “solubility modifying support”, and “anker” may be understood interchangeably herein as generally understood in the art. A tag may be any tag known for peptide chemistry in the art. At least one tag may be bound to each peptide chain. In some embodiments, more than one tag is bound to each peptide chain.

[0094] The tag may be, e.g., a polyethylene glycol (PEG) polymer, a ionic liquid, a hydrophobic polymer such as a polynorbornene support, a so-called polycarbon tag comprising long aliphatic chains such as alkoxy moieties, or a so-called GAP / SAP anchor comprising N-phosphoryl groups. Further examples and embodiments are detailed below.

[0095] In another embodiment, the acid-cleavable non-peptidic moiety is a solid support or a tag, which is linked with the peptide precursor via an acid-cleavable linker. In a preferred embodiment, such linker is selected from the group consisting of 2- chlorotrityl (2CT linker), 2-chlorotrityl amido-methyl (2CT-AM linker), 4-[9-Fmoc- amino-xanthen-3-yloxy]-butyryl)-4-methyl-benzhydrylamide (Sieber linker), 4- hydroxybenzylalcohol (Wang linker), Fmoc-2,4-dimethoxy-4’-(carboxymethyloxy)- benzhydrylamine (Rink Amide linker), Fmoc-suberol (5-Fmoc-amino-2- carboxymethoxy-10,11 -dihydro-5H-dibenzo[a,d]cycloheptene) (Ramage linker), and 5-Fmoc-amino-10,11 -dihydro-5H-dibenzo [a,d]cycloheptenyl-2-oxyacetyl-DL- Nle-4-methyl-benzhydrylamide (tricyclic amide linker) ), 2-Methoxy-4-alkoxy benzyl alcohol (Sasrin linker), 5-[4-(9-Fmoc)aminomethyl-3,5-dimethoxyphenoxy]- pentanoic acid (Fmoc-PAL linker), 4-(4-Formyl-3,5-dimethoxyphenoxy) (BAL linker), 4-Hydroxymethylphenoxyacetic acid (HMPA linker), and 4-(4-hydroxymethyl-3- methoxyphenoxy)butyric acid (HMPB linker). Bachem Holding AG, 16 BAC75006PC1

[0096] Novo Nordisk A / S September 29, 2025

[0097] The “peptide precursor”, which is conjugated to a solid support or to a tag, may further comprise one or more acid-cleavable protecting group(s), which may be the same or different. In some embodiments, at least one protecting group is a side chain protecting group. In other embodiments, at least one protecting group is a backbone protecting group, e.g. at the alpha-amino group of the peptide such as its N-terminus. In yet another embodiment, at least one protecting group is used as a side chain protecting group and as a protecting group at the alpha amino group of the peptide such as its N-terminus. In another embodiment, at least one protecting group is used at the carboxy group of the peptide such as its C-terminus. In yet another embodiment, at least one protecting group is used as a side chain protecting group and as a protecting group at the carboxy group of the peptide such as its C- terminus.

[0098] The term “protecting group” as used herein may be understood in the broadest sense as generally understood in the art. A protecting group may be present in the peptide precursor as a chemical modification of a functional group to block said group from reaction in subsequent process steps, e.g. to prevent side reactions of the peptide’s functional groups such as the amino acid side chains.

[0099] Examples of acid-cleavable protecting groups are tert-butyloxycarbonyl (Boc), tertbutyl (tBu), trityl (Trt), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 4-Methoxy-2,3,6- trimethylphenylsulfonyl (Mtr), 9-Xanthydryl (Xan), 3-methylpent-3-yl ester (OMpe), 4-methyltrityl (Mtt), monomethoxytrityl (Mmt), 2,4-dimethoxybenzyl (Dmb), and 1 ,2-diphenylmethyl (Dpm). Acid-cleavable protecting groups may be used to protect, e.g., amino acid side chains, or the peptide backbone. An example of the latter use is protection of a backbone amide group with 2,4-dimethoxybenzyl (Dmb). Furthermore, pseudoprolines may serve as an acid-cleavable protecting group. The term “pseudoproline dipeptides” as used herein refers to temporary proline mimics, which can be readily obtained from Ser and Thr by oxazolidine formation and from Cys by thiazolidine formation. The person skilled in the art is well-aware of such pseudoproline dipeptides, in particular of 2, 2-dimethyloxazolidines. Bachem Holding AG, 17 BAC75006PC1

[0100] Novo Nordisk A / S September 29, 2025

[0101] As indicated above, the one or more acid-cleavable non-peptidic moieties of the peptide precursor are cleavable by the cleavage cocktail of the present invention. As used herein, the terms “cleavage cocktail”, and “cleavage reagent” may be understood in the broadest sense as a composition that allows cleavage of an acid- cleavable non-peptidic moiety. The cleavage cocktail may contain any contents and components suitable for this purpose. Unless specified otherwise, the terms “cleavage cocktail” and “cleavage reagent” are used herein for the composition before it is contacted with the peptide precursor. It will be understood that a cleavage cocktail is a liquid composition, preferably a homogeneous liquid composition. As indicated above, the cleavage cocktail comprises trichloroacetic acid, and one or more solvents and / or scavengers. Optionally, the cleavage cocktail may contain one or more further components.

[0102] As used herein, the term “cleavage composition” is used to refer to the composition, which is obtained by combining and optionally incubating the “cleavage cocktail” and the peptide precursor. Hence, the “cleavage composition” will normally comprise products of the cleavage reaction at some point in time.

[0103] In one embodiment, the cleavage cocktail (essentially) consists of:

[0104] (b1 ) trichloroacetic acid,

[0105] (b2) one or more solvents and / or scavengers, and (b3) optionally a salt.

[0106] According to the first aspect of the invention, and preferably according to the second aspect of the invention, the amount of trichloroacetic acid in the cleavage cocktail, based on the total weight of the cleavage cocktail, is at least 60% by weight, e.g. from 60 to 91 % by weight, from 60 to 85% by weight, from 65 to 91 % by weight, more preferably from 68 to 88% by weight, more preferably from 70 to 88% by weight, more preferably from 75 to 86% by weight.

[0107] Moreover, according to the first aspect of the invention, and preferably according to the second aspect of the invention, the amount of trichloroacetic acid in the cleavage cocktail, relative to the one or more solvents (b2), is in the range from 2 to 12 g, preferably from 3 to 10 g, more preferably from 4 to 8 g, more preferably from 6 to Bachem Holding AG, 18 BAC75006PC1

[0108] Novo Nordisk A / S September 29, 2025

[0109] 8 g per 1 mL of the one or more solvents (b2) selected from the group consisting of diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole, wherein the volume of the solvent is the volume at 25 °C.

[0110] In some embodiments, the cleavage cocktail comprises 5.0 M or more, e.g. from 5.0 to 8.7 M, from 5.5 to 8.5 M, from 6.0 to 8.4 M, from 6.5 to 8.2 M, or from 7.0 to 7.9 M, more preferably from 6.3 to 8.7 M, more preferably from 6.5 to 7.7 M of trichloroacetic acid. It was found that the higher the concentration of trichloroacetic acid used in steps (i) and (ii), the better the reaction may be in terms of completeness versus side products. However, higher concentrations may require higher reaction temperature in step (ii) to avoid trichloroacetic acid crystallizing and / or precipitating. Furthermore, in particular when conducted on a large scale, higher temperatures may not be desired because of higher energy consumption and / or in view of an optional subsequent precipitation step (iii) which may be conducted with a cold liquid (e.g., cold diisopropylether). It was surprisingly found that there are particularly beneficial temperature ranges that allow a beneficial balance of low energy consumption and completeness of the cleavage step.

[0111] The cleavage cocktail may comprise at least 5.0 M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5 M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6.0 M, 6.1 M, 6.2 M, 6.3 M, 6.4 M, 6.5 M, 6.6 M, 6.7 M, 6.8 M, 6.9 M, 7.0 M of trichloroacetic acid. The cleavage cocktail may comprise from at least 5.0 M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5 M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6.0 M, 6.1 M, 6.2 M, 6.3 M, 6.4 M, 6.5 M, 6.6 M, 6.7 M, 6.8 M, 6.9 M, or 7.0 M of trichloroacetic acid, each up to (i.e., equal to or less than) 8.8M of trichloroacetic acid. The cleavage cocktail may comprise from at least 5.0 M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5 M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6.0 M, 6.1 M, 6.2 M, 6.3 M, 6.4 M, 6.5 M, 6.6 M, 6.7 M, 6.8 M, 6.9 M, or 7.0 M of trichloroacetic acid, each up to (i.e., equal to or less than) 7.7M of trichloroacetic acid.

[0112] The molar concentration of trichloroacetic acid in the cleavage cocktail the present context may be calculated using a molar weight of trichloroacetic acid of 163.39 g / mol and a density of trichloroacetic acid of 1.629 g / mL (The Merck Index, 13thedition 2001 ), assuming that the volumes of individual components are additive and Bachem Holding AG, 19 BAC75006PC1

[0113] Novo Nordisk A / S September 29, 2025 any changes in density within the relevant temperature range can be neglected. This allows estimating the molar concentration of trichloroacetic acid in the cleavage cocktail using an approximate concentration of neat trichloroacetic acid of 10 M (mol / L)

[0114] For example, the approximate molar concentration of trichloroacetic acid may be calculated by using the following equation, from which the amounts of individual components required for obtaining a given concentration can be determined: mTCA

[0115] 1.629 wherein

[0116] CTCA is the approximate molar concentration of trichloroacetic acid in the cleavage cocktail mTCA is the mass of trichloroacetic acid (in g) used for preparing the cleavage cocktail;

[0117] Vsoivent is the volume at 25 °C of the solvent (in mL) used for preparing the cleavage cocktail;

[0118] Nsoiid is the sum of weight to volume (W / V) percentages of any scavengers in the cleavage cocktail, which are solid at 25 °C; and

[0119] Niiquid is the sum of weight to volume (V / V) percentages of any scavengers in the cleavage cocktail, which are liquid at 25 °C.

[0120] Preferably, the cleavage cocktail is prepared by first combining a mass of trichloroacetic acid and a volume (as measured at 25 °C) of the one or more solvents (b2), so that the amount of trichloroacetic acid in the cleavage cocktail is in the range from 2 to 12 g per 1 mL of said one or more solvents (b2), and heating the obtained mixture until a liquid forms. Any further components, such as an optional salt and / or optional scavengers, may be combined directly with the obtained heated mixture, or the obtained mixture may be cooled to a temperature in the range from 20 to 45°C, preferably 25 to 30 °C, or, alternatively, to the temperature used in step (ii), before any further components of the cleavage cocktail are added. In some embodiments, the cleavage cocktail is prepared by placing a desired amount of components such Bachem Holding AG, 20 BAC75006PC1

[0121] Novo Nordisk A / S September 29, 2025 as salts and / or scavengers in a volumetric vessel, and filling the volumetric vessel to the desired volume with the mixture of trichloroacetic acid and the solvent (b2), which may be the heated mixture or the cooled mixture, preferably the cooled mixture. The contacting of the peptide precursor with the cleavage cocktail may also be conducted using the obtained heated mixture (optionally including any further components) or the cooled mixture (optionally including any further components).

[0122] The one or more scavengers may be any scavengers which are at least partly mixable with trichloroacetic acid (TCA). In a preferred embodiment, the one or more scavengers are fully miscible with TCA in concentration ranges of at least up to 80% of TCA by weight, at least up to 85% of TCA by weight, at least up to 90% of TCA by weight, at least up to 95% of TCA by weight at the temperature of the cleavage reaction.

[0123] In a preferred embodiment, the one or more solvents and / or scavengers do not comprise a per- or polyfluorinated substance (PFAS). Preferably, the entire cleavage cocktail does not comprise a per- or polyfluorinated substance. More preferably, the entire cleavage cocktail does not comprise any per- or polyhalogenated substance other than trichloroacetic acid

[0124] A solvent may be used as only solvent or in combination with one or more other solvents and / or one or more scavenges.

[0125] Solvents as usable in the context of the first aspect of the invention are selected from the group consisting of: diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole (preferably thioanisole and a monohalogenated thioanisole, more preferably thioanisole and a 2-halo-thioanisole, in particular 2- chlorothioanisole), and a combination of two or more thereof.

[0126] Solvents as usable in the context of the second aspect of the invention, and preferably in the first aspect of the invention, are selected from the group consisting Bachem Holding AG, 21 BAC75006PC1

[0127] Novo Nordisk A / S September 29, 2025 of: diphenylsulfide, a monohalogenated thiophenol and a monohalogenated thioanisole (preferably a monohalogenated thioanisole, more preferably a 2-halo- thioanisole), a combination of two or more thereof, and a combination of one or more thereof with thiophenol, thioanisole or both (preferably thioanisole).

[0128] Preferably, the cleavage cocktail does not comprise more than 20% by volume, preferably does not comprise more than 10% by volume, more preferably does not comprise more than 5% by volume, based on the total volume of solvents and scavengers, of solvents and scavengers other than diphenylsulfide, thiophenol, thioanisole, monohalogenated thiophenols, monohalogenated thioanisoles, water, thiol scavengers and silane scavengers. In a preferred embodiment, the cleavage cocktail does not comprise any solvents or scavengers other than diphenylsulfide, thiophenol, thioanisole, monohalogenated thiophenols, monohalogenated thioanisoles, water, thiol scavengers and silane scavengers.

[0129] In a preferred embodiment of the first aspect of the invention, the one or more solvents comprise or consist of thioanisole or 2-chlorothioanisole.

[0130] In a preferred embodiment, the one or more solvents comprise or consist of thiophenol, diphenylsulfide, thioanisole, 2-bromothioanisole, 2-fluorothioanisole or 2-chlorothioanisole.

[0131] In the context of the present application, the term “scavenger” is used to refer to a compound, which may be contacted with the peptide precursor to scavenge reactive intermediates (carbocations) and / or to suppress side reactions during cleavage of a peptide from an acid-cleavable non-peptidic moiety such as the solid support and / or the tag. The person skilled in the art is well-aware of a large variety of scavengers usable.

[0132] In a preferred embodiment, the one or more scavengers comprise or consist of water, one or more thiol scavengers (e.g., 1 ,2-ethanedithiol (EDT), dithioerythritol (DTE), ditiothreitol (DTT), 3,6-dioxa-1 ,8-octanedithiol (DODT), beta- Bachem Holding AG, 22 BAC75006PC1

[0133] Novo Nordisk A / S September 29, 2025 mercaptoethanol) and / or one or more silane scavengers (e.g., triethylsilane (TES), triisopropylsilane (TIS)). Additionally or alternatively, a scavenger is selected from the group consisting of ethyl methyl sulfide, anisole, m-or p-cresol, 2-Me-indole, Ac- Trp-OMe, and / or tryptamine.

[0134] Preferably, the cleavage cocktail comprises one or more scavengers different from the one or more solvents (b2), preferably in a total amount of from 1 % to 15%, preferably from 2% to 10%, more preferably from 4% to 8%, wherein the % are volume to volume (V / V) for scavengers liquid at 25 °C and weight to volume (W / V) for scavengers solid at 25 °C, based on the total volume of the cleavage cocktail at 25 °C.

[0135] Preferably, the one or more scavengers are selected from the group consisting of water, thiol scavengers and silane scavengers, preferably water or one or more scavengers from the group consisting of thiol scavengers and silane scavengers, preferably 1 ,2-ethanedithiol, dithioerythritol, dithiothreitol, 3,6-dioxa-1 ,8- octanedithiol, beta-mercaptoethanol, 1 ,4-benzenedimethanethiol, 1 ,3- benzenedimethanethiol, 1 ,2-benzenedimethanethiol, triethylsilane, triisopropylsilane.

[0136] More preferably, the one or more scavengers comprise water. More preferably, the one or more scavengers comprise water and one or more further scavengers from the group consisting of thiol scavengers and silane scavengers, preferably 1 ,2- ethanedithiol, dithioerythritol, dithiothreitol, 3,6-dioxa-1 ,8-octanedithiol, beta- mercaptoethanol, 1 ,4-benzenedimethanethiol, 1 ,3-benzenedimethanethiol, 1 ,2- benzenedimethanethiol, triethylsilane, triisopropylsilane.

[0137] Preferably, the thiol scavenger comprises or consists of 1 ,2-ethanedithiol. Preferably, the silane scavenger comprises or consists of triisopropylsilane.

[0138] Preferably, the cleavage cocktail comprises from 0.5 to 10%, from 0.5 to 6%, from 1 to 5.5%, from 1 .5 to 5%, or from 2 to 4.5% (V / V), based on the total volume of the cleavage cocktail, of water, and further preferably, from 0.5 to 10 % from 0.5 to 6%, from 1 to 5.5%, from 1 .5 to 5%, from 2 to 4.5% (V / V), based on the total volume of Bachem Holding AG, 23 BAC75006PC1

[0139] Novo Nordisk A / S September 29, 2025 the cleavage cocktail, of one or more scavengers selected from 1 ,2-ethanedithiol, 3,6-dioxa-1 ,8-octanedithiol, beta-mercaptoethanol, triethylsilane and triisopropylsilane, 1 ,3-benzenedimethanethiol and / or from 0.5 to 6%, from 1 to 5.5%, from 1.5 to 5%, from 2 to 4.5% (W / V), based on the total volume of the cleavage cocktail, of one or more scavengers selected from dithioerythritol, dithiothreitol, 1 ,4- benzenedimethanethiol, or 1 ,2-benzenedimethanethiol.

[0140] More preferably, the cleavage cocktail comprises from 0.5 to 10%, from 0.5 to 6%, from 1 to 5.5%, from 1 .5 to 5%, or from 2 to 4.5% (V / V), based on the total volume of the cleavage cocktail, of water, and from 0.5 to 6%, from 1 to 5.5%, from 1.5 to 5%, or from 2 to 4.5% (V / V), based on the total volume of the cleavage cocktail, of one or more scavengers selected from 1 ,2-ethanedithiol and triisopropylsilane.

[0141] Preferably, the cleavage cocktail is a liquid, more preferably a homogeneous liquid under the conditions of incubation in step (ii), more preferably at a temperature in the range from 15 to 65 °C, preferably from 20 to 60 °C, more preferably from 25 to 55 °C, more preferably from 25 to 45 °C. Preferably, the cleavage cocktail is a homogeneous liquid at a temperature in the range from 20 to 45 °C, more preferably 25 to 45 °C. More preferably, the cleavage cocktail remains a homogeneous liquid for at least 12, preferably at least 24 hours after being prepared and kept at the specified temperature, based on visual assessment.

[0142] In some embodiments, the cleavage cocktail comprises trichloroacetic acid, one or more solvents selected from the group consisting of diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole, water, and one or more scavengers selected from thiol scavengers and silane scavengers, wherein: the amount of trichloroacetic acid in the cleavage cocktail is in the range from 4 to 8 g per 1 mL of said one or more solvents (b2); the amount of trichloroacetic acid in the cleavage cocktail, based on the total weight of the cleavage cocktail, is at least 60% by weight; the volume of water in the cleavage cocktail is 0.5 to 10 %, based on the total volume of the cleavage cocktail; and Bachem Holding AG, 24 BAC75006PC1

[0143] Novo Nordisk A / S September 29, 2025 the volume of scavenger is in the range from 0.5 to 10%, based on the total volume of the cleavage cocktail; wherein all volumes are the volumes at 25°C. Preferably, said cleavage cocktail is a homogeneous liquid at a temperature in the range from 20 to 45 °C, more preferably 25 to 45 °C , 30 to 45 °C, or 25 to 30 °C.

[0144] In some embodiments of the method, cleavage of protecting groups from the peptide occurs in addition to cleavage of the peptide from the acid-cleavable solid support or from the at least one acid-cleavable tag. This may lead to a globally deprotected peptide, which is cleaved from the solid support or the tag and which is free from acid cleavable protecting groups.

[0145] The method of the present invention is suitable for any type of common peptide synthesis. The peptide precursor is obtained from a method involving a solid support or a tag and optionally additional acid-cleavable protecting groups. It may be used for solid phase peptide synthesis (SPPS) or tag-assisted peptide synthesis, or a combination thereof with liquid phase peptide synthesis (LPPS).

[0146] In a preferred embodiment, the peptide precursor is obtained from:

[0147] (a) solid phase peptide synthesis (SPPS) and is covalently bound to an acid- cleavable solid support;

[0148] (b) tag-assisted peptide synthesis (TAPS) and is covalently bound to an acid- cleavable tag; or

[0149] (c) combination of two or more of (a), (b) and liquid phase peptide synthesis (LPPS).

[0150] As used herein, solid-phase peptide synthesis (SPPS) may be understood in the broadest sense as generally understood in the art. SPPS may be understood as any synthesis on a solid support to which the growing peptide precursors covalently bound. Typically, the solid support has a size that allows removal from the synthesis mixture by physical means such as, e.g., filtration and / or sieving and / or centrifugation. Bachem Holding AG, 25 BAC75006PC1

[0151] Novo Nordisk A / S September 29, 2025

[0152] In a preferred embodiment, the solid support is a swellable polystyrene resin or a grafted polystyrene resin which may be grafted with polyethylene glycol (PEG). It will be understood that such solid support will preferably be linked to the peptide precursor via an acid-cleavable linker.

[0153] Various linkers, including acid-cleavable linkers, are known in the art (cf., for instance, Brochure “Solid phase peptide synthesis”, Bachem May 2020, published by Global Marketing, Bachem AG). For example, a solid support may be based on a polystyrene-based bead such as, e.g., a polystyrene-based bead selected from the group consisting of: swellable polystyrene beads crosslinked with 1 -2% divinyl benzene; and swellable polystyrene beads with grafted hydrophilic compounds, e.g. Tentagel.

[0154] As used herein, liquid-phase peptide synthesis (LPPS) may be understood in the broadest sense as generally understood in the art. LPPS may be understood as any peptide synthesis in a liquid phase. In other words, LPPS may be any peptide synthesis without involving a solid support, i.e. , which is not SPPS. When LPPS is conducted without any solubility-modifying tag, it may also be designated as “classical LPPS” or “classical solution peptide synthesis” or “CSPS”.

[0155] As used herein, tag-assisted peptide synthesis (TAPS) may be understood in the broadest sense as generally understood in the art. It is based on at least one tag, most typically at a carboxyl group, in particular the C-terminus, of the peptide precursor that allows shifting solubility to a desired range. Tags may comprise one or more benzylalcohol and / or amine derivatives. Sometimes, TAPS is considered as a variety of LPPS.

[0156] The tag may be a polyethylene glycol (PEG) polymer, a ionic liquid, a hydrophobic polymer such as a polynorbornene support, a so-called polycarbon tag comprising long aliphatic chains such as alkoxy moieties, or a so-called GAP / SAP anchor comprising N-phosphoryl groups. Such tags may be bound to the peptide moiety via one of the acid cleavable linkers known for SPPS as detailed above.

[0157] In a preferred embodiment, the tag is selected from the group consisting of substituted benzyl alcohols, substituted benzyl amines, substituted 1 ,1 - Bachem Holding AG, 26 BAC75006PC1

[0158] Novo Nordisk A / S September 29, 2025 diphenylmethanols, and substituted 1 ,1 -diphenylmethylamines. Such tags may be substituted with 1 to 3 alkoxy moieties per aromatic ring, e.g., with 1 to 3 alkoxy moieties having at least 8 carbon atoms each, e.g. having each between 8 and 30 carbon atoms. Linear or branched alkoxy moieties such as phytyl groups may be used. Some examples of benzyl alcohol tags are 3,4,5-Tri-substituted, 3,5-Di- substituted, or 2,4-Di-substituted; some examples of benzyl amine tags are 2,4-Di- substituted. Some examples of benzyl alcohol tags are 3,4,5-Tri-alkoxy substituted, 3,5-Di-alkoxy substituted, or 2,4-Di-alkoxy substituted; some examples of benzyl amine tags are 2,4-Di-substituted, in particular 2,4-Di-alkoxy substituted. Some examples of 1 ,1 -diphenylmethanol tags and of 1 , 1 -diphenylmethylamines tags are 4,4’ substituted. Some examples of 1 ,1 -diphenylmethanol tags and of 1 ,1 - diphenylmethylamines tags are 4,4’ alkoxy substituted. A peptide may be attached directly to such tags via an ester bond between the tag’s hydroxyl group and a carboxyl group of the peptide or via an amide bond between the tag’s amine group and a carboxyl group of the peptide. Fig. 14 of Ferrazano et al. (Green Chem., 2022, 24, 975-1020) and Figure 11 of Sharma et al. (Chem. Rev. 2022, 122, 13516-13546) give examples of commonly used tags.

[0159] In a preferred embodiment, the peptide precursor is characterized in that:

[0160] (a) it has a C- and / or N-terminus protected with an acid-cleavable group, and / or is bound to a solid support, and / or is bound to a tag, preferably wherein the tag is selected from the group consisting of substituted benzyl alcohols, substituted benzyl amines, substituted 1 ,1 -diphenylmethanols, and substituted 1 ,1 -diphenylmethylamines, and / or wherein the peptide is bound to a solid support or to a tag via an acid-cleavable linker, preferably wherein the linker is selected from the group consisting of 2-ch lorotrity I (2CT linker), 2-chlorotrityl amido-methyl (2CT-AM linker), 4-[9-Fmoc-amino-xanthen-3- yloxy]-butyryl)-4-methyl-benzhydrylamide (Sieber linker), 4- hydroxybenzylalcohol (Wang linker), Fmoc-2,4-dimethoxy-4’- (carboxymethyloxy)-benzhydrylamine (Rink Amide linker), Fmoc-suberol (5- Fmoc-amino-2-carboxymethoxy-10, 11 -dihydro-5H- dibenzo[a,d]cycloheptene) (Ramage linker), and 5-Fmoc-amino-10,11 - dihydro-5H-dibenzo [a,d]cycloheptenyl-2-oxyacetyl-DL-Nle-4-methyl- benzhydrylamide (tricyclic amide linker); 2-Methoxy-4-alkoxy benzyl alcohol Bachem Holding AG, 27 BAC75006PC1

[0161] Novo Nordisk A / S September 29, 2025

[0162] (Sasrin linker), 5-[4-(9-Fmoc)aminomethyl-3,5-dimethoxyphenoxy]- pentanoic acid (Fmoc-PAL linker), 4-(4-Formyl-3,5-dimethoxyphenoxy) (BAL linker), 4-Hydroxymethylphenoxyacetic acid (HMPA linker), and 4-(4- hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB linker) and / or

[0163] (b) the peptide precursor comprises one or more of the following structural elements: a 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf) side chain protecting group moiety, 2,2,5,7,8-pentamethylchromanyl-6-sulfonyl (Pmc) side chain protecting group moiety, in particular an Arg(Pbf) moiety, an intra-chain and / or C-terminal beta-hydroxy amino acid, preferably Ser or Thr,

[0164] Asn, Gin, Asp, Cys, Met, Trp, a hydrocarbon chain >Ce as a substituent, and / or a polyethylene glycol (PEG) moiety with two or more oxyethylene repeating units as a substituent.

[0165] It will be understood that the acid-cleavable linkers as noted above may partly be deprotected before conjugation of a peptide, because in some cases the designated forms refer to the storable form thereof.

[0166] In a preferred embodiment, the peptide precursor is obtained from SPPS and is covalently bound to an acid-cleavable solid support and further comprises one or more acid-cleavable protecting groups, in particular side chain protecting groups or backbone protecting groups. In an alternative embodiment, the peptide precursor is obtained from SPPS involving one or more acid-cleavable protecting groups, wherein the peptide precursor is bound to a solid support via an acid-cleavable linker.

[0167] In a preferred embodiment, the peptide precursor is obtained from SPPS using the Fmoc / tBu synthesis and is covalently bound to an acid-cleavable solid support. The Fmoc protecting group at the alpha-amino group is cleaved off in a last step. Then, the cleaving step (ii) of the present invention preferably allows cleaving off the solid support and concomitantly cleaving off the acid-cleavable protecting groups, in Bachem Holding AG, 28 BAC75006PC1

[0168] Novo Nordisk A / S September 29, 2025 particular side chain protecting groups, of the peptide and thereby providing the fully deprotected peptide.

[0169] In a preferred embodiment, cleavage of protecting groups from the peptide occurs in addition to cleavage of the peptide from the acid-cleavable support during step (ii) of the methods of the present invention, thereby providing the fully deprotected peptide. The acid-cleavable protecting groups removed may be sidechain protecting groups or backbone protecting groups, where the latter include protecting groups located at the N- or C-terminus of the peptide. Typical such protecting groups comprise those mentioned above, in particular tert-butyloxycarbonyl (Boc), tert-butyl (tBu), trityl (Trt), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), and 2,2-dimethyloxazolidines.

[0170] A fully deprotected peptide as used herein may be a peptide substantially free from acid-cleavable protecting groups, i.e. , the peptide sample comprises less than 10%, preferably less than 5%, 1 %, 0.5% or 0.01 % (mol / mol) of peptides containing at least one acid cleavable protecting group. As an example, if a peptide sample comprises 100 mmol of peptide species and 1 mmol of peptide species still comprise an acid-cleavable protecting group, corresponding to 1 % (mol / mol) of peptides, this would be considered a fully deprotected peptide.

[0171] In another preferred embodiment, the peptide precursor is obtained from LPPS and is covalently bound to an acid-cleavable tag and further involves one or more acid- cleavable protecting groups, in particular side chain protecting groups.

[0172] C- and / or N-terminus protected with acid-cleavable groups may be in particular present in LPPS.

[0173] The step (ii) of the method of the present invention may be conducted at any temperature at or above 20 °C. The step (ii) of the method of the present invention may be conducted at a temperature in the range of 20 to 70 °C, preferably 20 to 65 °C, more preferably of 25 to 60 °C, more preferably of 25 to 55 °C, more preferably of 25 to 45 °C, more preferably of 25 to 40 °C. It was found that even at low temperatures (e.g., 25 °C), the cleavage cocktail - depending on its specific Bachem Holding AG, 29 BAC75006PC1

[0174] Novo Nordisk A / S September 29, 2025 composition - may remain a homogeneous liquid. Further, it was found that at temperatures above 70 °C, side reactions rates increase.

[0175] In a preferred embodiment, the step (ii) is conducted at a temperature in the range of 25 °C to 70 °C, 25 °C to 60 °C, 25 °C to 55 °C, 25 °C to 50 °C, 25 °C to 45 °C, 25 °C to 40 °C, 30 °C to 70 °C, 30 °C to 60 °C, 30 °C to 55 °C, 30 °C to 50 °C, 30 °C to 45 °C, 35 °C to 70 °C, 35° to 60 °C, 35 °C to 55 °C, or 35 °C to 50 °C

[0176] In a preferred embodiment, the step (ii) is conducted at a temperature in the range of 15 °C to 45 °C, of 20 °C to 40 °C, of 25 °C to 60 °C, of 25 °C to 55 °C, of 25 °C to 50 °C, of 25 °C to 45 °C, of 30 °C to 60 °C, of 30 °C to 55 °C, or of 30 to 50 °C.

[0177] The step (ii) of the method of the present invention may be conducted at any temperature at or above 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, or 69 °C. The step (ii) of the method of the present invention may be conducted at any temperature at or above 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 °C. The step (ii) of the method of the present invention may be conducted at any temperature at or above 25 °C or 40 °C.

[0178] It is assumable that when the specified amounts of one or more solvents and / or scavengers are admixed, the cleavage cocktail is liquid at a temperature of 40 °C, often even at 30 °C or 25 °C.

[0179] The step (ii) may be conducted for any period of time. The time needed for the cleavage reaction of step (ii) may be varied and may depend on, e.g., the properties of the peptide of interest, the concentration of trichloroacetic acid (TCA) used and the temperature at which step (ii) is conducted.

[0180] It will be understood that an increased reaction temperature may accelerate the cleavage and may thus decrease the required reaction time. In a preferred embodiment, the step (ii) is conducted until the desired degree of cleavage of the one or more acid cleavable support, preferably acid-cleavable solid support or tag Bachem Holding AG, 30 BAC75006PC1

[0181] Novo Nordisk A / S September 29, 2025 is achieved. In a preferred embodiment, the step (ii) is conducted until the cleavage reaction is (essentially) finalized. In a preferred embodiment, the step (ii) is conducted until (essentially) all acid-cleavable protecting groups and supports, preferably solid supports or tags of interest have been cleaved.

[0182] In a preferred embodiment, the step (ii) is conducted for at least 1 hour, preferably for at least 2 hours, more preferably for at least 10 hours. In another preferred embodiment, the step (ii) is conducted for 24 hours or less, preferably for 20 hours or less. Preferably, the step (ii) is conducted for 1 to 24 hours, for 2 to 24 hours, for 3 to 24 hours, for 4 to 24 hours, for 5 to 24 hours, for 6 to 24 hours, for 7 to 24 hours, for 8 to 24 hours, for 9 to 24 hours, for 10 to 24 hours, for 11 to 24 hours, for 12 to 24 hours, for 13 to 24 hours, for 14 to 24 hours, for 15 to 24 hours, for 16 to 24 hours, for 17 to 24 hours, for 18 to 24 hours, for 19 to 24 hours, for 20 to 24 hours, for 21 to 24 hours, for 22 to 24 hours or for 23 to 24 hours.

[0183] In another preferred embodiment, the step (ii) is conducted for 1 to 23 hours, for 1 to 22 hours, for 1 to 21 hours, for 1 to 20 hours, for 1 to 19 hours, for 1 to 18 hours, for 1 to 17 hours, for 1 to 16 hours, for 1 to 15 hours, for 1 to 14 hours, for 1 to 13 hours, for 1 to 12 hours, for 1 to 11 hours, for 1 to 10 hours, for 1 to 9 hours, for 1 to 8 hours, for 1 to 7 hours, for 1 to 6 hours, for 1 to 5 hours, for 1 to 4 hours, for 1 to 3 hours or for 1 to 2 hours.

[0184] In another preferred embodiment, the step (ii) is conducted for at least 10 min, at least 30 min, at least 1 hour, at least 2 hours, at least 5 hours, at least 12 hours, at least 24 hours, at least 36 hours, or at least 48 hours.

[0185] In a preferred embodiment, the step (ii) is conducted for 1 to 6 hours, 2 to 6 hours, 1 to 5 hours, 3 to 6 hours, or 3 to 5 hours.

[0186] In a preferred embodiment, with reaction temperatures when conducting step (ii) at or below 45 °C, the reaction time may be >=8 h, e.g., more than 9, 10, 11 , 12, 13, 14, 15, or 16 hours. In a preferred embodiment, with temperatures when conducting step (ii) at or above 50 °C, the reaction time may be <= 8 h, e.g., less than 7, 6, 5, 4, 3, 2, or 1 hour. In a preferred embodiment, the step (ii) is conducted for 1 to 24 Bachem Holding AG, 31 BAC75006PC1

[0187] Novo Nordisk A / S September 29, 2025 hours, 3 to 8 hours, 4 to 8 hours, 3 to 6 hours, or 18 to 24 hours at a temperature of 30 to 45 °C.

[0188] In some embodiments according to the invention, the reaction conditions of trichloroacetic acid (TCA) and temperature are selected from the group consisting of the following combinations of temperature and concentration:

[0189] 30 °C to 70 °C with TCA concentration in range of 7.0 M to 8.7 M;

[0190] 30 °C to 70 °C with TCA concentration in range of 7.0 M to 8.2 M;

[0191] 30 °C to 70 °C with TCA concentration in range of 7.0 M to 7.9 M;

[0192] 30 °C to 50 °C with TCA concentration in range of 7.3 M to 8.7 M;

[0193] 30 °C to 50 °C with TCA concentration in range of 7.3 M to 8.2 M;

[0194] 30 °C to 50 °C with TCA concentration in range of 7.3 M to 7.9 M;

[0195] 25 °C to 55 °C with TCA concentration in range of 7.3 M to 8.7 M;

[0196] 25 °C to 55 °C with TCA concentration in range of 7.3 M to 8.2 M;

[0197] 25 °C to 55 °C with TCA concentration in range of 7.3 M to 7.9 M;

[0198] 25 °C to 45 °C with TCA concentration in range of 7.0 M to 8.4 M;

[0199] 25 °C to 45 °C with TCA concentration in range of 7.0 M to 8.2 M;

[0200] 25 °C to 45 °C with TCA concentration in range of 7.0 M to 7.9 M;

[0201] 25 °C to 45 °C with TCA concentration in range of 5.6 M to 8.1 M

[0202] 25 °C to 45 °C with TCA concentration in range of 6.0 M to 8.1 M

[0203] 25 °C to 45 °C with TCA concentration in range of 6.5 M to 8.1 M

[0204] 25 °C to 45 °C with TCA concentration in range of 6.5 M to 6.7 M

[0205] 30 °C to 70 °C with TCA concentration in range of 7.3 M to 8.9 M;

[0206] 30 °C to 70 °C with TCA concentration in range of 7.3 M to 8.8 M;

[0207] 30 °C to 70 °C with TCA concentration in range of 7.3 M to 8.7 M;

[0208] 30 °C to 60 °C with TCA concentration in range of 7.0 M to 8.9 M;

[0209] 30 °C to 60 °C with TCA concentration in range of 7.0 M to 8.8 M; and

[0210] 30 °C to 60 °C with TCA concentration in range of 7.0 M to 8.7 M.

[0211] The composition of the cleavage cocktail may optionally be adapted to the peptide of interest. Bachem Holding AG, 32 BAC75006PC1

[0212] Novo Nordisk A / S September 29, 2025

[0213] In some embodiments of the second aspect of the invention, the cleavage cocktail comprises or consists of:

[0214] (b1 ) trichloroacetic acid at a concentration of 7.0 to 8.9 M, and

[0215] (b2) 0.1 to 30% by volume, preferably 20 to 30% by volume, referred to the total volume of the cleavage cocktail, of one or more solvents and / or scavengers; and

[0216] (b3) optionally one or more salts.

[0217] In some embodiments of the second aspect of the invention, the cleavage cocktail consists of:

[0218] (b1 ) trichloroacetic acid at a concentration of 7.0 to 8.9 M,

[0219] (b2) 0.1 to 30% by volume, preferably 20 to 30% by volume, referred to the total volume of the cleavage cocktail, of one or more solvents and / or scavengers, and

[0220] (b3) optionally a salt.

[0221] In some embodiments of the second aspect of the invention, a ratio R as expressed by the following Formula (I): is at least 5, wherein:

[0222] X is the unitless numeric value of the temperature in degrees Celsius at which step (ii) is conducted, and

[0223] Y is the unitless numeric value of the molar content of trichloroacetic acid in the cleavage cocktail.

[0224] In some embodiments of the second aspect of the invention, the ratio R is at least 6, at least 7, at least 8 or at least 9, in the range of 5 to 13, of 6 to 12, of 7 to 11 , or of 7 to 10.

[0225] In a preferred embodiment, the method comprises:

[0226] (i) contacting Bachem Holding AG, 33 BAC75006PC1

[0227] Novo Nordisk A / S September 29, 2025

[0228] (A) a peptide precursor that is covalently bound to at least one acid cleavable support, preferably at least one acid-cleavable solid support and / or at least one acid-cleavable tag; with

[0229] (B) a cleavage cocktail comprising:

[0230] (b1 ) trichloroacetic acid,

[0231] (b2) one or more solvents selected from the group consisting of diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole and

[0232] (b3) optionally a salt; and

[0233] (ii) incubating the components of step (i) at a temperature in the range of 25 °C to 70 °C, preferably for 1 to 24 hours, thereby cleaving the at least one acid- cleavable solid support and / or the at least one acid-cleavable tag off the peptide, wherein the amount of trichloroacetic acid in the cleavage cocktail is in the range from 2 to 12 g per 1 mL of said one or more solvents (b2) (when the volume of the solvent is the volume at 25 °C), and the amount of trichloroacetic acid in the cleavage cocktail, based on the total weight of the cleavage cocktail, is at least 60% by weight, wherein the cleavage cocktail further comprises from 1 % to 15% of one or more scavengers, wherein the % are volume to volume (V / V) for scavengers liquid at 25 °C and weight to volume (W / V) for scavengers solid at 25 °C, based on the total volume of the cleavage cocktail at 25 °C, wherein at least one scavenger is water.

[0234] In a preferred embodiment, step (ii) results in a cleavage composition in which the peptide is dissolved. It will be understood that a method of preparing a peptide may further comprise one or more further procedural steps of further processing the peptide. For instance, the peptide may be separated from the cleavage composition and / or may be purified.

[0235] In a preferred embodiment, the method further comprises one or more further steps:

[0236] (iii) separating the peptide obtained in step (ii) from:

[0237] (iii-a) non-dissolved components, in particular from a solid support if present, and optionally washing the non-dissolved components once or more often, preferably with the cleavage cocktail, and / or Bachem Holding AG, 34 BAC75006PC1

[0238] Novo Nordisk A / S September 29, 2025

[0239] (iii-b) one or more other non-peptidic residues, in particular a tag;

[0240] (iv) precipitating the peptide obtained in any one of steps (ii) or (iii), preferably by mixing with anti-solvent, preferably diisopropyl ether, preferably at a temperature of not more than 10 °C, and optionally washing the peptide with one or more resuspension cycles in the anti-solvent;

[0241] (v) purifying the peptide obtained in any one of steps (ii), (iii), (iv), or (vi) by liquid chromatography,

[0242] (vi) chemically modifying the peptide obtained in any one of steps (ii), (iii), (iv), or (v), preferably by oxidative formation of disulfide bonds, or by conjugation to another moiety.

[0243] (vii) dissolving or suspending the precipitated peptide obtained in any one of steps

[0244] (iv), (v), or (vi) in a suitable solvent, preferably an evaporable solvent;

[0245] (viii) drying the composition obtained from any of steps (iii), (iv), (v), (vi), or (vii); and / or

[0246] (ix) dissolving or suspending the peptide obtained from any of steps (ii), (iii), (iv),

[0247] (v), (vi), (vii), or (viii) in a buffer suitable for a purpose of interest, in particular a pharmaceutically acceptable buffer.

[0248] An optional step (iii) of separating the peptide obtained in step (ii) may be performed by any means. It may be separating the peptide from non-dissolved components and optionally washing the non-dissolved components once or more often. Preferably, the non-dissolved components are not soluble in the cleavage cocktail under the conditions used in step (ii). Such non-dissolved components may in particular be a solid support that may be cleaved off the peptide precursor.

[0249] Removal of the non-dissolved components, in particular the solid support, may be performed by any means. For instance, it may be performed by centrifugation, filtration or a combination thereof. Optionally, the separated non-dissolved components, in particular the solid support, may be washed once or more often such as, e.g., by the cleavage cocktail. This may help to increase the peptide yield. For this purpose, a centrifugation and / or filtration step may be repeated.

[0250] Further, a step (iii) of separating the peptide obtained in step (ii) may be separating the peptide from one or more other non-peptidic residues, in particular a tag. This Bachem Holding AG, 35 BAC75006PC1

[0251] Novo Nordisk A / S September 29, 2025 may be performed by any means such as, e.g., by filtration, nanofiltration, extraction, precipitation, or by chromatographic means such as affinity chromatography selectively binding the tag or hydrophobicity chromatography or size exclusion chromatography (molecular sieve chromatography) separating the tag from the solution containing the peptide of interest.

[0252] An optional step (iv) of precipitating the peptide obtained in any one of steps (ii) or (iii) may be performed by any means. In a preferred embodiment, of precipitating the peptide is performed by mixing with an anti-solvent. As used herein, the term “anti-solvent” may be understood in the broadest sense as any solvent in which the peptide is not or poorly soluble any thus precipitates when mixed with the antisolvent. In a preferred embodiment, at least one, in particular all, of the cleaved reaction products of the protecting groups have a higher solubility in the anti-solvent than the peptide. In a preferred embodiment, the mass ratio of solubility of [cleaved off protecting groups] : [peptide] is at least 2 : 1 , at least 5 : 1 , at least 10 : 1 , or at least 100 : 1. In a preferred embodiment, step (ii) results in cleavage composition, in which the peptide is dissolved, and a solution of the peptide comprising cleavage products and components of the cleavage cocktail can be obtained by removing any particulate matter, in particular the solid support. In a preferred embodiment, mixing the anti-solvent with this solution is performed in a volume ratio of [antisolvent] : [solution] of at least 1 : 1 , of at least 2 : 1 , or of at least 3 : I .Then, the peptide is preferably precipitated. The mixing may be performed by either adding the peptide to the solution or vice versa. An anti-solvent may be any solvent usable for this purpose. In a preferred embodiment, the anti-solvent is an ether such as, e.g., diisopropyl ether or diethylether, in particular diisopropyl ether. In a preferred embodiment, the anti-solvent is a cold solvent, in particular of a temperature of not more than 20 °C, of not more than 15 °C, of not more than 10 °C, or of not more than 5 °C. In a preferred embodiment, the anti-solvent is cold diisopropyl ether, in particular of a temperature of not more than 20 °C, of not more than 15 °C, of not more than 10 °C, or of not more than 5 °C. Optionally, the precipitated peptide may be washed once or more often such as, e.g., by the anti-solvent. This may be performed by one or more resuspension cycles in the anti-solvent.

[0253] An optional step (v) of purifying the peptide obtained in any one of steps (ii), (iii), (iv) or (vi) by liquid chromatography may be performed by any means. For instance, it Bachem Holding AG, 36 BAC75006PC1

[0254] Novo Nordisk A / S September 29, 2025 may be performed by conducting high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC, also: LIPLC). This may be conducted by using any chromatographic material, including reverse phase (RP) solid phase such as silica particles coated with C4-C24-alkyl chains such as for instance, RP-C4, RP-C8 or RP-C18 material. The choice of the materials may be adapted to the hydrophobicity of the peptide. The liquid / mobile phase may optionally contain water and an organic modifier such as acetonitrile (ACN) and optionally one or more further solvents and / or one or more acids and / or one or more salts. The person skilled in the art knows that a gradient in the organic modifier may be used to separate the peptide from other components. The peptide peak obtained may be collected any may contain the purified peptide. Optionally, the obtained peptide may be precipitated. The optional step (v) may be performed or repeated after the peptide has been chemically modified as described in optional step (vi).

[0255] Optionally, in a step (vi), the peptide obtained in any one of steps (ii), (iii), (iv), or (v) may be subjected to chemical modification. For example, the peptide may be subjected to oxidative formation of disulfide bridges, to a condensation reaction between the N- and the C-terminus (“head to tail cyclization”) or be conjugated to any other moiety, e.g. a (suitable derivative of a) polyethylene glycol (PEG) moiety, a dye, or a pharmaceutically active compound. Optionally, the obtained modified peptide may be precipitated.

[0256] Optionally, in a step (vii), the peptide obtained in any one of steps (iv), (v), or (vi) may be dissolved or suspended in a suitable solvent, preferably an evaporable solvent. For instance, such evaporable solvent may be water, optionally containing an evaporable buffer, and / or acetonitrile.

[0257] Optionally, in a step (viii), the composition obtained from any of steps (iii), (iv), (v), (vi), or (vii) may be dried. As used herein, the term “drying” may be understood in the broadest sense as any means for removing solvent from the compositions used in steps (iii), (iv), (v), (vi), or (vii). For instance, drying may be drying at an elevated temperature and / or drying in vacuum. In a preferred embodiment, drying is spraydrying. In a preferred embodiment, drying is lyophilizing. As used herein, the terms Bachem Holding AG, 37 BAC75006PC1

[0258] Novo Nordisk A / S September 29, 2025

[0259] “lyophilizing” and “freeze-drying” may be understood interchangeably in the broadest sense. A lyophilized peptide is typically a dry foam or powder.

[0260] The peptide obtained from any of steps (ii), (iii), (iv), (v), (vi), (vii) or (viii) may optionally be dissolved or suspended in a buffer suitable for a purpose of interest, in particular a pharmaceutically acceptable buffer. The choice of such buffer may depend on the intended purpose.

[0261] As used herein, the term “pharmaceutically acceptable buffer” may be understood in the broadest sense as any buffer that may be used in a pharmaceutical context. Preferably, it is an aqueous buffer such as, e.g., saline. As used herein, the term “aqueous buffer” may be understood in the broadest sense as any buffer solution that mainly (i.e. , of more than 50% by weight) comprises water. It may have any pH suitable for this purpose such as a pH in the range of from pH 5 to 9, of from pH 6 to 8, of from pH 6.5 to 7.5, of from pH 6.9 to 7.4, of from pH 7.0 to 7.4, of pH 7.0, of pH 7.1 , of pH 7.2, of pH 7.3, or of pH 7.4.

[0262] A pharmaceutically acceptable buffer may exemplarily contain one or more ingredients selected from the list consisting of a dimethyl sulfoxide (DMSO), ethanol, vegetable oil, paraffin oil or combinations of two or more thereof. Furthermore, the pharmaceutically acceptable buffer may optionally contain one or more detergent(s), one or more foaming agent(s) (e.g., sodium lauryl sulfate (SLS), sodium dodecyl sulfate (SDS)), one or more coloring agent(s) (e.g., food coloring), one or more vitamin(s), one or more salt(s) (e.g., sodium, potassium, calcium, zinc salts), one or more humectant(s) (e.g., sorbitol, glycerol, mannitol, propylene glycol, polydextrose), one or more enzyme(s), one or more preserving agent(s) (e.g., benzoic acid, methylparaben), one or more antioxidant(s), one or more herbal and plant extract(s), one or more stabilizing agent(s), and / or one or more chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)).

[0263] When a pharmaceutically acceptable buffer is added, a pharmaceutical composition may be obtained. The terms “pharmaceutical composition” and “pharmaceutical formulation” may be understood interchangeably. Such pharmaceutical composition Bachem Holding AG, 38 BAC75006PC1

[0264] Novo Nordisk A / S September 29, 2025 may be ready to use and may preferably be a liquid formulation such as an injection portion or an inhalable portion or an orally administerable portion.

[0265] The present invention also relates to a dosage unit of the peptide obtainable by the method of the present invention. Exemplarily, a single dose container or to a multiple dosage form of the peptide may be prepared in a yet further step. A storage form may be a liquid or a dried form (e.g. a powder such as a powder comprising dried or freeze-dried peptide) or may be a paste or a syrup. Optionally, a dried form, paste or syrup may be dissolved or emulsified prior to being administered to a subject.

[0266] It will be noticed that the method of the present invention may be conducted for the synthesis of any peptide.

[0267] In a preferred embodiment, the peptide is 2 to 100 amino acid moieties in length, 5 to 90 amino acid moieties in length, 5 to 10 amino acid moieties in length, 5 to 20 amino acid moieties in length, 8 to 10 amino acid moieties in length, 10 to 80 amino acid moieties in length, 15 to 70 amino acid moieties in length, 20 to 60 amino acid moieties in length, 25 to 50 amino acid moieties in length, 20 to 40 amino acid moieties in length, 10 to 30 amino acid moieties in length, or 30 to 40 amino acid moieties in length.

[0268] In a preferred embodiment, the peptide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 47, 48, 49, 50 or more than 50 amino acid moieties in length.

[0269] In one embodiment, the peptide is selected from the group consisting of:

[0270] Tyr-Lys-Arg-Glu-Asp-Arg-Leu-Trp-Pro (in one-letter code: YKREDRLWP;

[0271] SEQ ID NO: 1 );

[0272] Tyr-Lys-Arg-Glu-Asn-Arg-Leu-Trp-Pro (in one-letter code: YKRENRLWP;

[0273] SEQ ID NO: 2)

[0274] Tyr-Lys-Arg-Glu-Asn-Arg-Leu-Trp-Pro-NH2 (in one-letter code: YKRENRLWP with C-terminal amide; SEQ ID NO: 3); Bachem Holding AG, 39 BAC75006PC1

[0275] Novo Nordisk A / S September 29, 2025

[0276] Tyr-Lys-Arg-Glu-Asn-Arg-Leu-Trp-Thr-Ser-Pro (in one-letter code: YKRENRLWTSP; SEQ ID NO: 4);

[0277] Dasiglucagon (His-Ser-GIn-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp- Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr; in one-letter code: HSQGTFTSDYSKYLDXARAEEFVKWLEST, wherein X is Aib (a-aminoisobutyric acid aka. a-methylalanine); SEQ ID NO: 5);

[0278] Glucagon (His-Ser-GIn-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser- Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr; in one-letter code: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT; SEQ ID NO: 6); Ala-Cys-Asp-Glu-Phe-Gly-His-lle-Lys-Leu-Met-Asn-Pro-GIn-Arg-Ser-Thr-Val-Trp- Tyr-Arg-Asp-Thr-Phe-Glu-Ala-Asn-Arg-Gln-NH2 (in one-letter code: ACDEFGHIKLMNPQRSTVWYRDTFEANRQ with C-terminal amide;

[0279] SEQ ID NO: 7);

[0280] HO2C-(CH2)i6-CO-YGIu-YGIu-YGIu-YGIu-yGlu-Gln-Glu-His-Pro-Gln-Ala-Arg-Lys- Tyr-Lys-Gly-Ala-GIn-Lys-Lys-Glu-Leu-Ser-Ser-Gly-Cys-Phe-Gly-Leu-Pro-Leu-Asp- Arg-lle-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys (in one-letter code: XXXXXQEHPQARKYKGAQKKELSSGCFGLPLDRIGSLSGLGC, wherein X are gamma-glutamyl residues and the N-terminus is conjugated with a 16-(tert-butoxy)- 16-oxohexadecanoyl moiety; SEQ ID NO: 8);

[0281] Dasiglucagon Amide (His-Ser-GIn-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu- Asp-Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr-NH2; in one-letter code: HSQGTFTSDYSKYLDXARAEEFVKWLEST, wherein X is Aib (a- aminoisobutyric acid aka. a-methylalanine) and the C-terminus is amidated; SEQ ID NO: 9); and lle-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr- Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Tyr-Arg-Gln-Arg-Tyr-NH2; in one-letter code: IKPEAPGEDASPEELNRYYASLRHYLNLVYRQRY with C-terminal amide; SEQ ID NO: 10).

[0282] As indicated above, a cleavage cocktail of the present invention bears special technical characteristics. Thus, a further aspect refers to a cleavage cocktail, as defined above for the first or second aspects of the invention.

[0283] In some embodiments, the cleavage cocktail of the present invention comprises: Bachem Holding AG, 40 BAC75006PC1

[0284] Novo Nordisk A / S September 29, 2025

[0285] (b1 ) trichloroacetic acid,

[0286] (b2) one or more solvents selected from the group consisting of diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole, and scavengers, and

[0287] (b3) optionally a salt; wherein the amount of trichloroacetic acid in the cleavage cocktail is in the range from 2 to 12 g per 1 mL of said one or more solvents (b2) (when the volume of the solvent is the volume at 25 °C), and the amount of trichloroacetic acid in the cleavage cocktail, based on the total weight of the cleavage cocktail, is at least 60%, optionally at least 65%, by weight.

[0288] In some embodiments, the cleavage cocktail of the present invention comprises:

[0289] (b1 ) trichloroacetic acid,

[0290] (b2) one or more solvents selected from the group consisting of diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole, and scavengers, and

[0291] (b3) optionally a salt; wherein the amount of trichloroacetic acid in the cleavage cocktail is in the range from 3 to 8 g per 1 mL of said one or more solvents (b2) (when the volume of the solvent is the volume at 25 °C), and the amount of trichloroacetic acid in the cleavage cocktail, based on the total weight of the cleavage cocktail, is at least 60%, optionally at least 65%, by weight.

[0292] In some embodiments, the cleavage cocktail of the present invention comprises:

[0293] (b1 ) trichloroacetic acid,

[0294] (b2) one or more solvents selected from the group consisting of diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole, and / or scavengers, and

[0295] (b3) optionally a salt; wherein Bachem Holding AG, 41 BAC75006PC1

[0296] Novo Nordisk A / S September 29, 2025 the amount of trichloroacetic acid in the cleavage cocktail is in the range from 4 to 8 g per 1 mL of said one or more solvents (b2) (when the volume of the solvent is the volume at 25 °C), and the amount of trichloroacetic acid in the cleavage cocktail, based on the total weight of the cleavage cocktail, is at least 60%, optionally at least 65%, by weight.

[0297] In each of the above embodiments, the amount of trichloroacetic acid in the cleavage cocktail, based on the total weight of the cleavage cocktail, may be less than 92%.

[0298] In some embodiments, the cleavage cocktail comprises:

[0299] (b1 ) trichloroacetic acid,

[0300] (b2) one or more solvents selected from the group consisting of diphenylsulfide, monohalogenated thiophenols, and monohalogenated thioanisoles, and

[0301] (b3) optionally a salt;

[0302] Preferably, the cleavage cocktail further comprises one or more scavengers different from the one or more solvents (b2), preferably in a total amount of from 1 % to 15%, preferably from 2% to 10%, more preferably from 4% to 8%, wherein the % are volume to volume (V / V) for scavengers liquid at 25 °C and weight to volume (W / V) for scavengers solid at 25 °C, based on the total volume of the cleavage cocktail at 25 °C. More preferably, the one or more scavengers are selected from the group consisting of water, thiol scavengers and silane scavengers, preferably water and optionally one or more further scavengers from the group consisting of thiol scavengers and silane scavengers, preferably 1 ,2-ethanedithiol, dithioerythritol, dithiothreitol, 3,6-dioxa-1 ,8-octanedithiol, beta-mercaptoethanol, 1 ,4-benzenedimethanethiol, 1 ,3-benzenedimethanethiol, 1 ,2- benzenedimethanethiol, triethylsilane, triisopropylsilane.

[0303] In some embodiments, the cleavage cocktail of the present invention as defined for the first or second aspect of the invention comprises:

[0304] (b1 ) 70% to 95% by weight, related to the total mass of the cleavage cocktail, of trichloroacetic acid,

[0305] (b2) one or more solvents and / or scavengers, preferably as defined above, and Bachem Holding AG, 42 BAC75006PC1

[0306] Novo Nordisk A / S September 29, 2025

[0307] (b3) optionally a salt.

[0308] It will be understood that the definitions and embodiments as laid out in the context of the method of the present invention mutatis mutandis apply to the cleavage cocktail of the present invention.

[0309] In a preferred embodiment, the cleavage cocktail comprises:

[0310] (b1 ) trichloroacetic acid in an amount as specified above,

[0311] (b2) one or more solvents selected from the group consisting of diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole, and

[0312] (b3) optionally a salt, wherein the cleavage cocktail comprises water and one or more scavengers selected from the group consisting of 1 ,2-ethanedithiol, dithioerythritol, dithiothreitol , 3,6-dioxa-1 ,8-octane-dithiol, beta-mercaptoethanol, triethylsilane and triisopropylsilane.

[0313] In another preferred embodiment, the cleavage cocktail comprises:

[0314] (b1 ) trichloroacetic acid in an amount as specified above,

[0315] (b2) 2-chlorothioanisole, and

[0316] (b3) one or more scavenger selected from the group consisting of silane scavengers, EDT and water, preferably consisting of triethylsilane, triisopropylsilane, EDT, and water.

[0317] In a preferred embodiment, the cleavage cocktail contains from 65 to 91 % by weight, more preferably from 68 to 90% by weight, more preferably from 70 to 88% by weight, more preferably from 75 to 86% by weight, related to the total mass of the cleavage cocktail, of trichloroacetic acid. In a preferred embodiment, the cleavage cocktail contains from 65 to 92 %, from 60 to 84 %, from 79 to 88 %, or 71 to 81 % by weight, related to the total mass of the cleavage cocktail, of trichloroacetic acid. In some embodiments of the cleavage cocktail of the invention as defined for the first and second aspect of the invention, the cleavage cocktail contains from 70% to 95% by weight, 75% to 94% by weight, 75% to 90% by weight, 80% to 90% by Bachem Holding AG, 43 BAC75006PC1

[0318] Novo Nordisk A / S September 29, 2025 weight, or 85% to 95% by weight, related to the total mass of the cleavage cocktail, of trichloroacetic acid.

[0319] The cleavage cocktail may be used for any purpose. In particular, the cleavage cocktail may be used for cleaving an acid-cleavable non-peptidic group off a peptide precursor.

[0320] A further aspect refers to the use of a cleavage cocktail of the present invention for cleaving an acid-cleavable solid or solubility-modifying support and / or an acid- cleavable protecting group off a peptide precursor.

[0321] Additional aspects of the present disclosure are defined by the following embodiments, to which the definitions disclosed above for the different aspects of the invention apply accordingly.

[0322] Embodiment 1 A method of preparing a peptide, wherein the method comprises the steps of:

[0323] (i) contacting

[0324] (A) a peptide precursor that is covalently bound to at least one acid- cleavable non-peptidic moiety; with

[0325] (B) a cleavage cocktail comprising:

[0326] (b1 ) trichloroacetic acid,

[0327] (b2) one or more solvents and / or scavengers, and

[0328] (b3) optionally a salt; and

[0329] (ii) incubating the components of step (i) at a temperature in the range of 30°C to 70°C, thereby cleaving off acid-cleavable non-peptidic moiety from the peptide precursor resulting in the peptide.

[0330] Embodiment 2. The method of embodiment 1 , wherein the one or more solvents and / or scavengers do not comprise a per- or polyfluorinated substance.

[0331] Embodiment 3. The method of one of embodiments 1 or 2, wherein the cleavage cocktail comprises 7.0 to 10 M of trichloroacetic acid. Bachem Holding AG, 44 BAC75006PC1

[0332] Novo Nordisk A / S September 29, 2025

[0333] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the acid- cleavable non-peptidic moiety is a solid support and / or a tag and / or a protecting group.

[0334] Embodiment 5. The method of any one of embodiments 1 to 4, wherein:

[0335] (a) the one or more solvents comprise or are consisted of one or more solvents selected from the group consisting of acetonitrile, an aromatic solvent, acetic acid, dichloroacetic acid, and an aliphatic nitro group- containing solvent, in particular wherein the one or more solvents comprise or are consisted of one or more solvents selected from the group consisting of acetonitrile, anisole, 1 ,3-dimethoxybenzene, thioanisole, acetic acid, dichloroacetic acid, 1 -nitropropane, 2-nitropropane, dichloromethane and nitromethane; and / or

[0336] (b) one or more scavengers are selected from the group consisting of 1 ,2- ethanedithiol, dithioerythritol, dithiothreitol, 3,6-dioxa-1 ,8- octanedithiol, beta-mercaptoethanol, triethylsilane, triisopropylsilane, ethyl methyl sulfide, thioanisole, anisole, m- or p-cresol, 2-Me-indole, Ac-Trp-OMe, water, phenol, and tryptamine.

[0337] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the peptide precursor is obtained from:

[0338] (a) solid phase peptide synthesis (SPPS) and is covalently bound to an acid-cleavable solid support, preferably wherein the solid support is a swellable polystyrene resin or a grafted polystyrene resin which may be grafted with polyethylene glycol (PEG);

[0339] (b) tag-assisted peptide synthesis (TAPS) and is covalently bound to an acid-cleavable tag, preferably wherein the tag is selected from the group consisting of substituted benzyl alcohols, substituted benzyl amines, substituted 1 ,1 -diphenylmethanols, and substituted 1 ,1 -diphenylmethylamines;

[0340] (c) liquid-phase peptide synthesis (LPPS); or

[0341] (d) a combination of two or more of (a), (b) and / or (c). Bachem Holding AG, 45 BAC75006PC1

[0342] Novo Nordisk A / S September 29, 2025

[0343] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the peptide precursor is characterized in that:

[0344] (a) it has a C- and / or N-terminus protected with an acid-cleavable group, and / or is bound to a solid support, and / or is bound to a tag, preferably wherein the tag is selected from the group consisting of substituted benzyl alcohols, substituted benzyl amines, substituted 1 ,1 - diphenylmethanols, and substituted 1 ,1 -diphenylmethylamines, and / or wherein the peptide is bound to a solid support or to a tag via an acid- cleavable linker, preferably wherein the linker is selected from the group consisting of 2-chlorotrityl (2CT linker), 2-chlorotrityl amidomethyl (2CT-AM linker), 4-[9-Fmoc-amino-xanthen-3-yloxy]-butyryl)- 4-methyl-benzhydrylamide (Sieber linker), 4-hydroxybenzylalcohol (Wang linker), Fmoc-2,4-dimethoxy-4’-(carboxymethyloxy)- benzhydrylamine (Rink Amide linker), Fmoc-suberol (5-Fmoc-amino- 2-carboxymethoxy-10, 11 -dihydro-5H-dibenzo[a,d]cycloheptene) (Ramage linker), 5-Fmoc-amino-10,11 -dihydro-5H-dibenzo [a,d]cycloheptenyl-2-oxyacetyl-DL-Nle-4-methyl-benzhydrylamide (tricyclic amide linker), 2-Methoxy-4-alkoxy benzyl alcohol (Sasrin linker), 5-[4-(9-Fmoc)aminomethyl-3,5-dimethoxyphenoxy]-pentanoic acid (Fmoc-PAL linker), 4-(4-Formyl-3,5-dimethoxyphenoxy) (BAL linker), 4-Hydroxymethylphenoxyacetic acid (HMPA linker), and 4-(4- hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB linker); and / or

[0345] (b) the peptide precursor comprises one or more of the following structural elements:

[0346] A 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf) side chain protecting group moiety, 2,2,5,7,8-pentamethylchroman-6- sulfonyl (Pmc) side chain protecting group moiety, in particular an Arg(Pbf) moiety, an intra-chain and / or C-terminal beta-hydroxy amino acid moiety, preferably Ser or Thr,

[0347] Asn, Gin, Asp, Cys, Met, Trp, a hydrocarbon chain >Ce as a substituent, and / or a polyethylene glycol (PEG) moiety with two or more oxyethylene repeating units as a substituent. Bachem Holding AG, 46 BAC75006PC1

[0348] Novo Nordisk A / S September 29, 2025

[0349] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the step (ii) is conducted at a temperature in the range of 35°C to 60°C, of 35°C to 55°C, of 35°C to 50°C, of 40°C to 60°C, of 40°C to 55°C, or of 40 to 50°C.

[0350] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the step (ii) is conducted for at least 2 hours, in particular for 2 to 24 hours.

[0351] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the cleavage cocktail comprises or consists of: (b1 ) 7.0 to 10 M of trichloroacetic acid,

[0352] (b2) 0.1 to 20% by volume, preferably 1 to 10% by volume, referred to the total volume of the cleavage cocktail, of one or more solvents and / or scavengers; and

[0353] (b3) optionally one or more salts.

[0354] Embodiment 11 . The method of any one of embodiments 1 to 10, wherein a ratio R as expressed by the following Formula (I): is at least 5, wherein:

[0355] X is the unitless numeric value of the temperature in degrees Celsius at which step (ii) is conducted, and

[0356] Y is the unitless numeric value of the molar content of trichloroacetic acid in the cleavage cocktail, preferably wherein R is at least 6, at least 7, at least 8 or at least 9, in the range of 5 to 13, of 6 to 12, of 7 to 11 , or of 7 to 10.

[0357] Embodiment 12. The method of any one of embodiments 1 to 11 , wherein the method comprises: (i) contacting

[0358] (A) a peptide precursor that is covalently bound to at least one acid- cleavable solid support and / or at least one acid-cleavable tag and / or at least one acid-cleavable protecting group; with Bachem Holding AG, 47 BAC75006PC1

[0359] Novo Nordisk A / S September 29, 2025

[0360] (B) a cleavage cocktail comprising:

[0361] (b1 ) 7.0 to 10 M of trichloroacetic acid,

[0362] (b2) one or more solvents and / or scavengers that do not comprise a per- or polyfluorinated substance, and

[0363] (b3) optionally a salt; and

[0364] (ii) incubating the components of step (i) at a temperature in the range of 30°C to 70°C, preferably for 1 to 24 hours, thereby cleaving the at least one acid-cleavable solid support and / or the at least one acid-cleavable tag and / or the at least one acid-cleavable protecting group off the peptide.

[0365] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the method further comprises one or more further steps:

[0366] (iii) separating the peptide obtained in step (ii) from:

[0367] (iii-a) non-dissolved components, in particular from a solid support if present, and optionally washing the non-dissolved components once or more often, preferably with the cleavage cocktail, and / or

[0368] (iii-b) one or more other non-peptidic residues, in particular a tag;

[0369] (iv) precipitating the peptide obtained in any one of steps (ii) or (iii), preferably by mixing with anti-solvent, preferably diisopropyl ether, preferably at a temperature of not more than 10°C, and optionally washing the peptide with one or more resuspension cycles in the antisolvent;

[0370] (v) purifying the peptide obtained in any one of steps (ii), (iii), (iv), or (vi) by liquid chromatography;

[0371] (vi) chemically modifying the peptide obtained in any one of steps (ii), (iii), (iv), or (v), preferably by oxidative formation of disulfide bonds, or by conjugation to another moiety;

[0372] (vii) dissolving or suspending the precipitated peptide obtained in any one of steps (iv), (v), or (vi) in a suitable solvent, preferably an evaporable solvent;

[0373] (viii) drying the composition obtained from any of steps (iii), (iv), (v), (vi), or (vii); and / or Bachem Holding AG, 48 BAC75006PC1

[0374] Novo Nordisk A / S September 29, 2025

[0375] (ix) dissolving or suspending the peptide obtained from any of steps (ii), (iii), (iv), (v), (vi), (vii), or (viii) in a buffer suitable for a purpose of interest, in particular a pharmaceutically acceptable buffer.

[0376] Embodiment 14. A cleavage cocktail comprising:

[0377] (b1 ) 70% to 95% by weight, related to the total mass of the cleavage cocktail, of trichloroacetic acid, and

[0378] (b2) one or more solvents and / or scavengers, preferably as defined in embodiment 5, and

[0379] (b3) optionally a salt.

[0380] Embodiment 15. Use of a cleavage cocktail of embodiment 14 for cleaving an acid-cleavable solid or solubility-modifying support and / or an acid-cleavable protecting group off a peptide precursor, preferably wherein the use is defined according to any one of embodiments 1 to 13.

[0381] Embodiment 16. A method of preparing a peptide, wherein the method comprises the steps of:

[0382] (i) contacting

[0383] (A) a peptide precursor that is covalently bound to an acid- cleavable solid support or to at least one acid-cleavable tag; with

[0384] (B) a cleavage cocktail comprising:

[0385] (b1 ) trichloroacetic acid at a concentration of 7.0 to 10 M, (b2) one or more solvents and / or scavengers, and (b3) optionally a salt; and

[0386] (ii) incubating the components of step (i) at a temperature in the range of 30°C to 70°C, thereby cleaving off the acid-cleavable solid support or the at least one acid-cleavable tag from the peptide precursor resulting in the peptide. Bachem Holding AG, 49 BAC75006PC1

[0387] Novo Nordisk A / S September 29, 2025

[0388] Embodiment 17. The method of embodiment 16, wherein the one or more solvents and / or scavengers do not comprise a per- or polyfluorinated substance.

[0389] Embodiment 18. The method of one of embodiments 16 or 17, wherein cleavage of protecting groups from the peptide occurs in addition to cleavage of the peptide from the acid-cleavable solid support or from the at least one acid- cleavable tag.

[0390] Embodiment 19. The method of any one of embodiments 16 to 18, wherein:

[0391] (a) the one or more solvents comprise or are consisted of one or more solvents selected from the group consisting of acetonitrile, an aromatic solvent, acetic acid, dichloroacetic acid, and an aliphatic nitro group- containing solvent, in particular wherein the one or more solvents comprise or are consisted of one or more solvents selected from the group consisting of acetonitrile, anisole, 1 ,3-dimethoxybenzene, thioanisole, acetic acid, dichloroacetic acid, 1 -nitropropane, 2-nitropropane, dichloromethane and nitromethane; and / or

[0392] (b) one or more scavengers are selected from the group consisting of 1 ,2- ethanedithiol, dithioerythritol, dithiothreitol, 3,6-dioxa-1 ,8- octanedithiol, beta-mercaptoethanol, triethylsilane, triisopropylsilane, ethyl methyl sulfide, thioanisole, anisole, m- or p-cresol, 2-Me-indole, Ac-Trp-OMe, water, phenol, and tryptamine.

[0393] Embodiment 20. The method of any one of embodiments 16 to 19, wherein the solvent is selected from the group consisting of diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole.

[0394] Embodiment 21 . The method of any one of embodiments 16 to 20, wherein the peptide precursor is obtained from:

[0395] (a) solid phase peptide synthesis (SPPS) and is covalently bound to an acid-cleavable solid support, Bachem Holding AG, 50 BAC75006PC1

[0396] Novo Nordisk A / S September 29, 2025 preferably wherein the solid support is a swellable polystyrene resin or a grafted polystyrene resin which may be grafted with polyethylene glycol (PEG);

[0397] (b) tag-assisted peptide synthesis (TAPS) and is covalently bound to an acid-cleavable tag, preferably wherein the tag is selected from the group consisting of substituted benzyl alcohols, substituted benzyl amines, substituted 1 ,1 -diphenylmethanols, and substituted 1 ,1 -diphenylmethylamines; or

[0398] (c) a combination of two or more of (a), (b) and liquid-phase peptide synthesis (LPPS).

[0399] Embodiment 22. The method of any one of embodiments 16 to 21 , wherein the peptide precursor is characterized in that:

[0400] (a) it has a C- and / or N-terminus protected with an acid-cleavable group, and / or is bound to a solid support, and / or is bound to a tag, preferably wherein the tag is selected from the group consisting of substituted benzyl alcohols, substituted benzyl amines, substituted 1 ,1 - diphenylmethanols, and substituted 1 ,1 -diphenylmethylamines, and / or wherein the peptide is bound to a solid support or to a tag via an acid- cleavable linker, preferably wherein the linker is selected from the group consisting of 2-chlorotrityl (2CT linker), 2-chlorotrityl amidomethyl (2CT-AM linker), 4-[9-Fmoc-amino-xanthen-3-yloxy]-butyryl)- 4-methyl-benzhydrylamide (Sieber linker), 4-hydroxybenzylalcohol (Wang linker), Fmoc-2,4-dimethoxy-4’-(carboxymethyloxy)- benzhydrylamine (Rink Amide linker), Fmoc-suberol (5-Fmoc-amino- 2-carboxymethoxy-10, 11 -dihydro-5H-dibenzo[a,d]cycloheptene) (Ramage linker), 5-Fmoc-amino-10,11 -dihydro-5H-dibenzo [a,d]cycloheptenyl-2-oxyacetyl-DL-Nle-4-methyl-benzhydrylamide (tricyclic amide linker), 2-Methoxy-4-alkoxy benzyl alcohol (Sasrin linker), 5-[4-(9-Fmoc)aminomethyl-3,5-dimethoxyphenoxy]-pentanoic acid (Fmoc-PAL linker), 4-(4-Formyl-3,5-dimethoxyphenoxy) (BAL linker), 4-Hydroxymethylphenoxyacetic acid (HMPA linker), and 4-(4- hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB linker); and / or Bachem Holding AG, 51 BAC75006PC1

[0401] Novo Nordisk A / S September 29, 2025

[0402] (b) the peptide precursor comprises one or more of the following structural elements:

[0403] A 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf) side chain protecting group moiety, 2,2,5,7,8-pentamethylchroman-6- sulfonyl (Pmc) side chain protecting group moiety, in particular an Arg(Pbf) moiety, an intra-chain and / or C-terminal beta-hydroxy amino acid moiety, preferably Ser or Thr,

[0404] Asn, Gin, Asp, Cys, Met, Trp, a hydrocarbon chain >Ce as a substituent, and / or a polyethylene glycol (PEG) moiety with two or more oxyethylene repeating units as a substituent.

[0405] Embodiment 23. The method of any one of embodiments 16 to 22, wherein the step (ii) is conducted at a temperature in the range of 35°C to 60°C, of 35°C to 55°C, of 35°C to 50°C, of 35°C to 45°C, of 30°C to 45°C, of 30°C to 40°C, of 40°C to 60°C, of 40°C to 55°C, or of 40 to 50°C.

[0406] Embodiment 24. The method of any one of embodiments 16 to 23, wherein the step (ii) is conducted for at least 2 hours, in particular for 2 to 24 hours.

[0407] Embodiment 25. The method of any one of embodiments 16 to 24, wherein the cleavage cocktail comprises or consists of: (b1 ) 7.0 to 10 M of trichloroacetic acid,

[0408] (b2) 0.1 to 30% by volume, preferably 20 to 30% by volume, referred to the total volume of the cleavage cocktail, of one or more solvents and / or scavengers; and

[0409] (b3) optionally one or more salts.

[0410] Embodiment 26. The method of any one of embodiments 16 to 25, wherein a ratio R as expressed by the following Formula (I): Bachem Holding AG, 52 BAC75006PC1

[0411] Novo Nordisk A / S September 29, 2025 is at least 5, wherein:

[0412] X is the unitless numeric value of the temperature in degrees Celsius at which step (ii) is conducted, and

[0413] Y is the unitless numeric value of the molar content of trichloroacetic acid in the cleavage cocktail, preferably wherein R is at least 6, at least 7, at least 8 or at least 9, in the range of 5 to 13, of 6 to 12, of 7 to 11 , or of 7 to 10.

[0414] Embodiment 27. The method of any one of embodiments 16 to 26, wherein the method further comprises one or more further steps:

[0415] (iii) separating the peptide obtained in step (ii) from:

[0416] (iii-a) non-dissolved components, in particular from a solid support if present, and optionally washing the non-dissolved components once or more often, preferably with the cleavage cocktail, and / or

[0417] (iii-b) one or more other non-peptidic residues, in particular a tag;

[0418] (iv) precipitating the peptide obtained in any one of steps (ii) or (iii), preferably by mixing with anti-solvent, preferably diisopropyl ether, preferably at a temperature of not more than 10°C, and optionally washing the peptide with one or more resuspension cycles in the antisolvent;

[0419] (v) purifying the peptide obtained in any one of steps (ii), (iii), (iv), or (vi) by liquid chromatography;

[0420] (vi) chemically modifying the peptide obtained in any one of steps (ii), (iii), (iv), or (v), preferably by oxidative formation of disulfide bonds, or by conjugation to another moiety;

[0421] (vii) dissolving or suspending the precipitated peptide obtained in any one of steps (iv), (v), or (vi) in a suitable solvent, preferably an evaporable solvent;

[0422] (viii) drying the composition obtained from any of steps (iii), (iv), (v), (vi), or (vii); and / or

[0423] (ix) dissolving or suspending the peptide obtained from any of steps (ii), (iii), (iv), (v), (vi), (vii), or (viii) in a buffer suitable for a purpose of interest, in particular a pharmaceutically acceptable buffer. Bachem Holding AG, 53 BAC75006PC1

[0424] Novo Nordisk A / S September 29, 2025

[0425] Embodiment 28. A cleavage cocktail comprising:

[0426] (b1 ) trichloroacetic acid at a concentration of 7.0 to 10 M,

[0427] (b2) one or more solvents selected from the group consisting of diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole, and

[0428] (b3) one or more scavengers selected from the group consisting of 1 ,2- ethanedithiol, dithioerythritol, dithiothreitol, 3,6-dioxa-1 ,8-octane- dithiol, beta-mercaptoethanol, triethylsilane, triisopropylsilane, and water.

[0429] Embodiment 29. The cleavage cocktail of embodiment 28, wherein:

[0430] (b2) one solvent is 2-chlorothioanisole; and

[0431] (b3) one scavenger is ethanedithiol, triisopropylsilane or triethylsilane.

[0432] Embodiment 30. Use of a cleavage cocktail of embodiment 28 or 29 for cleaving an acid-cleavable solid or solubility-modifying support off a peptide precursor, preferably wherein the use is defined according to any one of embodiments 16 to 27.

[0433] It will be understood that the definitions and embodiments as laid out in the context of the method and the cleavage cocktail of the present invention mutatis mutandis apply to the use of the cleavage cocktail of the present invention. In a preferred embodiment, the use is defined as laid out in the context of the method above.

[0434] Bachem Holding AG, 54 BAC75006PC1

[0435] Novo Nordisk A / S September 29, 2025

[0436] List of abbreviations

[0437] 2Me-THF - 2-methyltetrahydrofuran

[0438] Aib - a-aminoisobutyric acid aka. a-methylalanine

[0439] ACN - acetonitrile

[0440] AcOH - acetic acid

[0441] DCM - dichloromethane

[0442] EDT - ethane-1 ,2-dithiol

[0443] HPLC I UHPLC - high performance liquid chromatography I ultra high performance liquid chromatography

[0444] MSA - methanesulfonic acid

[0445] RT or rt - room temperature

[0446] TCA - trichloroacetic acid

[0447] TFA - trifluoroacetic acid

[0448] TIS - triisopropylsilane

[0449] CTC - 2-chlorotrityl

[0450] DCA - dichloroacetic acid

[0451] TES - triethylsilane

[0452] DODT - 3,6-dioxa-1 ,8-octanedithiol

[0453] DTE - dithioerythritol

[0454] NMM - N-methylmorpholine

[0455] DMSO - dimethyl sulfoxide

[0456] FLP - full length peptide

[0457] Pbf - 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl

[0458] DTT - dithiothreitol

[0459] DIC - N,N’-diisopropylcarbodiimide

[0460] DMF - N,N-dimethylformamide yGlu - gamma glutamyl tBuOCO-(CH2)i6-CO (or tBuO2C-(CH2)i6-CO) - 16-(tert-butoxy)-16- oxohexadecanoyl moiety

[0461] SPPS - solid phase peptide synthesis

[0462] “Asp” - Asn deamidation product of FLP

[0463] Asi - Aspartimide rpm - Rounds per minute Bachem Holding AG, 55 BAC75006PC1

[0464] Novo Nordisk A / S September 29, 2025

[0465] IPE - Diisopropyl ether

[0466] TBTLI - 2-(1 H-Benzotriazole-1 -yl)-1 ,1 ,3,3-tetramethylaminium tetrafluoroborate TATLI - O-(7-Azabenzotriazole-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate DEPBT - (3-(diethoxyphosphoryloxy)-1 ,2,3-benzotriazin-4(3H)-one)

[0467] DIPEA - N,N-Diisopropylethylamine

[0468] Fmoc - Fluorenylmethyloxycarbonyl

[0469] EtOAc - Ethyl acetate

[0470] (V / V) - Volume to volume

[0471] (W / V) - Weight to volume

[0472] PS - Polystyrene tBu - tert-butyl

[0473] Boc - tert-butyloxycarbonyl

[0474] Mpe - 3-methyl-3-pentyl

[0475] HOPO - 2-Hydroxypyridine-N-oxide

[0476] Oxyma Pure - Ethyl cyano(hydroximo)acetate

[0477] Trt - trityl

[0478] Tricyclic amide linker resin (DL-form) - 5-Fmoc-amino-10,11 -dihydro-5H- dibenzo[a,d]-cycloheptenyl-2-oxyacetyl-DL-Nle-4-methyl-benzhydrylamide resin

[0479] The Figures and Experiments are intended to provide further embodiments of the invention. The Figures and Experiments should not have any limiting effect on the scope.

[0480] Brief description of the Figures

[0481] Figure 1 shows a model peptide II after cleavage and deprotection as described in Experiment 1 a. HPLC chromatograms recorded at 220 nm. *(FLP): product peak; Asi: aspartimide derivative of product; 1 Pbf-FLP: product with one Pbf protecting group uncleaved; 2Pbf-FLP: product with two Pbf protecting groups uncleaved; 2,3- Dihydro-2, 2, 4, 6, 7, -pentamethylbenzofuran is a product of Pbf cleavage. Inlay shows zoom-in onto product peak with resolution of FLP from Asi.

[0482] A) [TCA neat with 2.5% H2O + 5% EDT (VA / A / ) 30 min at 70 °C],

[0483] B) [4 M MSA in AcOH / ACN (1 / 1 ) with 2.5% H2O + 5% EDT (VA / A / ) 2 h at RT],

[0484] C) [TFA with 5% H2O + 5% EDT (VA / A / ) 2h at RT], Bachem Holding AG, 56 BAC75006PC1

[0485] Novo Nordisk A / S September 29, 2025

[0486] Figure 2 shows a model peptide I after cleavage and deprotection as described in Experiment 2a. HPLC chromatograms recorded at 220 nm. *) Product (aka. FLP) peak. 2) Product with one Pbf protecting group uncleaved (aka. 1 Pbf-FLP), 3) Product with two Pbf protecting groups uncleaved (aka. 2Pbf-FLP).

[0487] A) [TCA neat with 2.5 % H2O + 5% EDT (VA / A / ) 30 min at 70 °C],

[0488] B) [4 M MSA in AcOH / ACN (1 / 1 ) with 2.5% H2O + 5% EDT (VA / A / ) 2 h at RT]

[0489] C) [TFA with 5% H2O + 5% EDT (VA / A / ) 2 h at RT],

[0490] Figure 3 shows a model peptide III after cleavage and deprotection as described in Experiment 3a. HPLC chromatograms recorded at 220 nm. *) Product (aka. FLP) peak., 2) Product with one Pbf protecting group uncleaved (aka. 1 Pbf-FLP), 3) Product with two Pbf protecting groups uncleaved (aka. 2Pbf-FLP).

[0491] A) [TCA neat with 2.5% H2O + 5% EDT (VA / A / ) 30 min at 70 °C],

[0492] B) [4 M MSA in AcOH / DCM (1 / 1 ) with 2.5% H2O + 5% EDT (VA / A / ) 3 h at RT],

[0493] Figure 4 shows a model peptide VII after cleavage and deprotection as described in Experiment 4a. HPLC chromatograms recorded at 220 nm. *) Product (aka. FLP) peak. 2) Product with one Pbf protecting group uncleaved (aka. 1 Pbf-FLP), 3) Product with two Pbf protecting groups uncleaved (aka. 2Pbf-FLP).

[0494] A) [1 M MSA in DCA with 3% H2O + 3% TIS + 3% DTT (V / V / V / W) 2 h at RT],

[0495] B) [TCA neat with 2.5% H2O + 5% EDT (VA / A / ) 30 min at 70 °C],

[0496] Figure 5 shows a model peptide VII after cleavage, deprotection and isolation as described in Experiment 4b. HPLC chromatograms recorded at 220 nm. *) Product (aka. FLP) peak.

[0497] A) [1 M MSA in DCA with 3% H2O + 3% TIS + 3% DTT (V / V / V / W) 2 h at RT],

[0498] B) [TCA neat with 2.5% H2O + 5% EDT (VA / A / ) 30 min at 70 °C],

[0499] Figure 6 shows a Dasiglucagon after cleavage and deprotection as described in Experiment 5a. HPLC chromatograms recorded at 220 nm. *) Product (aka. FLP) peak.

[0500] A) [TCA neat with 2.5% H2O + 5% (VA / A / ) EDT 30 min at 70 °C],

[0501] B) [4 M MSA in AcOH with 2.5% H2O + 5% (VA / A / ) EDT 2 h at RT]

[0502] C) [TFA with 2.5% H2O + 5% EDT (VA / A / ) 2 h at RT], Bachem Holding AG, 57 BAC75006PC1

[0503] Novo Nordisk A / S September 29, 2025

[0504] Figure 7 shows a Glucagon after cleavage and deprotection as described in Experiment 6a. HPLC chromatograms recorded at 220 nm. *) Product (aka. FLP) peak.

[0505] A) [TCA neat with 2.5% H2O + 5% EDT (VA / A / ) 30 min at 70 °C],

[0506] B) [TFA with 2.5% H2O + 5% EDT (VA / A / ) 2 h at RT],

[0507] Figure 8 shows a 29-mer ABCEXT peptide after cleavage and deprotection as described in Experiment 7. HPLC chromatograms recorded at 214 nm. *) Product peak.

[0508] A) 8 M TCA in nitromethane with 2% H2O, 2% TIS, 2% DTT (V / V / V / W) at 40 °C for 24 h. B) 94% TFA with 2% H2O, 2% TIS, 2% DTT (V / V / V / W) at room temperature for 24 h.

[0509] Figure 9 shows a 43-mer peptide after cleavage and deprotection as described in Experiment 8. HPLC chromatograms recorded at 214 nm. *) Product peak.

[0510] A) 8 M TCA in nitromethane with 2% H2O, 2% TIS, 2% DTT (V / V / V / W) at 40 °C for 24 h. B) 94% TFA with 2% H2O, 2% TIS, 2% DTT (V / V / V / W) at room temperature for 24 h.

[0511] Figure 10 shows Dasiglucagon amide after cleavage and deprotection as described in Experiment 9. HPLC chromatograms recorded at 214 nm. *) Product peak.

[0512] A) 8 M TCA in nitromethane with 2% H2O, 2% TIS, 2% DTT (V / V / V / W) at 40 °C for 24 h.

[0513] B) 94% TFA with 2% H2O, 2% TIS, 2% DTT (V / V / V / W) at room temperature for 24 h.

[0514] Figure 11 shows a PYY3-36 peptide after cleavage and deprotection as described in Experiment 10. HPLC chromatograms recorded at 214 nm. *) Product peak.

[0515] A) 8 M TCA in nitromethane with 2% H2O, 2% TIS, 2% DTT (V / V / V / W) at 40 °C for 24 h.

[0516] B) 94% TFA with 2% H2O, 2% TIS, 2% DTT (V / V / V / W) at room temperature for 24 h.

[0517] Figure 12 shows a resolution of model peptide I Asn deamidation product (“Asp”) and model peptide I (“Asn”) as described in UHPLC Method A.

[0518] A) Mixture 1 / 1 of peptide I containing Asn and Peptide II containing Asp Bachem Holding AG, 58 BAC75006PC1

[0519] Novo Nordisk A / S September 29, 2025

[0520] B) Reference peptide II containing Asp

[0521] C) Reference peptide I containing Asn.

[0522] Examples

[0523] Both in the description above and the experiments below, if a weight to volume percentage is given, the value defines the amount of grams (g) of a substance in 100 milliliters (mL) of the total mixture. For example, n% (W / V) of Y in X corresponds to n g of Y in 100 mL of the mixture of Y and X, where the remainder ad 100 mL is X.

[0524] Expressions such as “X with n% Y + m% Z (V / V / W)” mean that n mL of Y and m g of Z are present in 100 mL total volume of the mixture, where the remainder ad 100 mL is X.

[0525] Similarly, if a weight to volume ratio X:Y (WA / ) between two components X and Y is given, the ratio is defined by the amount of X in g relative to the volume of Y in mL.

[0526] If not indicated, percentages of substances that are solid at room temperature are given as weight to volume percentages, and those of substances that are liquid at room temperature are given as volume to volume percentages.

[0527] Where solutions of a substance in a solvent are defined by an expression such as “n g / 1 mL”, or “n g substance I mL solvent” the numerical value n defines the amount of the substance in g present for each mL of the solvent, but not for each mL of the total solution. This applies accordingly for expressions such as “n mL substance I g resin”.

[0528] Moreover, unless otherwise stated, the volumes referred to are given for the respective substances or mixtures having a temperature of 25 °C. However, within the temperature ranges relevant for the invention, the temperature-dependent differences in volume are smaller than the accuracy-related deviations of the used volumetric measurement methods. Therefore, the volumes are considered to be constant within the range from 15 to 70 °C. Bachem Holding AG, 59 BAC75006PC1

[0529] Novo Nordisk A / S September 29, 2025

[0530] 1. General Procedures

[0531] 1.1. Analytical Methods

[0532] Samples were analyzed by UHPLC using the following protocols.

[0533] 1.1.1. UHPLC Method A:

[0534] Instrument: Dionex Ultimate 3000 RS

[0535] Column: Waters Acquity UPLC BEH130 C8, 1 .7 pm, 150 x 2.1 mm

[0536] Detection: UV (220 nm)

[0537] Eluent A: 0.05% (VA / ) TFA in H2O / ACN (99:1 VA / )

[0538] Eluent B: 0.05% (VA / ) TFA in ACN

[0539] Flow rate: 0.4 mL / min

[0540] Column temperature: 50 °C

[0541] Gradient:

[0542] The ability of the method to generate sufficient resolution to distinguish peptides and their post-cleavage side-products (especially aspartic acid formation from asparagine) was assessed. The separation of the product peaks resulting from Model Peptide I (containing asparagine) and peaks resulting from Model Peptide II (containing aspartic acid) with UHPLC Method A (cf. para. 1.1.1 ) was demonstrated (cf. Fig. 12).

[0543] 1.1.2. UHPLC Method B:

[0544] Instrument: Dionex Ultimate 3000 RS

[0545] Column: Waters Acquity UPLC BEH130 C8, 1.7 pm, 150 x 2.1 mm

[0546] Detection: UV (220 nm)

[0547] Eluent A: 0.05% (VA / ) TFA in H2O / ACN (99:1 VA / )

[0548] Eluent B: 0.05% (VA / ) TFA in ACN

[0549] Flow rate: 0.4 mL / min

[0550] Column temperature: 50 °C Bachem Holding AG, 60 BAC75006PC1

[0551] Novo Nordisk A / S September 29, 2025

[0552] Gradient:

[0553] 1.1.3. General procedure 1 for sample preparation of cleavage solutions for UHPLC analysis

[0554] 20 pL of the corresponding reaction mixture (cleavage solution) were quenched in a 2 mL UHPLC vial containing 200 pL of NMM cooled to 5 °C (+-3 °C). After quenching, the sample was diluted with 1 mL DMSO / H2O 90:10 (V / V)

[0555] 1.1.4. General procedure for sample preparation of isolated crude peptides for UHPLC analysis

[0556] 2 mg of the corresponding crude peptide were dissolved in 1 mL DMSO / H2O 90:10 (V / V)

[0557] 1.1.5. UPLC Method C

[0558] Instrument: Waters Acquity UPLC connected to an Acquity TUV detector

[0559] Column: Acquity UPLC BEH C18 130A. 1.7 pm, 100 x 2.1 mm

[0560] Eluent A: 0.1% (VA / ) TFA in H2O

[0561] Eluent B: 0.1% (V / V) TFA in ACN

[0562] Flow rate: 0.45 mL / min

[0563] Column temperature: 50 °C

[0564] Gradient:

[0565] 1.1.6. General procedure 2 for sample preparation of cleavage solutions for UHPLC analysis Bachem Holding AG, 61 BAC75006PC1

[0566] Novo Nordisk A / S September 29, 2025

[0567] Sample preparation: 20 pL of the corresponding reaction mixture (cleavage solution) were quenched with 200 pL 7 M NH4OAC solution and diluted with 800 pL DMSO / water 90: 10 (VA / ) 1.2. General procedures for cleavage / global deprotection experiments

[0568] 1.2.1. General procedure A (for cleavage and global deprotection of resin-bound peptides): Bachem Holding AG, 62 BAC75006PC1

[0569] Novo Nordisk A / S September 29, 2025 Bachem Holding AG, 63 BAC75006PC1

[0570] Novo Nordisk A / S September 29, 2025 Bachem Holding AG, 64 BAC75006PC1

[0571] Novo Nordisk A / S September 29, 2025

[0572] 1.2.2. General procedure B (for cleavage and global deprotection of resin-bound peptides with isolation of the crude peptide) Bachem Holding AG, 65 BAC75006PC1

[0573] Novo Nordisk A / S September 29, 2025

[0574] 1.2.3. General procedure C (for cleavage and global deprotection of resin-bound peptides): 1.2.4. General procedure D (for global deprotection of soft cleaved model peptide II): Bachem Holding AG, 66 BAC75006PC1

[0575] Novo Nordisk A / S September 29, 2025

[0576] 2. Experiment 1 : Cleavage of model peptide II with various reagents

[0577] Model peptide II (H-Tyr(tBu)-Lys(Boc)-Arg(Pbf)-Glu(tBu)-Asp(OMpe)-Arg(Pbf)-Leu- Trp(Boc)-Pro-OH, which is the protected form of Tyr-Lys-Arg-Glu-Asp-Arg-Leu-Trp- Pro (SEQ ID NO: 1 ), was synthesized on 2-chloro-trityl (CTC) PS resin (loading 0.64 Bachem Holding AG, 67 BAC75006PC1

[0578] Novo Nordisk A / S September 29, 2025 mmol / g) using standard Fmoc sidechain protected amino acid derivatives. The synthesis was performed on a Sonata instrument on 60 mmol scale using DMF as solvent. Couplings were performed with Oxyma Pure™ and DIC, and Fmoc-removal was carried out with 20% (VA / ) piperidine in DMF using standard SPPS conditions.

[0579] Experiment 1a

[0580] Samples of the obtained resin-bound model peptide II were subjected to cleavage and the resulting cleavage solution was analyzed by UHPLC according to steps #1 and 2 of general procedure A (cf. para. 1.2.1 ), and UHPLC Method A (cf. paras. 1.1.1 and 1.1.3). The composition of the cleavage solutions and reaction conditions used are indicated in Table 1 a.

[0581] Fig. 1 shows an overlay of three chromatograms of the cleavage solutions obtained, namely for the experiment reported in entries 1.2, 1.6, and 1.10 of Table 1 a. One can see that the fully deprotected peptide (aka. FLP, asterisk) was resolved from incompletely deprotected peptide carrying one or two Pbf protecting groups and from the aspartimide by-product. Aspartimide formation can occur during piperidine- mediated N-a-Fmoc cleavage and in the presence of TFA during global deprotection I release. By using suitable Asp sidechain protecting groups and mild standard cleavage conditions like concentrated TFA at ambient temperature for 2 h, the extent of aspartimide formation can be kept minimal. The difference of aspartimide formation observed and aspartimide formation after TFA standard conditions can therefore be attributed to the effect of a cleavage cocktail at indicated temperature and time. In order to quantitatively compare the results of the different experiments reported in Table 1 a, the peak areas were determined and the amount of aspartimide (Asi) was expressed as the peak area of the aspartimide peak divided by the sum of the area of the aspartimide peak plus the area of the FLP peak (cf. Table 1 a, col. 5). This reflects the fact that the sum of both species is a good approximation of the total amount of peptide cleaved, at least for reasonably complete cleavages. Moreover, the extinction coefficients of FLP and the aspartimide at 220 nm can be approximated to be the same, due to their similar chemical structures. Similarly, the amount of FLP with one residual Pbf group (1 Pbf- FLP) was expressed as the peak area of the 1 Pbf-FLP peaks divided by the sum of the area of the aspartimide peak plus the area of the FLP peak (cf. Table 1 a, col. 6: “1 Pbf-FLP / (FLP+Asi)”); and the amount of FLP with two residual Pbf groups (2Pbf- Bachem Holding AG, 68 BAC75006PC1

[0582] Novo Nordisk A / S September 29, 2025

[0583] FLP) was expressed as the peak area of the 2Pbf-FLP peak divided by the sum of the area of the aspartimide peak plus the area of the FLP peak (of. Table 1 a, col. 7: “2Pbf-FLP / (FLP+Asi)”). This way, the amount of each of the three unwanted cleavage products Asi, 1 Pbf-FLP, and 2Pbf-FLP formed in the various experiments can be compared to between experiments. In other words: One can conclude based on Table 1 a, that, e.g., the amount of aspartimide formed in experiment #1 .3 (using neat TFA with 2.5% H2O and 5% EDT as scavengers for 30 min at 70 °C) was similar to the amount of aspartimide formed in experiment #1.10 (using neat TCA with 2.5% H2O and 5% EDT). However, as the extinction coefficients of Asi, 1 Pbf- FLP, and 2Pbf-FLP at 220 nm are expected to differ from each other, the values between each of the columns cannot be directly compared. In other words: It would not be accurate to conclude based on entry 1 .7 of Table 1 a only that less 1 Pbf-FLP was formed compared to 2Pbf-FLP in this experiment. Table 1a: Cleavage of model peptide II from CTC resin with various reagents Bachem Holding AG, 69 BAC75006PC1

[0584] Novo Nordisk A / S September 29, 2025 Bachem Holding AG, 70 BAC75006PC1

[0585] Novo Nordisk A / S September 29, 2025 Bachem Holding AG, 71 BAC75006PC1

[0586] Novo Nordisk A / S September 29, 2025

[0587] 1’TCA concentration in cleavage cocktail ca. 9.3 M (9.22 M or 9.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0588] 2’TCA concentration in cleavage cocktail ca. 7.3 M (7.25 M or 7.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively) Bachem Holding AG, 72 BAC75006PC1

[0589] Novo Nordisk A / S September 29, 2025

[0590] 3)TCA concentration in cleavage cocktail ca.9.3 M (9.23 M or 9.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0591] 4)TCA concentration in cleavage cocktail ca. 7.7 M (7.66 M or 7.8 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0592] #Entry 1 .23: Additional kinetics experiments with soft-cleaved, protected peptide showed that the reaction is incomplete after 5 h at 40 °C (data not shown).

[0593] $ Value not available, as peak overlaps with signal from solvent and cannot be integrated.

[0594] * Entry 1.15: The mixture of TCA and Phenol in the presence of peptide resin remained solid at RT; after the specified reaction duration the whole mixture was solubilized in 1 mL ACN, filtered and the UHPLC sample prepared as described in General procedure for sample preparation of cleavage solutions for UHPLC analysis (cf. para. 1.1.3) and analyzed using UHPLC Method A (cf. para. 1.1.1 ). It cannot be ascertained whether the cleavage observed occurred during incubation or during sample work-up in solution.a)reference exampleb)comparative example

[0595] As a reference, cleavage and deprotection of peptide II was tested with TFA at room temperature (experiments # 1.1 , 1.2) and found to give complete deprotection and low levels of aspartimide formation. If the TFA deprotection was performed at 70 °C, slightly higher extent of Asi formation was observed (experiment # 1 .3).

[0596] The DCA containing reagent (experiment # 1.9) required prolonged heating at elevated temperature for full deprotection but resulted in formation of too much aspartimide. The deprotection could be accelerated by addition of MSA in 1 M concentration, giving complete deprotection and an acceptable aspartimide content (experiment # 1 .8).

[0597] Higher MSA concentrations were tested in a variety of solvents (experiments # 1 .4 - 1.7) resulted in complete or well advanced deprotection and high degree of aspartimide formation.

[0598] TCA with either EDT / H2O or phenol as additive allowed at 70 °C for 30 min complete deprotection and to keep aspartimide formation on an acceptable level (experiments # 1.10, 1.14). Dilution of TCA with suitable solvents (experiments # 1.12, 1.13; 1 .26 to 1.38, 1.57, 1.59) allowed advanced deprotection again with low concomitant Bachem Holding AG, 73 BAC75006PC1

[0599] Novo Nordisk A / S September 29, 2025 aspartimide formation at lower temperature. However, TCA with phenol at room temperature resulted at most in far incomplete deprotection of the peptide, even after 24 hours (experiment # 1.15).

[0600] TCA containing mixtures and MSA in DCA were therefore further investigated. In a first step, it was examined whether peptide could be isolated from the cleavage solutions and the total yield of target peptide II was determined.

[0601] Experiment 1b

[0602] Samples of the obtained resin-bound model peptide II were subjected to cleavage and the peptide in the resulting cleavage solution was isolated according to general procedure B (cf. para. 1.2.2), and UHPLC Method A (cf. paras. 1.1.1 and 1.1.4). The composition of the cleavage solutions and reaction conditions used were as indicated in Table 1 b. Table 1b: Cleavage of model peptide II from CTC resin and isolation of crude peptide by precipitation Bachem Holding AG, 74 BAC75006PC1

[0603] Novo Nordisk A / S September 29, 2025 Bachem Holding AG, 75 BAC75006PC1

[0604] Novo Nordisk A / S September 29, 2025 Bachem Holding AG, 76 BAC75006PC1

[0605] Novo Nordisk A / S September 29, 2025

[0606] 1’TCA concentration in cleavage cocktail ca. 9.3 M (9.22 M or 9.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0607] 2’TCA concentration in cleavage cocktail ca. 7.3 M (7.25 M or 7.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0608] 3’TCA concentration in cleavage cocktail ca.9.3 M (9.23 M or 9.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0609] 4)TCA concentration in cleavage cocktail ca. 7.7 M (7.66 M or 7.8 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)a)reference exampleb)comparative example

[0610] The total dried precipitate obtained by general procedure B (cf. para. 1 .2.2) is herein referred to as “crude peptide”. The total mass of this precipitate is indicated in column 6 of Table 1 b (“crude peptide mass [mg]”). The purity of this precipitate as determined by UHPLC method A is indicated as the percentage of the area of the product peptide peak divided by the total area of all peaks observed in column 5 of Table 1 b (“crude peptide purity [%]”).

[0611] Column 7 of Table 1 b gives an estimate of the total peptide contained in the crude peptide, i.e. of the mass of target peptide and peptide impurities contained in the Bachem Holding AG, 77 BAC75006PC1

[0612] Novo Nordisk A / S September 29, 2025 crude peptide. This value was determined based on the nitrogen content of the precipitate determined by elemental analysis: Total peptide mass [mg] = (peptide content determined by elemental analysis [%]) / 100*(crude peptide mass [mg]).

[0613] Column 8 of Table 1 b gives an estimate of the total target peptide contained in the crude peptide, i.e. of the mass of full length, deprotected peptide II. This value was calculated as: Target peptide mass [mg] = crude peptide purity [%] / 100 * total peptide mass [mg].

[0614] It was concluded that, while TFA cleavage set the standard in terms of crude peptide purity and target peptide mass isolated, TCA stood out amongst the alternative cleavage reagents. In particular, cleavage reagents comprising TCA in either nitroalkyl solvents, thioether or aromatic thiol solvents gave attractive results at conditions amenable to large scale manufacturing (i.e. 40 °C, for either 4, 5, or 16 h or for 20 h at 25 °C). The mass of target peptide isolated using some of these TCA cleavage reagents was comparable to that obtained using the standard TFA reagent (cf. entries 1.45-1.49, 1.53, 1.58, 1.60).

[0615] 3. Experiment 2: Cleavage of model peptide I with various reagents

[0616] Model peptide I H-Tyr(tBu)-Lys(Boc)-Arg(Pbf)-Glu(tBu)-Asn(Trt)-Arg(Pbf)-Leu- Trp(Boc)-Pro-OH, which is the protected form of Tyr-Lys-Arg-Glu-Asn-Arg-Leu-Trp- Pro (SEQ ID NO: 2), was synthesized on 2-chloro-trityl (CTC) PS resin (loading 0.60 mmol / g) using standard Fmoc sidechain protected amino acid derivatives. The synthesis was performed on a Sonata instrument on 30 mmol scale using DMF as solvent. Couplings were performed with Oxyma Pure™ and DIC, and Fmoc-removal was carried out with 20% (VA / ) piperidine in DMF using standard SPPS conditions.

[0617] Experiment 2a

[0618] Samples of the obtained resin-bound model peptide I were subjected to cleavage and the resulting cleavage solution was analyzed by UHPLC according to steps #1 and 2 of general procedure A (cf. para. 1.2.1 ), and UHPLC Method A (cf. paras. 1.1.1 and 1.1.3). The composition of the cleavage solutions used, and reaction conditions used were as indicated in Table 2a.

[0619] Fig. 2 shows an overlay of three chromatograms of the cleavage solutions obtained, namely for the experiment reported in entries 2.1 , 2.4, and 2.5 of Table 2a. The fully Bachem Holding AG, 78 BAC75006PC1

[0620] Novo Nordisk A / S September 29, 2025 deprotected peptide (aka. FLP, asterisk) could be resolved from the FLP containing an aspartic acid residue instead of asparagine (cf. Fig 12), which is formed by hydrolysis of the asparagine residue, and from incompletely deprotected peptide species carrying one or two Pbf protecting groups. In order to quantitatively compare the results of the different experiments reported in Table 2a, the peak areas were determined, and the amount of FLP-aspartic acid (“Asp”) was expressed as the peak area of the Asp peak divided by the sum of the area of the “Asp” peak plus the area of the FLP peak (cf. Table 2a, col. 5: ““Asp7(“Asp”+FLP)”). This reflects the fact that the sum of FLP-aspartic acid plus FLP is a good approximation of the total amount of peptide cleaved, at least for reasonably complete cleavages. Moreover, the extinction coefficients of FLP and the FLP-aspartic acid at 220 nm can be approximated to be the same, due to their similar chemical structures. Similarly, the amount of FLP with one residual Pbf group (1 Pbf-FLP) was expressed as the peak area of the 1 Pbf-FLP peaks divided by the sum of the area of the “Asp” peak plus the area of the FLP peak (cf. Table 2a, col. 6: “1 Pbf-FLP / (FLP+“Asp”)”); and the amount of FLP with two residual Pbf groups (2Pbf-FLP) was expressed as the peak area of the 2Pbf-FLP peak divided by the sum of the area of the “Asp” peak plus the area of the FLP peak (cf. Table 2a, col. 7: “2Pbf-FLP / (FLP+“Asp”)”). This way, the amount of each of the three unwanted cleavage products “Asp”, 1 Pbf-FLP, and 2Pbf-FLP formed in the various experiments can be compared between experiments. For example, one can conclude based on Table 2a, that, e.g., the amount of “Asp” (i.e., FLP-aspartic acid) formed in experiment # 2.1 (using neat TFA with 5% H2O and 5% EDT as scavengers for 2 h at rt) was only slightly lower than the amount of “Asp” formed in experiment # 2.5 (using neat TCA with 2.5% H2O and 5% EDT for 30 min at 70 °C). However, as the extinction coefficients of “Asp”, 1 Pbf- FLP, and 2Pbf-FLP at 220 nm are expected to differ from each other, the values between each of the columns cannot be compared within the same line directly and without corrections, analogous to the explanations provided in experiment 1 .

[0621] Table 2a: Cleavage of model peptide from CTC resin with various reagents Bachem Holding AG, 79 BAC75006PC1

[0622] Novo Nordisk A / S September 29, 2025

[0623] 1)TCA concentration in cleavage cocktail ca. 9.3 M (9.22 M or 9.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0624] 2)TCA concentration in cleavage cocktail ca. 7.7 M (7.66 M or 7.8 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)3)TCA concentration in cleavage cocktail ca. 7.3 M (7.25 M or 7.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)a)reference exampleb)comparative example Bachem Holding AG, 80 BAC75006PC1

[0625] Novo Nordisk A / S September 29, 2025

[0626] It was concluded that the conditions of experiments # 2.2 to # 2.8 gave a low, acceptable degree of asparagine deamidation, confirming MSA and TCA as suitable cleavage reagents for model peptide I.

[0627] Experiment 2b

[0628] Samples of the obtained resin-bound model peptide I were subjected to cleavage and the peptide in the resulting cleavage solution was isolated according to general procedure B (cf. para. 1.2.2), and UHPLC Method A (cf. paras. 1.1.1 and 1.1.4). The composition of the cleavage solutions and reaction conditions used were as indicated in Table 2b.

[0629] The total dried precipitate obtained by general procedure B (cf. para. 1 .2.2) is herein referred to as “crude peptide”. The total mass of this precipitate is indicated in column 5 of Table 2b (“crude peptide mass [mg]”). The purity of this precipitate as determined by UHPLC method A is indicated as the percentage of the area of the product peptide peak divided by the total area of all peaks observed in column 4 of Table 2b (“crude peptide purity [%]”).

[0630] Column 6 of Table 2b gives an estimate of the total peptide contained in the crude peptide, i.e. of the mass of target peptide and peptide impurities contained in the crude peptide. This value was determined based on the nitrogen content of the precipitate determined by elemental analysis: Total peptide mass [mg] = (peptide content determined by elemental analysis [%]) / 100*(crude peptide mass [mg]).

[0631] Column 7 of Table 2b gives an estimate of the total target peptide contained in the crude peptide, i.e. of the mass of full length, deprotected peptide I. This value was calculated as: Target peptide mass [mg] = crude peptide purity [%] / 100 * total peptide mass [mg].

[0632] Table 2b: Cleavage of model peptide I from CTC resin and isolation of crude peptide by precipitation Bachem Holding AG, 81 BAC75006PC1

[0633] Novo Nordisk A / S September 29, 2025 a)Scavenger: 5% EDT, 2.5% H2Ob)comparative examplec)reference example

[0634] It was concluded that TCA allowed to obtain up to 85% of the target peptide mass obtained with the standard TFA cleavage reagent.

[0635] 4. Experiment 3: Cleavage of model peptide III with various reagents

[0636] Model peptide III H-Tyr(tBu)-Lys(Boc)-Arg(Pbf)-Glu(tBu)-Asn(Trt)-Arg(Pbf)-Leu- Trp(Boc)-Pro-NH2, which is the protected form of Tyr-Lys-Arg-Glu-Asn-Arg-Leu-Trp- Pro-NH2(SEQ ID NO: 3), was synthesized on tricyclic amide linker resin (DL-form) (loading 0.64 mmol / g) using standard Fmoc sidechain protected amino acid derivatives. The synthesis was performed on a Sonata instrument on 60 mmol scale using DMF as solvent. Couplings were performed with Oxyma Pure™ and DIC, and Fmoc-removal was carried out with 20% (V / V) piperidine in DMF using standard SPPS conditions.

[0637] Example 3a

[0638] Samples of the obtained resin-bound model peptide III were subjected to cleavage and the resulting cleavage solution was analyzed by UHPLC according to steps #1 and 2 of general procedure A (cf. para. 1.2.1 ), and UHPLC Method A (cf. paras. Bachem Holding AG, 82 BAC75006PC1

[0639] Novo Nordisk A / S September 29, 2025

[0640] 1.1.1 and 1.1.3). The composition of the cleavage solutions used, and reaction conditions used were indicated in Table 3a.

[0641] Fig. 3 shows an overlay of two chromatograms of the cleavage solutions obtained, namely for the experiments reported in entries 3.3 and 3.6 of Table 3a. Due to peak tailing, the fully deprotected peptide (aka. FLP, asterisk) could not be resolved from the FLP containing an aspartic acid residue instead of asparagine (“Asp”) in the case of model peptide III. Qualitatively, however, very little “Asp”, if any, appeared to be formed. By contrast, incompletely deprotected peptide species carrying one or two Pbf protecting groups could be clearly identified. In order to quantitatively compare the results of the different experiments reported in Table 3a, the peak areas were determined and the amount of FLP with one residual Pbf group (1 Pbf- FLP) was expressed as the peak area of the 1 Pbf-FLP peaks divided by the area of the FLP peak (cf. Table 3a, col. 5: “1 Pbf-FLPZ(FLP)”). Similarly, the amount of FLP with two residual Pbf groups (2Pbf-FLP) was expressed as the peak area of the 2Pbf-FLP peak divided by the area of the FLP peak (cf. Table 3a, col. 6: “2Pbf- FLP / (FLP)”). This way, the amount each of the unwanted cleavage products 1 Pbf- FLP and 2Pbf-FLP formed in the various experiments can be compared between experiments. In other words: One can conclude based on Table 3a, that, e.g., the amount 1 Pbf-FLP formed in experiment # 3.1 (using neat TFA with 5% H2O and 5% EDT as scavengers for 3 h at RT) was lower than the amount of 1 Pbf-FLP formed in experiment # 3.6 (using neat TCA with 2.5% H2O and 5% EDT). However, as the extinction coefficients of 1 Pbf-FLP and 2Pbf-FLP at 220 nm are expected to differ from each other, the values between each of the columns cannot be compared within the same line directly and without corrections, analogous to the explanations provided in experiment 1 .

[0642] Table 3a: Cleavage of model peptide III from Tricyclic amide resin with various reagents Bachem Holding AG, 83 BAC75006PC1

[0643] Novo Nordisk A / S September 29, 2025 Bachem Holding AG, 84 BAC75006PC1

[0644] Novo Nordisk A / S September 29, 2025

[0645] # value not available, as peak overlaps with signal from solvent and cannot be integrated.

[0646] 1)TCA concentration in cleavage cocktail ca. 9.3 M (9.22 M or 9.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0647] 2)TCA concentration in cleavage cocktail ca. 7.3 M (7.25 M or 7.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)a)reference exampleb)comparative example

[0648] It was concluded that also on the more difficult to cleave Tricyclic amide resin, solutions of MSA (entries 3.2 and 3.3) as well as TCA (entry 3.6) gave well advanced or complete deprotection. Cleavage reagents comprising TCA in thiophenol or thioanisole gave such results under conditions amenable to large scale manufacturing (40 °C, for 5 h or 25 °C for 20 h).

[0649] Experiment 3b

[0650] Samples of the obtained resin-bound model peptide III were subjected to cleavage and the peptide in the resulting cleavage solution was isolated according to general procedure B (cf. para. 1.2.2), and UHPLC Method A (cf. paras. 1.1.1 and 1.1.4). The composition of the cleavage solutions and reaction conditions used were as indicated in Table 3b.

[0651] The total dried precipitate obtained by general procedure B (cf. para. 1 .2.2) is herein referred to as “crude peptide”. The total mass of this precipitate is indicated in column 5 of Table 3b (“crude peptide mass [mg]”). The purity of this precipitate as determined by UHPLC method A is indicated as the percentage of the area of the product peptide peak divided by the total area of all peaks observed in column 4 of Table 3b (“crude peptide purity [%]”).

[0652] Column 6 of Table 3b gives an estimate of the total peptide contained in the crude peptide, i.e. of the mass of target peptide and peptide impurities contained in the crude peptide. This value was determined based on the nitrogen content of the precipitate determined by elemental analysis: Total peptide mass [mg] = (peptide content determined by elemental analysis [%]) / 100*(crude peptide mass [mg]).

[0653] Column 7 of Table 3b gives an estimate of the total target peptide contained in the crude peptide, i.e. of the mass of full length, deprotected peptide III. This value was Bachem Holding AG, 85 BAC75006PC1

[0654] Novo Nordisk A / S September 29, 2025 calculated as: Target peptide mass [mg] = crude peptide purity [%] / 100 * total peptide mass [mg].

[0655] Table 3b: Cleavage of model peptide III from Tricyclic amide resin and isolation of crude peptide by precipitation a)Scavenger: 5% EDT, 2.5% H2O;

[0656] 1)TCA concentration in cleavage cocktail ca. 9.3 M (9.22 M or 9.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0657] 2)TCA concentration in cleavage cocktail ca. 7.3 M (7.25 M or 7.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)b)comparative examplec)reference example Bachem Holding AG, 86 BAC75006PC1

[0658] Novo Nordisk A / S September 29, 2025

[0659] The mass of target peptide isolated using some of the TCA cleavage reagents were comparable to that obtained using the standard TFA reagent.

[0660] 5. Experiment 4: Cleavage of model peptide VII with various reagents

[0661] Model peptide VII H-Tyr(tBu)-Lys(Boc)-Arg(Pbf)-Glu(tBu)-Asn(Trt)-Arg(Pbf)-Leu- Trp(Boc)-Thr(tBu)-Ser(tBu)-Pro-OH, which is the protected form of Tyr-Lys-Arg- Glu-Asn-Arg-Leu-Trp-Thr-Ser-Pro (SEQ ID NO: 4), was synthesized on 2-chloro- trityl (CTC) resin (loading 0.64 mmol / g) using standard Fmoc sidechain protected amino acid derivatives. The synthesis was performed on a Sonata instrument on 60 mmol scale using DMF as solvent. Couplings were performed with Oxyma Pure™ and DIC, and Fmoc-removal was carried out with 20% (VA / ) Piperidine in DMF using standard SPPS conditions.

[0662] Experiment 4a

[0663] Samples of the obtained resin-bound model peptide VII were subjected to cleavage and the resulting cleavage solution was analyzed by UHPLC according to steps #1 to #6 of general procedure A (cf. para. 1 .2.1 ), and UHPLC Method A (cf. paras. 1.1.1 and 1.1.3). The composition of the cleavage solutions used, and reaction conditions used were as indicated in Table 4a.

[0664] Fig. 4 shows an overlay of two chromatograms of the cleavage solutions obtained, namely for the experiment reported in entries 4.2 and 4.4 of Table 4a. The fully deprotected peptide (aka. FLP, asterisk) was resolved from incompletely deprotected peptide species carrying one or two Pbf protecting groups. No product of asparagine deamidation -i.e., no FLP-aspartic acid (“Asp”)- could be detected in any of the samples. In order to quantitatively compare the results of the different experiments reported in Table 4a, the peak areas were determined and the amount of FLP with one residual Pbf group (1 Pbf-FLP) was expressed as the peak area of the 1 Pbf-FLP peaks divided by the area of the FLP peak (cf. Table 4a, col. 6: “1 Pbf- FLP / (FLP)”); and the amount of FLP with two residual Pbf groups (2Pbf-FLP) was expressed as the peak area of the 2Pbf-FLP peak divided by the area of the FLP peak (cf. Table 4a, col. 7: “2Pbf-FLP / (FLP)”). This way, the amount each of the unwanted cleavage products 1 Pbf-FLP and 2Pbf-FLP formed in the various experiments can be compared between experiments. Column 5 of Table 4a further Bachem Holding AG, 87 BAC75006PC1

[0665] Novo Nordisk A / S September 29, 2025 gives a qualitative indication of the amount of FLP found in the second, TFA mediated cleavage. This reflects the completeness of the first cleavage reaction: Any peptide not cleaved in the first cleavage reaction would have to be observed in the second, TFA mediated cleavage.

[0666] Table 4a: Cleavage of model peptide VII from CTC resin with various reagents Bachem Holding AG, 88 BAC75006PC1

[0667] Novo Nordisk A / S September 29, 2025

[0668] 1)TCA concentration in cleavage cocktail ca. 9.3 M (9.22 M or 9.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0669] 2)TCA concentration in cleavage cocktail ca. 7.3 M (7.25 M or 7.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)a)reference exampleb)comparative example

[0670] Both TCA and MSA containing cocktails allowed essentially complete cleavage of peptide from resin. TCA allowed clean formation of FLP (entry 4.2; Fig. 4 B) whereas the presence of additional signals using MSA cocktails (entry 4.4; Fig 4 A) suggested peptide degradation. This conclusion was corroborated by peptide isolation.

[0671] Experiment 4b

[0672] Samples of the obtained resin-bound model peptide VII were subjected to cleavage and the peptide in the resulting cleavage solution was isolated according to general procedure B (cf. para. 1.2.2), and UHPLC Method A (cf. paras. 1.1.1 and 1.1.4). The corresponding chromatograms for experiments #4.6. and 4.7 are shown in Fig. 5. The composition of the cleavage solutions and reaction conditions used were as indicated in Table 4b.

[0673] The total dried precipitate obtained by general procedure B (cf. para. 1 .2.2) is herein referred to as “crude peptide”. The total mass of this precipitate is indicated in column 5 of Table 4b (“crude peptide mass [mg]”). The purity of this precipitate as determined by UHPLC method A is indicated as the percentage of the area of the product peptide peak divided by the total area of all peaks observed in column 4 of Table 4b (“crude peptide purity [%]”).

[0674] Column 6 of Table 4b gives an estimate of the total peptide contained in the crude peptide, i.e. of the mass of target peptide and peptide impurities contained in the crude peptide. This value was determined based on the nitrogen content of the Bachem Holding AG, 89 BAC75006PC1

[0675] Novo Nordisk A / S September 29, 2025 precipitate determined by elemental analysis: Total peptide mass [mg] = (peptide content determined by elemental analysis [%]) / 100*(crude peptide mass [mg]).

[0676] Column 7 of Table 4b gives an estimate of the total target peptide contained in the crude peptide, i.e. of the mass of full length, deprotected peptide VII. This value was calculated as: Target peptide mass [mg] = crude peptide purity [%] / 100 * total peptide mass [mg].

[0677] It was concluded that TCA (neat) allowed to obtain up to 76% of the target peptide mass obtained with the standard TFA cleavage reagent. Combinations of TCA and thioanisoles allowed to obtain up to 91 % of the target peptide mass obtained with the standard TFA cleavage reagent. By contrast, only 13% of the target peptide mass could be obtained with 1 M MSA in DCA, confirming the suspicion of target peptide degradation by this reagent.

[0678] Table 4b: Cleavage of model peptide VII from CTC resin and isolation of crude peptide by precipitation Bachem Holding AG, 90 BAC75006PC1

[0679] Novo Nordisk A / S September 29, 2025 a)Scavenger: 5% EDT, 2.5% H2Ob)Scavenger: 3% DTT, 3% H2O, 3% TIS

[0680] 1)TCA concentration in cleavage cocktail ca. 9.3 M (9.22 M or 9.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0681] 2)TCA concentration in cleavage cocktail ca. 7.3 M (7.25 M or 7.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)c)comparative exampled)reference example

[0682] 6. Experiment 5: Cleavage of Dasiglucagon from CTC resin

[0683] Dasiglucagon, His-Ser-GIn-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp- Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr (SEQ ID NO: 5), was synthesized on 2-chloro-trityl (CTC) resin (loading 0.42 mmol / g) using standard Fmoc sidechain protected amino acid derivatives. The synthesis was performed on a PeptiPilot instrument on 1.2 mmol scale using DMF as solvent. Couplings were performed with Oxyma PureTM / DIC / HOPO, and Fmoc-removal was carried out with 20% (V / V) piperidine in DMF using standard SPPS conditions.

[0684] Experiment 5a

[0685] Samples of the obtained resin-bound Dasiglucagon were subjected to cleavage and the resulting cleavage solution was analyzed by UHPLC according to steps #1 to #6 of general procedure A (cf. para. 1.2.1 ), and UHPLC Method B (cf. paras. 1.1.2 and 1.1.3).

[0686] Five parallel experiments were conducted with the cleavage conditions listed in Table 5a. Fig. 6 shows the chromatograms obtained for experiments # 5.1 -5.3. While the TCA containing cleavage reagent gave an overall cleavage pattern very Bachem Holding AG, 91 BAC75006PC1

[0687] Novo Nordisk A / S September 29, 2025 similar to the standard TFA mediated cleavage, 4M MSA in acetic acid appeared to lead to degradation of the peptide: Nearly no full-length peptide could be observed, but only a large variety of peaks most likely representing peptide degradation products.

[0688] Table 5a: Cleavage of Dasiglucagon from CTC resin with various reagents a)reference exampleb)comparative example Dasiglucagon was degraded during deprotection with MSA solutions as well as with DCA (experiments # 5.3 - 5.5). Surprisingly, TCA (experiment # 5.2) at 70 °C resulted in completed deprotection within 30 min, with an impurity profile well comparable (Fig. 6) to that obtained in deprotection with TFA (experiment # 5.1 ). Experiment 5b

[0689] Samples of the obtained resin-bound Dasiglucagon were subjected to cleavage and the peptide in the resulting cleavage solution was isolated according to general procedure B (cf. para. 1.2.2), and UHPLC Method B (cf. paras. 1.1.2 and 1.1.4). The Bachem Holding AG, 92 BAC75006PC1

[0690] Novo Nordisk A / S September 29, 2025 composition of the cleavage solutions and reaction conditions used were as indicated in Table 5b.

[0691] The total dried precipitate obtained by general procedure B (cf. para. 1 .2.2) is herein referred to as “crude peptide”. The total mass of this precipitate is indicated in column 5 of Table 5b (“crude peptide mass [mg]”). The purity of this precipitate as determined by UHPLC method B is indicated as the percentage of the area of the product peptide peak divided by the total area of all peaks observed in column 4 of Table 5b (“crude peptide purity [%]”).

[0692] Column 6 of Table 5b gives an estimate of the total peptide contained in the crude peptide, i.e. of the mass of target peptide and peptide impurities contained in the crude peptide. This value was determined based on the nitrogen content of the precipitate determined by elemental analysis: Total peptide mass [mg] = (peptide content determined by elemental analysis [%]) / 100*(crude peptide mass [mg]).

[0693] Column 7 of Table 5b gives an estimate of the total target peptide contained in the crude peptide, i.e. of the mass of full length, deprotected Dasiglucagon. This value was calculated as: Target peptide mass [mg] = crude peptide purity [%] / 100 * total peptide mass [mg].

[0694] It was concluded that TCA allowed to obtain up to 78% of the target peptide mass obtained with the standard TFA cleavage reagent.

[0695] Table 5b: Cleavage of Dasiglucagon from CTC resin and isolation of crude peptide by precipitation Bachem Holding AG, 93 BAC75006PC1

[0696] Novo Nordisk A / S September 29, 2025 a> Scavenger 5% EDT, 2.5% H2O

[0697] * Dasiglucagon resin batch 1 ; ** Dasiglucagon resin batch 2

[0698] 1)TCA concentration in cleavage cocktail ca. 9.3 M (9.22 M or 9.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)2)TCA concentration in cleavage cocktail ca. 7.3 M (7.25 M or 7.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)b)comparative examplec)reference example 7. Experiment 6: Cleavage of Glucagon from Wang resin

[0699] Glucagon, His-Ser-GIn-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-

[0700] Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr ((SEQ ID NO 6), was Bachem Holding AG, 94 BAC75006PC1

[0701] Novo Nordisk A / S September 29, 2025 synthesized on Wang resin (loading 0.61 mmol / g) using standard Fmoc sidechain protected amino acid derivatives. The synthesis was performed on a 150 L SPPS- Reactor instrument on 3 mol scale using DMF as solvent. Couplings were performed with Oxyma PureTM / DIC / DEPBT, and Fmoc-removal was carried out with 20% (V / V) piperidine in DMF using standard SPPS conditions.

[0702] Experiment 6a

[0703] Samples of the obtained resin-bound glucagon were subjected to cleavage and the resulting cleavage solution was analyzed by UHPLC according to steps #1 to #6 of general procedure A (cf. para. 1.2.1 ), and UHPLC Method B (cf. paras. 1.1.2 and 1.1.3).

[0704] Two parallel experiments were conducted with the cleavage conditions listed in Table 6a. Fig. 7 shows the chromatograms obtained. The TCA containing cleavage reagent gave an overall cleavage pattern very similar to the standard TFA mediated cleavage.

[0705] Table 6a: Cleavage of Glucagon from Wang resin with various reagents a)reference exampleb)comparative example

[0706] It was concluded from these results that TCA containing cleavage reagents resulted in the successful cleavage of the peptide when using Wang Linkers.

[0707] Experiment 6b:

[0708] Samples of the obtained resin-bound glucagon were subjected to cleavage and the peptide in the resulting cleavage solution was isolated according to general procedure B (cf. para. 1.2.2), and UHPLC Method B (cf. paras. 1.1.2 and 1.1.4). The Bachem Holding AG, 95 BAC75006PC1

[0709] Novo Nordisk A / S September 29, 2025 composition of the cleavage solutions and reaction conditions used were as indicated in Table 6b.

[0710] The total dried precipitate obtained by general procedure B (cf. para. 1 .2.2) is herein referred to as “crude peptide”. The total mass of this precipitate is indicated in column 5 of Table 6b (“crude peptide mass [mg]”). The purity of this precipitate as determined by UHPLC method B is indicated as the percentage of the area of the product peptide peak divided by the total area of all peaks observed in column 4 of Table 6b (“crude peptide purity [%]”).

[0711] Column 6 of Table 6b gives an estimate of the total peptide contained in the crude peptide, i.e. of the mass of target peptide and peptide impurities contained in the crude peptide. This value was determined based on the nitrogen content of the precipitate determined by elemental analysis: Total peptide mass [mg] = (peptide content determined by elemental analysis [%]) / 100*(crude peptide mass [mg]).

[0712] Column 7 of Table 6b gives an estimate of the total target peptide contained in the crude peptide, i.e. of the mass of full length, deprotected Glucagon. This value was calculated as: Target peptide mass [mg] = crude peptide purity [%] / 100 * total peptide mass [mg].

[0713] It was concluded that TCA allowed to obtain up to 68% of the target glucagon peptide mass obtained with the standard TFA cleavage reagent, showing that the TCA cleavage reagents of the present invention give acceptable yields with a difficult-to-cleave, sensitive peptide like glucagon.

[0714] Table 6b: Cleavage of glucagon from Wang resin and isolation of crude peptide by precipitation Bachem Holding AG, 96 BAC75006PC1

[0715] Novo Nordisk A / S September 29, 2025 a)Scavenger: 5% EDT, 2.5% H2Ob)Scavenger: 2.5% EDT, 2.5% H2O, 2.5% TIS

[0716] 1)TCA concentration in cleavage cocktail ca. 9.3 M (9.22 M or 9.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)2)TCA concentration in cleavage cocktail ca. 7.3 M (7.25 M or 7.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0717] 3)TCA concentration in cleavage cocktail ca. 7.3 M (7.25 M or 7.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)c)comparative exampled)reference example

[0718] 8. Experiment 7: Cleavage of 29-mer ABCEXT peptide from Rink amide resin PS resin

[0719] Model peptidyl-resin XX (H-Ala-Cys(Trt)-Asp(tBu)-Glu(tBu)-Phe-Gly-His(Trt)-lle- Lys(Boc)-Leu-Met-Asn(Trt)-Pro-Gln(Trt)-Arg(Pbf)-Ser(tBu)-Thr(tBu)-Val-Trp(Boc)-

[0720] Tyr(tBu)-Arg(Pbf)-Asp(tBu)-Thr(tBu)-Phe-Glu(tBu)-Ala-Asn(Trt)-Arg(Pbf)-Gln(Trt)- Bachem Holding AG, 97 BAC75006PC1

[0721] Novo Nordisk A / S September 29, 2025

[0722] Rink amide PS), which is the protected form of Ala-Cys-Asp-Glu-Phe-Gly-His-lle- Lys-Leu-Met-Asn-Pro-GIn-Arg-Ser-Thr-Val-Trp-Tyr-Arg-Asp-Thr-Phe-Glu-Ala-Asn- Arg-GIn (SEQ ID NO: 7), was synthesized on Rink amide PS resin (loading 0.74 mmol / g) using standard Fmoc sidechain protected amino acid derivatives. The synthesis was performed on a Symphony X instrument on 0.45 mmol scale using 30% DMSO in EtOAc (v / v) as solvent. Couplings were performed with Oxyma Pure™ and DIC, and Fmoc-removal was carried out with piperidine using standard SPPS conditions.

[0723] Samples of the obtained resin-bound peptide were subjected to cleavage either with TCA in nitromethane solution or with TFA according to general procedure C (cf. paras. 1 .2.3). The resulting solutions were analyzed by UHPLC according to UHPLC method C (cf. paras. 1.1.5 and 1.1.6). A comparison of the chromatograms obtained is depicted in Fig. 8.

[0724] 9. Experiment 8: Cleavage of 43-mer peptide from CTC PS resin

[0725] Model peptidyl-resin XXI (tBuO2C-(CH2)i6-CO-YGIu(tBu)-YGIu(tBu)-yGlu(tBu)- YGIu(tBu)-YGIu(tBu)-Gln(Trt)-Glu(tBu)-His(Trt)-Pro-Gln(Trt)-Ala-Arg(Pbf)-Lys(Boc)- Tyr(tBu)-Lys(Boc)-Gly-Ala-Gln(Trt)-Lys(Boc)-Lys(Boc)-Glu(tBu)-Leu-Ser(tBu)- Ser(4jPro)-Gly-Cys(Trt)-Phe-Gly-Leu-Pro-Leu-Asp(tBu)-Arg(Pbf)-lle-Gly- Ser(4jPro)-Leu-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-CTC-resin), which is the protected form of HO2C-(CH2)i6-CO-YGIu-YGIu-YGIu-YGIu-YGIu-Gln-Glu-His-Pro-Gln-Ala-Arg- Lys-Tyr-Lys-Gly-Ala-GIn-Lys-Lys-Glu-Leu-Ser-Ser-Gly-Cys-Phe-Gly-Leu-Pro-Leu- Asp-Arg-lle-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys (SEQ ID NO: 8), was synthesized on 2-chlorotrityl (CTC) PS resin (loading 0.75 mmol / g, pre-loaded with Cys(Trt)) using standard Fmoc sidechain protected amino acid derivatives. The synthesis was performed on a CS-BIO instrument on 40 mmol scale using DMF as solvent. Couplings were performed with Oxyma Pure™ and DIC, and Fmoc-removal was carried out with piperidine using standard SPPS conditions.

[0726] Samples of the obtained resin-bound peptide were subjected to cleavage either with TCA in nitromethane solution or with TFA according to general procedure C (cf. paras. 1 .2.3). The resulting solutions were analyzed by UHPLC according to UHPLC method C (cf. paras. 1 .1 .5 and 1.1.6). A comparison of the chromatograms obtained is depicted in Fig. 9. Bachem Holding AG, 98 BAC75006PC1

[0727] Novo Nordisk A / S September 29, 2025

[0728] 10. Experiment 9: Cleavage of Dasiglucagon amide from Rink amide resin PS resin

[0729] Model peptidyl-resin XXII (H-His(Trt)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)- Ser(tBu)-Asp(tBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(tBu)-Aib-Ala- Arg(Pbf)-Ala-Glu(tBu)-Glu(tBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Glu(tBu)-Ser(tBu)- Thr(tBu)-Rink amide PS), which is the protected form of His-Ser-GIn-Gly-Thr-Phe- Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp- Leu-Glu-Ser-Thr-NH2 (SEQ ID NO: 9), was synthesized on Rink amide PS resin (loading 0.74 mmol / g) using standard Fmoc sidechain protected amino acid derivatives. The synthesis was performed on a CS-BIO instrument on 5 mmol scale using DMF as solvent. Couplings were performed with Oxyma Pure™ and DIC, and Fmoc-removal was carried out with piperidine using standard SPPS conditions.

[0730] Samples of the obtained resin-bound peptide were subjected to cleavage either with TCA in nitromethane solution or with TFA according to general procedure C (cf. paras. 1 .2.3). The resulting solutions were analyzed by UHPLC according to UHPLC method C (cf. paras. 1 .1 .5 and 1.1.6). A comparison of the chromatograms obtained is depicted in Fig. 10.

[0731] 11. Experiment 10: Cleavage of PYY3.36 peptide from Rink amide resin PS resin Model peptidyl-resin XXIII (H-lle-Lys(Boc)-Pro-Glu(tBu)-Ala-Pro-Gly-Glu(tBu)- Asp(tBu)-Ala-Ser(tBu)-Pro-Glu(tBu)-Glu(tBu)-Leu-Asn(Trt)-Arg(Pbf)-Tyr(tBu)- Tyr(tBu)-Ala-Ser(tBu)-Leu-Arg(Pbf)-His(Trt)-Tyr(tBu)-Leu-Asn(Trt)-Leu-Val- Tyr(tBu)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Tyr(tBu)-Rink amide PS), which is the protected form of lle-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg- Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Tyr-Arg-Gln-Arg-Tyr-NH2 (SEQ ID NO: 10), was synthesized on Rink amide PS resin (loading 0.74 mmol / g) using standard Fmoc sidechain protected amino acid derivatives. The synthesis was performed on a Symphony X instrument on 0.45 mmol scale using 30% DMSO in EtOAc (v / v) as solvent. Couplings were performed with Oxyma Pure™ and DIC, and Fmoc-removal was carried out with piperidine using standard SPPS conditions.

[0732] Samples of the obtained resin-bound peptide were subjected to cleavage either with TCA in nitromethane solution or with TFA according to general procedure C (cf. paras. 1 .2.3). The resulting solutions were analyzed by UHPLC according to UHPLC Bachem Holding AG, 99 BAC75006PC1

[0733] Novo Nordisk A / S September 29, 2025 method C (of. paras. 1.1.5 and 1 .1 .6). A comparison of the chromatograms obtained is depicted in Fig. 11 .

[0734] 12. Experiment 11 : Kinetics of cleavage reaction - influence of solvent

[0735] Model peptide II (H-Tyr(tBu)-Lys(Boc)-Arg(Pbf)-Glu(tBu)-Asp(OMpe)-Arg(Pbf)-Leu- Trp(Boc)-Pro-OH, which is the protected form of Tyr-Lys-Arg-Glu-Asp-Arg-Leu-Trp- Pro (SEQ ID NO: 1 ), was synthesized on 2-chloro-trityl (CTC) PS resin (loading 0.64 mmol / g) using standard Fmoc sidechain protected amino acid derivatives. The synthesis was performed on a Sonata instrument on 60 mmol scale using DMF as solvent. Couplings were performed with Oxyma Pure™ and DIC, and Fmoc-removal was carried out with 20% (V / V) Piperidine in DMF using standard SPPS conditions. The peptide was cleaved from the resin with 1 .0% TFA in DCM (VA / ) to obtain soft cleaved peptide II, i.e. free peptide II with side chain protecting groups. Isolated, soft cleaved peptide II was subjected to global deprotection and the resulting cleavage solution was analyzed by UHPLC according to steps #1 to #3 of general procedure D (cf. para. 1.2.4). The composition of the cleavage solutions and reaction conditions used are indicated in Table 11 .

[0736] To quantitatively compare the results of the different experiments reported in Table 11 , the peak areas were determined and the amount of aspartimide (Asi) was expressed as the peak area of the aspartimide peak divided by the sum of the area of the aspartimide peak plus the area of the FLP peak (cf. Table 11 , col. 3). This reflects the fact that the sum of both species is a good approximation of the total amount of peptide cleaved, at least for reasonably complete cleavages. Moreover, the extinction coefficients of FLP and the aspartimide at 220 nm can be approximated to be the same, due to their similar chemical structures. Similarly, the amount of FLP with one residual Pbf group (1 Pbf-FLP) was expressed as the peak area of the 1 Pbf-FLP peaks divided by the sum of the area of the aspartimide peak plus the area of the FLP peak (cf. Table 11 , col. 4: “1 Pbf-FLP / (FLP+Asi)”); and the amount of FLP with two residual Pbf groups (2Pbf-FLP) was expressed as the peak area of the 2Pbf-FLP peak divided by the sum of the area of the aspartimide peak plus the area of the FLP peak (cf. Table 11 , col. 5: “2Pbf-FLP / (FLP+Asi)”).

[0737] This way, the amount of each of the three unwanted cleavage products Asi, 1 Pbf- FLP, and 2Pbf-FLP formed in the various experiments can be compared to between Bachem Holding AG, 100 BAC75006PC1

[0738] Novo Nordisk A / S September 29, 2025 experiments. In other words: One can conclude based on Table 11 , that, e.g., the amount of aspartimide formed in experiment #11.1 (using neat TFA with 5% H2O and 5% EDT as scavengers) was similar to the amount of aspartimide formed in experiment #11.5 (using TCA and 2-chlorothioanisole (8 g / 1 mL) with 2.5% H2O and 5% EDT).

[0739] Completion of cleavage was determined by visual inspection of the UHPLC traces. Virtual disappearance of the signals attributed to the 2Pbf-FLP and 1 Pbf-FLP intermediates suggested complete conversion.

[0740] Table 11: Cleavage reaction at 25 °C with cleavage cocktails containing different solvents Bachem Holding AG, 101 BAC75006PC1

[0741] Novo Nordisk A / S September 29, 2025

[0742] 1)TCA concentration in cleavage cocktail ca. 7.3 M (7.25 M or 7.4 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0743] 2)TCA concentration in cleavage cocktail ca. 6.6 M (6.55 M or 6.6 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)

[0744] 3)TCA concentration in cleavage cocktail ca. 7.7 M (7.66 M or 7.8 M, calculated with a density of 1 .629 g / mL or 1 .66 g / mL, respectively)a)reference exampleb)comparative examplec)values after 20 h

[0745] Cleavage cocktail for sample 11.1 contained 5% H2O and 5% EDT; cleavage was performed for 2 h at 25 °C.

[0746] Cleavage cocktails for samples 11.2, 11 .3, and 11 .6-11.10 contained 2.5% H2O and 5% EDT; cleavage was performed for 20 h at 25 °C; and continued for samples 11.6-11.10.

[0747] Cleavage cocktails for samples 11.4 and 11.5 contained 2.5% H2O and 5% EDT; cleavage was performed for 10 h at 25 °C.

[0748] It was concluded that the cleavage kinetics for cleavage cocktails made with 4 to 8 g of TCA per mL of thioanisole or 2-chlorothioanisole were surprisingly fast. Those Bachem Holding AG, 102 BAC75006PC1

[0749] Novo Nordisk A / S September 29, 2025 cocktails allowed for essentially complete cleavage with rather low aspartimide formation after 10 to 20 hours at 25 °C (cf. samples 11 .2-11 .5).

[0750] By contrast, cleavage cocktails made with 6 g of TCA per mL of other common solvents needed 48 h or more for completion of the reaction at 25 °C. At the 20 h time point, cleavage of Pbf protecting groups was insufficient; at the 48 h time point, cleavage was about complete, but aspartimide formation was unsatisfactorily high (cf. samples 11.6-11.10). Of note, these limitations were also observed for the solvents used in TCA containing cleavage cocktails of the prior art (cf. sample 11 .9 [de la Torre et al., Letters in Peptide Science 2002, 8, 331 -338 reported use of 3% TCA in DCM for N-alpha-detritylation]; sample 11.10 [US 4,800,166 reported use of 3% TCA / in DCE I anisole 9:1 (v / v), for removal of DMT groups from N“-amino groups of peptides bound to solid phase],

[0751] 13. Comparative Experiment 12: Cleavage of model peptide II with cleavage protocol according to CN1154071

[0752] Experiment 12a - Kinetics of cleavage reaction

[0753] Experiment 11 was repeated but using the cleavage cocktails of CN1154071. Isolated, soft cleaved peptide II was subjected to global deprotection and resulting cleavage solution was analyzed by UHPLC according to steps #1 to #3 of general procedure D (cf. para. 1.2.4). The composition of the cleavage solutions and reaction conditions used are indicated in Table 12a.

[0754] Table 12a: Cleavage reaction at 25 °C Bachem Holding AG, 103 BAC75006PC1

[0755] Novo Nordisk A / S September 29, 2025

[0756] Experiment 12b - Cleavage and crude peptide isolation

[0757] Experiment 1 b was essentially repeated, but the cleavage protocol of CN1154071 was used. Samples of the obtained resin-bound model peptide II were subjected to cleavage and the peptide in the resulting cleavage solution was isolated according to general procedure B (cf. para. 1 .2.2), and UHPLC Method A (cf. paras. 1.1.1 and 1 .1 .4). The composition of the cleavage solutions and reaction conditions used were as indicated in Table 12b.

[0758] Table 12b: Cleavage of model peptide II from CTC resin and isolation of crude peptide by precipitation Bachem Holding AG, 104 BAC75006PC1

[0759] Novo Nordisk A / S September 29, 2025

[0760] *) (TCA in DCM 50:50) I H2O I thioanisole I EDT I TES 100:5:5:2.5:2 (VA / A / A / A / )

[0761] It was concluded that the protocol of CN1154071 does not work: The cleavage reaction in the described cleavage cocktail is very slow and the protocol does barely yield any target peptide, i.e. , fully deprotected free peptide. In fact, from comparing the text of CN1154071 to that of the corresponding PCT application WO9533496 A1 , which was originally filed in English, the protocol of CN1154071 appears to contain a translation error. WO9533496 A1 does not mention TCA containing cleavage cocktails but teaches: “Resin-bound products were routinely cleaved using a solution comprised of trifluoroacetic acid or 50 / 50 trifluoroacetic acid / dichloromethane, optionally containing water, thioanisole, ethanedithiol, and triethylsilane, prepared in ratios of 100 : 5 5 2.5 : 2 for 1.5 - 3 h at room temperature / (WO9533496 A1 , p. 11 , I. 11-15).

[0762] 14. Comparative Experiment 13: Cleavage cocktails according to CN1150206 C and CN1253953 A

[0763] CN1 150206 C, p. 30, 1.27. teaches a cleavage cocktail composed of 10 mL of trichloroacetic acid, 0.5 mL of phenylthiomethane and 0.25 mL of ethanedithiol” to be used for 3 hours at room temperature for cleavage of an SPPS product from the resin and concomitant removal of protecting groups. CN1150206 C indicates the amount of trichloroacetic acid to be used as a volume, although the compound is solid at room temperature.

[0764] When the present inventors tried to put this protocol in practice, they found that a mixture of 10 mL TCA (16.3 g; calculated with d 1 .629 g / mL) + 0.5 mL thioanisole + 0.25 mL EDT (corresponding to 32.6 g TCA per 1 mL of thioanisole) was essentially a heterogenous mixture of solid TCA and a liquid phase. Upon heating to 70 °C, the mixture became liquid but solidified again upon cooling to room temperature. The inventors concluded that the protocol of CN1150206 C does not work. In fact, from comparing the text of CN1150206 C / CN1148393 A to that of the corresponding PCT application WO95 / 24422 A1 , which was originally filed in English, the protocol of CN1150206 C appears to contain a translation error. WO95 / 24422 A1 does not Bachem Holding AG, 105 BAC75006PC1

[0765] Novo Nordisk A / S September 29, 2025 mention TCA containing cleavage cocktails but teaches: “The resin was then treated with 10 mL trifluoroacetic acid containing 0.5 mL of thioanisole and 0. 5 mL of ethanedithiol. The reaction bubbled for 3 hours at room temperature.” A similar observation was made when trying to reproduce the cleavage cocktail of CN1253953 A (p. 9, I. 29 to p.10, I. 1 ). Just like CN1150206 C, CN1253953 A indicates the amount of trichloroacetic acid to be used as a volume, although the compound is solid at room temperature. When combining 10 mL TCA (16.3 g; calculated with d 1.629 g / mL) with 0.75 g phenol, 0.25 mL EDT, 0.5 mL thioanisole, and 0.5 mL water (corresponding to 32.6 g TCA per 1 mL of thioanisole), a heterogenous mixture of solid TCA and a liquid phase was obtained. Upon heating to 70 °C, the mixture became liquid and solidified again partially upon cooling to room temperature. Hence, it was not possible to execute the cleavage protocol of CN1253953 A, which requires cleavage for 1 .5 h at room temperature.

Claims

Bachem Holding AG, 106 BAC75006PC1Novo Nordisk A / S September 29, 2025Claims1 . A method of preparing a peptide, wherein the method comprises the steps of:(i) contacting(A) a peptide precursor that is covalently bound to at least one acid- cleavable support; with(B) a cleavage cocktail comprising:(b1 ) trichloroacetic acid,(b2) one or more solvents selected from the group consisting of diphenylsulfide, thiophenol, thioanisole, a monohalogenated thiophenol, and a monohalogenated thioanisole, and(b3) optionally a salt; and(ii) incubating the components of step (i), thereby cleaving off the acid- cleavable support from the peptide precursor resulting in the peptide, wherein the amount of trichloroacetic acid in the cleavage cocktail is in the range from 2 to 12 g per 1 mL of said one or more solvents (b2) (when the volume of the solvent is the volume at 25 °C), and the amount of trichloroacetic acid in the cleavage cocktail, based on the total weight of the cleavage cocktail, is at least 60% by weight.

2. The method of claim 1 , wherein the cleavage cocktail comprises one or more scavengers different from the one or more solvents (b2), preferably in a total amount of from 1 % to 15%, preferably from 2% to 10%, more preferably from 4% to 8%, wherein the % are volume to volume (VA / ) for scavengers liquid at 25 °C and weight to volume (W / V) for scavengers solid at 25 °C, based on the total volume of the cleavage cocktail at 25 °C.

3. The method of claim 2, wherein the one or more scavengers are selected from the group consisting of water, thiol scavengers and silane scavengers.Bachem Holding AG, 107 BAC75006PC1Novo Nordisk A / S September 29, 20254. The method of any one of claims 2 or 3, wherein the one or more scavengers comprise water and one or more further scavengers selected from the group consisting of thiol scavengers and silane scavengers, preferably from the group consisting of 1 ,2-ethanedithiol, dithioerythritol, dithiothreitol , 3,6-dioxa- 1 ,8-octanedithiol, beta-mercaptoethanol, 1 ,4-benzenedimethanethiol, 1 ,3- benzenedimethanethiol, 1 ,2-benzenedimethanethiol, triethy Isi lane, triisopropylsilane.

5. The method of any one of claims 1 to 4, wherein the cleavage cocktail does not comprise more than 20% by volume, preferably does not comprise more than 10% by volume, more preferably does not comprise more than 5% by volume, based on the total volume of solvents and scavengers, of solvents and scavengers other than diphenylsulfide, thiophenol, thioanisole, monohalogenated thiophenols, monohalogenated thioanisoles, water, thiol scavengers and silane scavengers.

6. The method of any one of claims 1 to 5, wherein the one or more solvents and / or scavengers, preferably the entire cleavage cocktail, do not comprise a per- or polyfluorinated substance.

7. The method of any one of claims 1 to 6, wherein cleavage of protecting groups from the peptide occurs in addition to cleavage of the peptide from the acid-cleavable support.

8. The method of any one of claims 1 to 7, wherein the peptide precursor is obtained from:(a) solid phase peptide synthesis (SPPS) and is covalently bound to an acid-cleavable solid support, preferably wherein the solid support is a swellable polystyrene resin, a resin comprising polyethylene glycol (PEG), or a crosslinked poly- epsilon-lysine resin;(b) tag-assisted peptide synthesis (TAPS) and is covalently bound to an acid-cleavable tag, preferably wherein the tag is selected from the group consisting ofBachem Holding AG, 108 BAC75006PC1Novo Nordisk A / S September 29, 2025 substituted benzyl alcohols, substituted benzyl amines, substituted 1 ,1 -diphenylmethanols, and substituted 1 ,1 -diphenylmethylamines; or (c) a combination of two or more of (a), (b) and liquid-phase peptide synthesis (LPPS).

9. The method of any one of claims 1 to 8, wherein the peptide precursor is characterized in that:(a) it has a C- and / or N-terminus protected with an acid-cleavable group, and / or is bound to a solid support, and / or is bound to a tag, preferably wherein the tag is selected from the group consisting of substituted benzyl alcohols, substituted benzyl amines, substituted 1 ,1 - diphenylmethanols, and substituted 1 ,1 -diphenylmethylamines, and / or wherein the peptide is bound to a solid support or to a tag via an acid- cleavable linker, preferably wherein the linker is selected from the group consisting of 2-chlorotrityl (2CT linker), 2-chlorotrityl amidomethyl (2CT-AM linker), 4-[9-Fmoc-amino-xanthen-3-yloxy]-butyryl)- 4-methyl-benzhydrylamide (Sieber linker), 4-hydroxybenzylalcohol (Wang linker), Fmoc-2,4-dimethoxy-4’-(carboxymethyloxy)- benzhydrylamine (Rink Amide linker), Fmoc-suberol (5-Fmoc-amino- 2-carboxymethoxy-10, 11 -dihydro-5H-dibenzo[a,d]cycloheptene) (Ramage linker), 5-Fmoc-amino-10,11 -dihydro-5H-dibenzo [a,d]cycloheptenyl-2-oxyacetyl-DL-Nle-4-methyl-benzhydrylamide (tricyclic amide linker), 2-Methoxy-4-alkoxy benzyl alcohol (Sasrin linker), 5-[4-(9-Fmoc)aminomethyl-3,5-dimethoxyphenoxy]-pentanoic acid (Fmoc-PAL linker), 4-(4-Formyl-3,5-dimethoxyphenoxy) (BAL linker), 4-Hydroxymethylphenoxyacetic acid (HMPA linker), and 4-(4- hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB linker); and / or(b) the peptide precursor comprises one or more of the following structural elements:A 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf) side chain protecting group moiety, 2,2,5,7,8-pentamethylchroman-6- sulfonyl (Pmc) side chain protecting group moiety, in particular an Arg(Pbf) moiety,Bachem Holding AG, 109 BAC75006PC1Novo Nordisk A / S September 29, 2025 an intra-chain and / or C-terminal beta-hydroxy amino acid moiety, preferably Ser or Thr,Asn, Gin, Asp, Cys, Met, Trp, a hydrocarbon chain >Ce as a substituent, and / or a polyethylene glycol (PEG) moiety with two or more oxyethylene repeating units as a substituent.

10. The method of any one of claims 1 to 9, wherein the step (ii) is conducted at a temperature in the range of 20 to 65 °C, preferably of 25 to 60 °C, more preferably of 25 to 55 °C, more preferably of 25 to 45 °C.11 . The method of any one of claims 1 to 10, wherein the step (ii) is conducted for 24 hours or less, preferably for 2 to 24 hours, more preferably for 10 to 24 hours.

12. The method of any one of claims 1 to 11 , wherein the amount of trichloroacetic acid in the cleavage cocktail is in the range from 4 to 8 g per 1 mL of said one or more solvents (b2) (when the volume of the solvent is the volume at 25 °C) and the step (ii) is conducted at a temperature in the range of 25 to 45 °C.

13. The method of any one of claims 1 to 12, wherein the method further comprises one or more further steps:(iii) separating the peptide obtained in step (ii) from:(iii-a) non-dissolved components, in particular from a solid support if present, and optionally washing the non-dissolved components once or more often, preferably with the cleavage cocktail, and / or(iii-b) one or more other non-peptidic residues, in particular a tag;(iv) precipitating the peptide obtained in any one of steps (ii) or (iii), preferably by mixing with anti-solvent, preferably diisopropyl ether, preferably at a temperature of not more than 10 °C, and optionally washing the peptide with one or more resuspension cycles in the antisolvent;Bachem Holding AG, 110 BAC75006PC1Novo Nordisk A / S September 29, 2025(v) purifying the peptide obtained in any one of steps (ii), (iii), (iv), or (vi) by liquid chromatography;(vi) chemically modifying the peptide obtained in any one of steps (ii), (iii), (iv), or (v), preferably by oxidative formation of disulfide bonds, or by conjugation to another moiety;(vii) dissolving or suspending the precipitated peptide obtained in any one of steps (iv), (v), or (vi) in a suitable solvent, preferably an evaporable solvent;(viii) drying the composition obtained from any of steps (iii), (iv), (v), (vi), or (vii); and / or(ix) dissolving or suspending the peptide obtained from any of steps (ii), (iii), (iv), (v), (vi), (vii), or (viii) in a buffer suitable for a purpose of interest, in particular a pharmaceutically acceptable buffer.

14. A cleavage cocktail as defined in any one of claims 1 to 6, preferably wherein: one solvent is 2-chlorothioanisole; and one scavenger is ethanedithiol, triisopropylsilane or triethylsilane.

15. Use of a cleavage cocktail of claim 14 for cleaving an acid-cleavable solid or solubility-modifying support off a peptide precursor, preferably wherein the use is defined according to any one of claims 1 to 13.

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