Novel stationary phases for large-scale reversed-phase HPLC purification.

Abstract

To provide a novel stationary phase for large scale reverse-phase HPLC purification, particularly for the purification of a modified conjugated peptide such as a GLP-1 receptor agonist or a GLP-2 analogs, and provide silica particles that can be used as stationary phases for large scale reverse-phase HPLC purification.SOLUTION: The invention relates to silica particles having a fine pore size of 1 to 250 Angstrom (Å) and having a particle size of 0.1 μm to 100 μm and comprising two groups which are each independently selected from the group consisting of alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl. The silica particles are suitable for purifying a modified conjugated peptide, such as a GLP-1 agonist or a GLP-2 analog.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to silica particles having pore sizes of 1 to 250 angstroms (Å) and containing silane groups, each containing two groups independently selected from the group consisting of alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl. The present invention further relates to a method for preparing such silica particles and the use of such silica particles as a stationary phase for purifying modified conjugated peptides, such as GLP-1 agonists or GLP-2 analogs. [Background technology]

[0002] Reverse-phase HPLC (High Performance Liquid Chromatography) is a commonly used technique for separating, identifying, and quantifying all components contained in a given mixture. In the pharmaceutical field, HPLC is particularly suitable for achieving the necessary high-grade purification of drugs, especially drugs with higher molecular weights, such as modified conjugated peptides. The basic principle of HPLC is that the mixture to be purified is pumped under pressure with a liquid solvent through a column packed with a solid adsorbent material, the so-called stationary phase, which is generally a silica-based material. Each component of the mixture to be separated usually interacts slightly differently with the adsorbent material due to more or less pronounced physical and chemical interactions between the component and the surface and / or the pore size of the adsorbent material. This interaction causes different flow rates for different components, resulting in the separation of the mixture components. The different components may then be collected in different fractions as they exit the column.

[0003] Many different adsorbent materials are available for HPLC columns. Particular attention has been paid to silica particles, which have proven highly suitable for separations in terms of cost and raw material availability. Particular focus in optimizing these silica particles for HPLC use has also been placed on particle size and particle pore size. However, some efforts have been made to modify silica particles with functional groups to fine-tune the physical and chemical interactions between the absorbent material in the stationary phase and any potential components in the mixture being separated. For example, U.S. Patent No. 9,670,067 B2 describes the modification of silica particles with organic groups containing halohydrin groups. Similarly, U.S. Patent No. 10,690,6365 B2 discloses silica particles modified with C8 or C18 groups. In U.S. Patent No. 7,537,703 B2, silica particles are modified with n-butyl groups.

[0004] Modified conjugate peptides have been found to be difficult to purify. This is due to their relatively high molecular weight, which is assumed to complicate the separation of the desired peptide from impurity peptides of somewhat similar molecular weight. The purification of such conjugate peptides usually requires two or more purification steps. However, from a commercial perspective, the number of purification steps should ideally be limited to only two, while achieving a commercially acceptable yield of highly purified conjugate peptides. In the pharmaceutical field, a purity, i.e., selectivity, of at least >99.5% is usually targeted. Therefore, commercially available silica suitable for the stationary phase should ideally be suitable for separating the components of a mixture into fractions containing only one pure component (i.e., having a purity of at least >99.5%), thereby achieving the same high selectivity and high recovery, i.e., overall yield of this one pure compound. In other words, for a commercially available suitable HPLC stationary phase, it may not be sufficient for some fractions to contain a highly pure compound. Rather, the overall yield of this compound with high purity is also an important factor. A commercially relevant overall yield is, for example, at least 70%, preferably at least 75%, and even more preferably at least 80%.

[0005] Examples of conjugated peptides of pharmaceutical interest are, for example, GLP-1 (glucagon-like peptide 1) receptor agonists and GLP-2 (glucagon-like peptide 2) analogues. These types of conjugated peptides are used in the treatment of obesity. A commercially relevant GLP-1 receptor agonist is, for example, semaglutide, an antidiabetic drug used in the treatment of type 2 diabetes and an antiobesity drug for long-term weight management. Another commercially relevant GLP-1 receptor agonist is liraglutide (N-ε-(γ-Glu(N-α-hexadecanoyl)))-Lys 26 Arg 34 -GLP-1(7-37), also known as NN2211. Liraglutide is approved for the treatment of type 2 diabetes and for the treatment of obesity in adults.

[0006] However, in addition to the problems generally encountered when purifying complex peptides already mentioned above, purification of glucagon-like peptides has been found to be particularly difficult due to their tendency to form aggregates, especially at acidic pH.

[0007] In summary, there remains a need for improved stationary phases for HPLC applications, particularly for the purification of pharmaceuticals such as high molecular weight molecules, for example, modified conjugated peptides such as GLP-1 receptor agonists and GLP-2 analogues, particularly liraglutide and semaglutide. In particular, there is a need for improved silica particles for HPLC applications that have improved separation performance in terms of selectivity (i.e., purity) and recovery (i.e., yield), and at the same time, ideally, are easily accessible, readily available, and environmentally harmless. Furthermore, there is a need to provide a method for preparing such improved silica particles that can be used in HPLC applications for the successful and efficient purification of modified conjugated peptides such as GLP-1 receptor agonists and GLP-2 analogues.

[0008] Accordingly, the present invention is directed to the discovery of such improved silica particles and methods for producing silica particles. It has been discovered that the above problems can be solved by improved silica particles having a micropore size of 1 to 250 angstroms (Å) and containing silane groups, each containing two groups independently selected from the group consisting of alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl. Summary of the Invention

[0009] In a first aspect, the present invention is directed to silica particles having a micropore size of 1 to 250 angstroms (Å) and comprising silane groups according to the formula: [ka] In the formula, R 1 and R 2are each independently selected from the group consisting of alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl; R is hydrogen or silica; and SiO is silica.

[0010] In a second aspect, the present invention provides a method for preparing silica particles, comprising: (i) providing silica particles having a micropore size of 1 to 250 angstroms (Å); and (ii) modifying silica particles with a compound of the formula: [ka] In the formula, R 1 and R 2 are each independently selected from the group consisting of alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl, and X 1 and X 2 is independently a halogen or an alkoxy group.

[0011] The present invention is further directed to the use of silica particles according to the first aspect, or obtained by the method according to the second aspect, as a stationary phase for purifying modified conjugated peptides, preferably GLP-1 agonists or GLP-2 analogues. [Brief explanation of the drawings]

[0012] [Figure 1a] FIG. 1a shows the chromatogram obtained in the first purification step of Example 2a). [Figure 1b] FIG. 1b shows the chromatogram obtained in the second purification step of Example 2a). [Figure 2a] FIG. 2a shows the chromatogram obtained in the first purification step of Example 2b). [Figure 2b] FIG. 2b shows the chromatogram obtained in the second purification step of Example 2b). [Figure 3a]FIG. 3a shows the chromatogram obtained in the first purification step of Example 3. [Figure 3b] FIG. 3b shows the chromatogram obtained in the second purification step of Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0013] In a first aspect, the present invention discloses novel stationary phases for large-scale reversed-phase HPLC purification, particularly for the purification of modified complex peptides such as GLP-1 receptor agonists and GLP-2 analogs. In particular, the present invention provides silica particles that can be used as stationary phases for large-scale reversed-phase HPLC purification.

[0014] The novel stationary phase according to the present invention comprises silica particles having a micropore size of 1 to 250 angstroms (Å) and containing silane groups according to the formula: [ka] In the formula, R 1 and R 2 are each independently selected from the group consisting of alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl; R is hydrogen or silica; and SiO is silica.

[0015] Surprisingly, it has been found that such modified silica particles can be used as the stationary phase in HPLC applications, and can achieve improved separation characteristics, particularly for modified conjugated peptides, such as GLP-1 receptor agonists and GLP-2 analogs, particularly modified conjugated peptides such as liraglutide and semaglutide. By using these novel modified silica particles as the stationary phase in HPLC applications, certain difficult impurities in crude active pharmaceutical ingredients, such as liraglutide and semaglutide, can be successfully removed in the first chromatographic step, for example, under alkaline conditions. This allows for the delivery of more purified active pharmaceutical ingredients per cycle and per hour to the second chromatographic step, which then increases the possibility of meeting the pharmaceutical industry's purity requirements of >99.5% and a single impurity content of <0.1% in the second HPLC purification step, while simultaneously achieving a commercially acceptable overall yield. In other words, the modified silica particles of the present invention significantly increase selectivity and recovery, resulting in significantly improved overall separation productivity.

[0016] In the above formula I, the remaining R 1 and R 2 are each independently selected from the group consisting of alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl.

[0017] The term "alkyl" refers to a straight-chain, branched-chain, and / or cyclic ("cycloalkyl") hydrocarbon having 1 to 30 (e.g., 1 to 20, or 1 to 4) carbon atoms. Alkyl moieties having 1 to 4 carbons are referred to as "lower alkyl." Examples of alkyl moieties include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, and dodecyl. Cycloalkyl moieties can be monocyclic or polycyclic, and examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl. Additional examples of alkyl moieties include straight-chain, branched, and / or cyclic moieties (e.g., 1-ethyl-4-methyl-cyclohexyl). As used herein, the term "alkyl" includes saturated and unsaturated hydrocarbons, preferably saturated hydrocarbons.

[0018] The term "aryl" refers to an aromatic ring or aromatic or partially aromatic ring system composed of carbon and hydrogen atoms. An aryl moiety may contain multiple rings bonded or fused together. Examples of aryl moieties include anthracenyl, azulenyl, biphenyl, fluorenyl, indan, indenyl, naphthyl, phenanthrenyl, phenyl, 1,2,3,4-tetrahydro-naphthalene, and tolyl.

[0019] The term "alkylaryl" refers to a moiety that includes at least one alkyl group and at least one aryl group, e.g., an alkyl moiety bonded to an aryl moiety, where the terms "alkyl" and "aryl" are defined above.

[0020] The term "heteroalkyl" refers to an alkyl group that also contains at least one heteroatom, such as a nitrogen, oxygen, phosphorus, sulfur, and / or boron atom, where the term "alkyl" is defined above.

[0021] The term "heteroaryl" refers to an aryl group that also contains at least one heteroatom, such as a nitrogen, oxygen, phosphorus, sulfur, and / or boron atom, where the term "aryl" is as defined above. For example, the term "heteroaryl" preferably refers to an aryl group in which at least one of its carbon atoms is replaced with nitrogen, oxygen, or sulfur. Examples of heteroaryl moieties include acridinyl, benzimidazolyl, benzofuranyl, benzisothiazolyl, benzisoxazolyl, benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furyl, imidazolyl, indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, phthalazinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolinyl, tetrazolyl, thiazolyl, and triazinyl.

[0022] The term "heteroalkylaryl" refers to a moiety that includes at least one alkyl group, at least one aryl group, and at least one heteroatom, such as a nitrogen, oxygen, phosphorus, sulfur, and / or boron atom, where the terms "alkyl" and "aryl" are defined above.

[0023] Any of the above groups, alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl, can be unsubstituted or further substituted.

[0024] The term "substituted," when used to describe a chemical structure or moiety, refers to a derivative of that structure or moiety in which one or more of its hydrogen atoms has been replaced with an atom, chemical moiety, or functional moiety, such as, but not limited to, alcohol, aldehyde, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl), alkynyl, alkylcarbonyloxy (-OC(O)alkyl), amido (-C(O)NH-alkyl- or -alkyl-NHC(O)alkyl), amidinyl (-C(NH)NH-alkenyl), alkyl, or -C(NR)NH2), amines (primary, secondary, and tertiary, e.g., alkylamino, arylamino, arylalkylamino), aroyl, aryl, aryloxy, azo, carbamoyl (-NHC(O)O-alkyl-, or -OC(O)NH-alkyl), carbamyl (e.g., CONH2, and CONH-alkyl, CONH-aryl, and CONH-arylalkyl), carbonyl, carboxyl, carboxylic acid, carboxylic acid anhydride, carboxylic acid chloride, cyano, ester, epoxide, ether (e.g., methoxy, ethoxy), guanidino, halo, haloalkyl (e.g., -CCl3, -CF3, -C(CF3)3), heteroalkyl, hemiacetal, imine (primary and secondary), ketone, nitrile, nitro, oxygen (i.e., providing an oxo moiety), phosphodiester, sulfide, sulfonamide (e.g., SO2NH2), sulfone, sulfonyl (including alkylsulfonyl, arylsulfonyl, and arylalkylsulfonyl), sulfoxide, thiol (e.g., sulfhydryl, thioether), and urea (-NHCONH-alkyl-).

[0025] In some embodiments, R 1 and R 2 and each independently contain 2 to 20 carbon atoms.

[0026] In some embodiments, R 1 and R 2 are preferably both independently selected from the group consisting of alkyl and heteroalkyl.1 and R 2 are both independently alkyl, e.g., C-C 20 Branched or unbranched alkyl, preferably C2-C 10 branched or unbranched alkyl, for example, C3-C 10 -branched or unbranched alkyl, or C3-C8-branched or unbranched alkyl, and even more preferably C4-C8-branched or unbranched alkyl, for example C4-branched or unbranched alkyl, C5-branched or unbranched alkyl, C6-branched or unbranched alkyl, C7-branched or unbranched alkyl, or C8-branched or unbranched alkyl, or for example C4-C6-branched or unbranched alkyl. Particularly preferred is unbranched alkyl, for example C2-C 20 -Unbranched alkyl, C2-C 10 -Unbranched alkyl, C3-C 10 -unbranched alkyl, C3-C8-unbranched alkyl, or further C4-C8-unbranched alkyl, for example C4-unbranched alkyl, C5-unbranched alkyl, C6-unbranched alkyl, C7-unbranched alkyl, or C8-unbranched alkyl, or for example C4-C6-unbranched alkyl. 1 and R 2 are both independently C4-, C6-, and / or C8-branched or unbranched alkyl, and even more preferably C4-, C6-, and / or C8-unbranched alkyl. 1 and R 2 are both the same. For example, in some embodiments, R 1 and R 2 are both C4-, C6-, or C8-branched or unbranched alkyl, e.g., C4- or C8-branched or unbranched alkyl, and even more preferably C4-, C6-, or C8-unbranched alkyl, e.g., C4- or C8-unbranched alkyl.

[0027] "SiO" in the above formula I means silica, which means that the silane group in formula I is bonded to the silica structure, i.e., silica particle, via an O-Si bond.

[0028] The silica or silica particles may be derived, for example, from precipitated silica, microsilica (fumed silica), pyrogenic silica (fumed silica), silica sol or silica gel, and mixtures thereof. The silica particles may be in the form of porous particles.

[0029] Suitably, the silica particles have a particle size of 20 to 1500, preferably 50 to 900, most preferably 70 to 800 m 2 / g. The specific surface area may be measured by titration with sodium hydroxide as described in Sears in Analytical Chemistry 28(1956), 12, 1981-1983 and U.S. Pat. No. 5,176,891. The given area therefore represents the average specific surface area of ​​the particles.

[0030] The remaining R in the above formula is either hydrogen or silica. "Silica" in this context means that the silane group of formula I is bonded to silica or silica particles via an additional O-Si bond.

[0031] The silica particles of the present invention have pore sizes of 1 to 250 angstroms (Å), such as 10 to 200 Å, 50 to 250 Å, 60 to 200 Å, or even 70 to 180 Å. Particularly preferred are pore sizes of 80 to 150 Å, such as 100 to 130 Å, and particularly about 100 Å or about 120 Å. It has been found that pore sizes greater than 250 Å tend to significantly impair the overall separation productivity of silica particles in HPLC columns, i.e., to result in lower overall yields of purified components. All references to pore size used herein refer to the average pore size. Methods for determining the pore size of silica particles are commonly known to those skilled in the art and include, for example, the Brunauer-Emmett-Teller (BET) method using, for example, a Micromeritics TriStar II Plus instrument.

[0032] The particle size of the silica particles of the present invention is not particularly limited. For example, the silica particles of the present invention may have an average particle size of 0.1 to 100 μm, preferably 1 to 50 μm. In some embodiments, they may further have an average particle size in the range of 1.8 to 25 μm. Methods for determining the average particle size of silica particles are generally known to those skilled in the art. For example, particle size may be calculated from the specific surface area and the equation relating particle size in Iller (The Chemistry of Silica, Wiley, 1979). As is conventional in silica chemistry, particle size refers to the average size of primary particles. Particle size may also be measured via the Coulter principle using a Beckman Coulter Multisizer 4e Coulter Counter.

[0033] The pore volume of the modified silica particles is suitably 0.1 to 4 ml / g, preferably 0.2 to 2 ml / g, and most preferably 0.3 to 1.2 ml / g.

[0034] The specific surface area (BET method) of the modified silica-containing particles is preferably 1 to 1000 m 2 / g, preferably 25 to 700m 2 / g, most preferably 50 to 500m 2 / g.

[0035] A preferred method for measuring surface area, pore volume, and average micropore diameter (from surface area and pore volume) is by the Brunauer-Emmett-Teller (BET) method, which is also based on nitrogen adsorption / desorption, and surface area is typically calculated from the linear portion of the isotherm. Examples of such methods are provided in ISO 9277:2010 (for BET) and ISO 15901-2:2006 (for gas adsorption / desorption). Preferably, surface area, pore volume, and average micropore diameter are measured using ISO 9277:2010.

[0036] In another aspect, the present invention provides a method for preparing silica particles, comprising: (i) providing silica particles having a micropore size of 1 to 250 angstroms (Å); and (ii) modifying silica particles with a compound of the formula: [ka] In the formula, R 1 and R 2 are each independently selected from the group consisting of alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl; and X 1 and X 2 are each independently a halogen or an alkoxy group.

[0037] In this way it is possible to obtain silica particles according to the first aspect of the invention described above. The present invention is therefore also directed to silica particles obtainable by the above-described method according to this aspect of the invention.

[0038] The silica particles provided in step (i) of the process according to the second aspect of the invention may be as described above and may have the same preferred pore sizes as described above in the context of the first aspect of the invention.

[0039] The compound according to formula II used in step (i)i of the method according to the second aspect of the present invention to modify the silica particles comprises two groups R each independently selected from the group consisting of alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl. 1 and R 2 and two further groups X which are halogen or alkoxy groups. 1 and X 2 The silane is a silane comprising:

[0040] base R 1 and R 2 may be the same as those described above in the context of the first aspect of the invention.

[0041] Group X 1 and X 2 are each independently a halogen or alkoxy group, preferably a halogen.

[0042] Halogens include fluorine, chlorine, bromine, and iodine, with chlorine being particularly preferred.

[0043] The term "alkoxy group" refers to an -O-alkyl moiety, where the alkyl group may be as defined above in the context of the first aspect of the invention. Preferably, the alkyl moiety has 1 to 8 carbon atoms, such as 1 to 4 carbon atoms. Examples of alkoxy moieties include -OCH3, -OCH2CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, and -O(CH2)5CH3.

[0044] Such modification reactions are known to those skilled in the art and may be adjusted as necessary. For example, the reaction of the compound of Formula II with the silica particles is carried out at a temperature suitable for carrying out the reaction, preferably 40 to 180°C, more preferably 60 to 160°C, and most preferably 80 to 140°C.

[0045] Preferably, the compound of formula II may be added to the silica particles at a controlled rate under stirring until a suitable amount of the compound of formula II has been added, and the reaction time may be from 1 to 24 hours.

[0046] The reaction of the compound of formula II with silica particles may be carried out in an organic solvent, preferably under stirring.The organic solvent is preferably an aprotic solvent.Examples of such an aprotic solvent may be acetonitrile, acetone, xylene, or toluene, preferably toluene, or a mixture thereof.

[0047] The silica particles may be added to the solvent, followed by the optional step of evaporating any water present. The compound of formula II may then be added to the solvent, which may then be added to the dispersion of the silica particles and solvent.

[0048] The proportion of the compound of formula II is 2 The amount of the solvent is preferably selected so that the amount of silica particles in the dispersion is 5 to 20% by weight.

[0049] The silanol surface moieties on the silica particles react with the compound of Formula II to form a covalent bond between the silica and the compound of Formula II.

[0050] When the dispersion of modified silica particles is formed, the dispersion may be cooled and purified, for example, by ultrafiltration or by washing, for example, with toluene, ethanol, or formic acid. The dispersion may then be dried, for example, at 40 to 100°C, preferably 60 to 90°C, for 2 to 30 hours, preferably 10 to 25 hours.

[0051] According to a further aspect, the present invention relates to a separation column for chromatography, comprising the silica particles of the present invention, i.e., the silica particles according to the first aspect of the present invention, or the silica particles obtained by the method according to the second aspect of the present invention. In this aspect, the silica particles of the present invention are packed in a separation column. Thus, in this aspect, the silica particles of the present invention are used as a stationary phase for chromatography. Thus, the silica particles may be used in a separation column for any type of chromatographic separation method, such as HPLC, supercritical fluid chromatography (SFC), and simulated moving bed chromatography (SMB).

[0052] According to a still further aspect, the present invention relates to the use of silica particles according to the first aspect of the invention or obtainable by the method according to the second aspect of the invention as a stationary phase for purifying modified conjugated peptides, preferably GLP-1 agonists or GLP-2 analogues. It is particularly preferred that the silica particles according to the first aspect of the invention or obtainable by the method according to the second aspect of the invention are used as a stationary phase for purifying semaglutide or liraglutide. [Example]

[0053] Example 1: Preparation of modified silica particles Different modified silica particles were prepared using the following commercially available Kromasil® particles (Nouryon BV) as starting materials:

[0054] Average particle size 5μm, average pore size 100Å, specific surface area 315m 2 Silica particles (Kromasil® KR-100-5 SIL, manufactured by Nouryon BV) having a .gamma. / g.

[0055] Average particle diameter 10μm, average pore diameter 100Å, specific surface area 320m 2 Silica particles (Kromasil® KR-100-10 SIL, manufactured by Nouryon BV) having a .gamma. / g.

[0056] Average particle size 5μm, average pore size 200Å, specific surface area 195m 2 Silica particles (Kromasil® KR-200-5 SIL, manufactured by Nouryon BV) having a .gamma. / g.

[0057] Average particle size 10μm, average pore size 300Å, specific surface area 105m 2 Silica particles (Kromasil® KR-300-10 SIL, manufactured by Nouryon BV) having a .gamma. / g.

[0058] Example 1a) 20 g of KR-100-5 SIL and 10.3 g of imidazole were added to 175 g of toluene. The resulting dispersion was heated to evaporate 20 g of toluene. After heating, the dispersion was cooled to 80°C. 8 g of dibutyldichlorosilane was added to the toluene, silica, and imidazole dispersion under stirring. The temperature was increased, and the dispersion was refluxed overnight. The reaction was cooled to 70°C, and 32 g of ethanol was added to deactivate the silane. The dispersion was washed with 87 g of toluene and 2 x 79 g of ethanol. The silica was dried overnight at 90°C in a laboratory oven. The resulting powder was white and is further referred to herein as Kromasil® 100-5-diC4. Elemental analysis of carbon yielded 8.3 wt% C.

[0059] Example 1b) KR-100-10 SIL was modified with dibutyldichlorosilane in the same manner as described above for KR-100-5 SIL in Example 1a). The resulting powder was white and is further referred to herein as Kromasil® 100-10-diC4. Elemental analysis of carbon yielded 8.4 wt.% C.

[0060] Example 1c) KR-100-10 SIL was modified in the same manner as described above in Example 1a), except that 5.7 g of butyldimethylchlorosilane and 4.1 g of trimethylchlorosilane were added as surface modifications instead of dibutyldichlorosilane. The resulting powder was white and is further referred to herein as Kromasil® 100-10-C4. Elemental analysis of carbon yielded 7.7 wt% C.

[0061] Example 1d) KR-200-5 SIL was modified with 5.0 g of dibutyldichlorosilane in the same manner as described above for KR-100-5 SIL in Example 1a). The resulting powder was white and is further referred to herein as Kromasil® 200-5-diC4. Elemental analysis of carbon yielded 5.2 wt.% C.

[0062] Example 1e) KR-100-5 SIL was modified in the same manner as described above in Example 1a), except that 12.4 g of dioctyldichlorosilane was used instead of dibutyldichlorosilane. The resulting powder was white and is further referred to herein as Kromasil® 100-5-diC8. Elemental analysis of carbon yielded 14.8 wt% C.

[0063] Example 1f) KR-100-5 SIL was modified in the same manner as described above in Example 1a), except that 10.2 g of dihexyldichlorosilane was used instead of dibutyldichlorosilane. The resulting powder was white and is further referred to herein as Kromasil® 100-5-diC6. Elemental analysis of carbon yielded 11.9 wt% C.

[0064] Example 1g) KR-300-10 SIL was modified with 2.7 g of dibutyldichlorosilane in the same manner as described above for KR-100-5 SIL in Example 1a). The resulting powder was white and is further referred to herein as Kromasil® 300-10-diC4. Elemental analysis of carbon yielded 3.1 wt% C.

[0065] Example 2: Two-step purification of liraglutide In all the following examples, the sample to be purified was 5.0 mg mL -1 of crude liraglutide (purity ≦10%), which was prepared by dissolving 80 mg of crude liraglutide in 16 mL of NH4HCO3 buffer (0.1 mol L -1 The solution was prepared by dissolving in 95:5 ethanol (pH 7.0) / acetonitrile, filtered through a 0.45 μm syringe filter, and injected directly into the column.

[0066] Example 2a): Two-step purification of liraglutide on Kromasil® 100-10-diC4 The first purification step was performed with ammonium bicarbonate at pH 7, and the second purification step was performed with ammonium acetate as buffer at pH 8, with acetonitrile used as the organic modifier in both systems. The purifications were performed on an Agilent 1260 system equipped with an autocollector. Both purification steps were performed on a 4.6 x 250 mm column packed with Kromasil® 100-10-diC4 from Example 1b).

[0067] First purification step: The crude liraglutide sample (purity ≤10%) was loaded onto a column (32 mg / g stationary phase), and the mobile phase consisted of 0.1 M ammonium bicarbonate (pH 7) and acetonitrile. Purification was performed under gradient elution according to the following sequence: 5% acetonitrile was loaded for 5 min between 5 and 138 min, with the acetonitrile concentration increasing linearly from 29.4% to 39.8%. After 128 min, the acetonitrile concentration was increased to 80% for 11 min to elute strongly adsorbed components. After washing the column, the acetonitrile concentration was reduced to 5% to equilibrate the column. Liraglutide was collected between 105 and 115 min, and the pooled fractions showed a purity of 97.1% with a recovery of 81%. The resulting chromatogram is shown in Figure 1a.

[0068] Second purification step: Pooled fractions from the first purification step were diluted to approximately 10% acetonitrile and loaded onto a column containing Kromasil® 100-10-diC4. The mobile phase consisted of 0.1 M ammonium acetate buffer (pH 8) and acetonitrile as an organic modifier. The purification was performed under gradient elution, with the acetonitrile concentration increasing linearly from 30.5% to 41.3% for 5 minutes, with 5% acetonitrile loaded between 5 and 95 minutes, according to the following sequence A: After 95 minutes, the acetonitrile concentration was increased to 80% over 11 minutes to elute strongly adsorbed components. After washing the column, the acetonitrile concentration was reduced to 5% to equilibrate the column. Liraglutide was collected between 79 and 86 minutes, and the pooled fractions showed a purity of ≥99.5% with a recovery of 98.2%. Therefore, the overall recovery rate over both purification steps was 79.6%. The resulting chromatogram is shown in Figure 1b.

[0069] Example 2b): Two-step purification of liraglutide on Kromasil® 100-5-diC8 The first purification step was performed at pH 7 using ammonium carbonate, and the second purification step was performed at pH 8 using ammonium acetate as the buffer; in both systems, acetonitrile was used as the organic modifier. The purifications were performed on an Agilent 1260 system equipped with an autocollector. Both purification steps were performed on a 4.6 x 150 mm column packed with Kromasil® 100-5-diC8 from Example 1e) above.

[0070] First purification step: The crude liraglutide sample (purity ≤10%) was loaded onto a column (12 mg / g stationary phase), and the mobile phase consisted of 0.1 M ammonium bicarbonate (pH 7) and acetonitrile. Purification was performed under gradient elution according to the following sequence: 5% acetonitrile was loaded for 5 min, and the acetonitrile concentration was linearly increased from 29.8% to 40.2%. After 45 min, the acetonitrile concentration was increased to 70% over 11 min to elute strongly adsorbed components. After washing the column, the acetonitrile concentration was reduced to 5% to equilibrate the column. Liraglutide was collected between 37 and 40 min, and the pooled fractions showed a purity of 96.4% with a recovery of 96%. The resulting chromatogram is shown in Figure 2a.

[0071] Second purification step: The pooled fractions from the first purification step were diluted to approximately 10% acetonitrile and loaded onto a column containing Kromasil® 100-5-diC8. The mobile phase consisted of 0.1 M ammonium acetate buffer (pH 8) and acetonitrile as an organic modifier. The purification was performed under gradient elution according to the following sequence: 5% acetonitrile was loaded for 5 min between 5 and 45 min, with the acetonitrile concentration increasing linearly from 29.8% to 40.2%. After 45 min, the acetonitrile concentration was increased to 70% over 11 min to elute strongly adsorbed components. After washing the column, the acetonitrile concentration was reduced to 5% to equilibrate the column. Liraglutide was collected between 43 and 45 min, and the pooled fractions showed a purity of 99.6%, with an overall recovery of 79% across both purification steps. The resulting chromatogram is shown in Figure 2b. The resulting chromatogram is shown in Figure 2b.

[0072] Example 2c): Two-step purification of liraglutide on Kromasil® 100-10-C4 (comparative example) The two-step purification of the liraglutide sample on Kromasil® 100-10-C4 from Example 1c) above was carried out in the same manner as described above in Example 2a) for the two-step purification of liraglutide on Kromasil® 100-10-diC4, using the same solvent gradient and instrument parameters.

[0073] In the first purification step, fractions were collected every minute between 100 and 125 minutes. Fractions 12 to 17 were pooled together for the second purification step. The pooled fractions showed a purity of 96.5% with a recovery of 87.4%.

[0074] In the second purification step, fractions were collected every 30 seconds between minutes 77 and 87. Pooled fractions 12-16 exhibited a purity of 99.5% with a recovery of 65.6%. Thus, the overall recovery across both purification steps was only 57.3%. This is significantly lower than the overall recovery achieved, for example, by using Kromasil® 100-10-diC4 (see Example 2a above), where an overall recovery of 79.6% was achieved.

[0075] Example 2d): Two-step purification of liraglutide on Kromasil® 300-10-diC4 (comparative example) The two-step purification of the liraglutide sample on Kromasil® 300-10-diC4 from Example 1g) above was carried out in the same manner as described above in Example 2a) for the two-step purification of liraglutide on Kromasil® 100-10-diC4, using the same solvent gradient and instrument parameters.

[0076] In the first purification step, fractions were collected every minute from 92 to 109 minutes. Fractions 3 to 7 were pooled together for the second purification step. The pooled fractions showed a purity of 94.5% with a recovery of 73.8%.

[0077] In the second purification step, fractions were collected every 30 seconds between minutes 71 and 86. Only fraction number 16 showed an acceptable purity of 99.5%, with a recovery of 4.7%. Thus, the overall recovery across both purification steps was only 3.4%. This is significantly lower than the overall recovery achieved, for example, by using Kromasil® 100-10-diC4 (see Example 2a above), where an overall recovery of 79.6% was achieved.

[0078] Example 3: Two-step purification of semaglutide on Kromasil® 100-10-diC4 The first purification step was carried out at pH 7 using ammonium bicarbonate, and the second purification was carried out at pH 8 with ammonium acetate as buffer, with acetonitrile used as the organic modifier in both systems. Purification was carried out on an Agilent 1260 system equipped with an automated fraction collector. Both purification steps were carried out on a 4.6 x 250 mm column packed with Kromasil® 100-10-diC4 from Example 1c). The purified semaglutide sample was 5.0 mg mL -1 of crude semaglutide (purity ≦60%), which was prepared by dissolving 80 mg of semaglutide in 16 mL of NH4HCO3 buffer (0.1 mol L -1 The solution was prepared by dissolving in 95:5 ethanol (pH 7.0) / acetonitrile, filtered through a 0.45 μm syringe filter, and injected directly into the column.

[0079] First purification step: The crude semaglutide sample (purity ≦60%) was loaded onto a column (34 mg / g stationary phase), and the mobile phase consisted of 0.1 M ammonium bicarbonate (pH 7) and acetonitrile. Purification was performed under gradient elution according to the following sequence: 5% acetonitrile was loaded for 5 min between 5 and 138 min, with the acetonitrile concentration increasing linearly from 30% to 40.5%. After 128 min, the acetonitrile concentration was increased to 80% for 11 min to elute strongly adsorbed components. After washing the column, the acetonitrile concentration was reduced to 5% to equilibrate the column. Semaglutide was collected between 50 and 70 min, and the pooled fractions showed a purity of 98.1% with a recovery of 95%. The resulting chromatogram is shown in Figure 3a.

[0080] Second purification step: Pooled fractions from the first purification step were diluted to approximately 10% acetonitrile and loaded onto a column containing Kromasil® 100-10-diC4. The mobile phase consisted of 0.1 M ammonium acetate buffer (pH 8) and acetonitrile as an organic modifier. Purification was performed under gradient elution according to the following sequence: 5% acetonitrile was loaded for 5 min between 5 and 95 min, with the acetonitrile concentration increasing linearly from 30.5% to 41.3%. After 95 min, the acetonitrile concentration was increased to 80% over 11 min to elute strongly adsorbed components. After washing the column, the acetonitrile concentration was reduced to 5% to equilibrate the column. Liraglutide was collected between 40 and 65 min, and the pooled fractions showed a purity of 99.6%, with an overall recovery of 76% across both purification steps. The resulting chromatogram is shown in Figure 3b.

[0081] Example 4: Further one-step purification study of liraglutide Further experiments were conducted to confirm that various silica particles of the present invention are already suitable for achieving commercially acceptable yields for the first purification step at first purification step purity levels of at least 70%, preferably at least 80%, more preferably at least 90%, such as at least 95% liraglutide. The liraglutide sample to be purified, the solvent gradient, and the instrument parameters were identical to those described above for Example 2a). The only difference was the silica particles used as the stationary phase in the HPLC column. Example 4a) used Kromasil® 100-5-diC4 from Example 1a) above. Example 4b) used Kromasil® 200-5-diC4 from Example 1d) above. Example 4c) used Kromasil® 100-5-diC6 from Example 1f) above.

[0082] Table 1 summarizes the results and also incorporates the results of Examples 2 and 3 above. [Table 1]

[0083] It is clear from Table 1 above that the silica particles of the present invention are particularly suitable for achieving greater than 95% purity already in the first purification step, with a commercially acceptable yield of at least 70%. Furthermore, the silica particles of the present invention also achieve an overall recovery (i.e., yield) of well over 75%, with a pharmaceutically relevant purity of at least 99.5%. The present invention includes the following aspects. Section 1. Silica particles having a micropore size of 1 to 250 angstroms (Å) and containing silane groups according to the following general formula: [ka] In the formula, R 1 and R 2 are each independently selected from the group consisting of alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl; R is hydrogen or silica, and SiO2 is silica, silica particles. Section 2. Item 2. The silica particles according to Item 1, wherein the micropore diameter is 50 to 150 angstroms (Å). Section 3. R 1 and R 2 Item 3. The silica particles according to Item 1 or 2, wherein are the same. Section 4. R 1 and R 2 is alkyl, preferably C2 to C 20 Item 4. Silica particles according to any one of items 1 to 3, which are branched or unbranched alkyl. Section 5. R 1 and R 2 But C2~C 10 Item 5. Silica particles according to any one of items 1 to 4, wherein the alkyl group is an unbranched alkyl, preferably a C4 to C8 unbranched alkyl. Section 6. Item 6. Silica particles according to any one of Items 1 to 5, wherein the silica particles have an average particle size of 0.1 to 100 μm, preferably 1 to 50 μm. Section 7. 1. A method for preparing silica particles, comprising: (i) providing silica particles having a micropore size of 1 to 250 angstroms (Å); and (ii) modifying silica particles with a compound of the formula: [ka] In the formula, R 1 and R 2 are each independently selected from the group consisting of alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl; and X 1 and X 2 are each independently a halogen or an alkoxy group. Section 8. Item 8. The method according to Item 7, wherein the micropore diameter is 50 to 150 angstroms (Å). Section 9. R 1 and R 2 and (c) are the same. Section 10. R 1 and R 2 is alkyl, preferably C2 to C 20 Branched or unbranched alkyl, more preferably C2-C 10 Item 10. The method according to any one of Items 7 to 9, wherein the alkyl is unbranched, and more preferably C4 to C8 unbranched alkyl. Section 11. X 1 and X 2 11. The method according to any one of Items 7 to 10, wherein is chlorine. Section 12. Item 12. Silica particles obtained by the method according to any one of Items 7 to 11. Section 13. A separation column for chromatography, comprising the silica particles according to any one of Items 1 to 6, or the silica particles obtained by the method according to any one of Items 7 to 11. Section 14. Use of silica particles according to any one of items 1 to 6 or obtained by the method according to any one of items 7 to 11 as a stationary phase for purifying a modified conjugated peptide, preferably a GLP-1 agonist or a GLP-2 analogue. Section 15. Item 15. The use according to Item 14, wherein the modified conjugated peptide is semaglutide or liraglutide.

Claims

1. Silica particles having a micropore size of 80 to 250 angstroms (Å) and containing silane groups according to the following general formula: 【Chemical Formula 1】 In the formula, R １ and R ２ are each independently a C 2 -C 20 branched or unbranched alkyl; R is hydrogen or silica; SiO ２ is silica, and Silica particles, wherein the pore size is measured using ISO 9277:2010.

2. 2. The silica particles according to claim 1, wherein the micropore diameter is 80 to 150 angstroms (Å).

3. R １ and R ２ The silica particles according to claim 1 , wherein are the same.

4. R １ and R ２ 2. The silica particles of claim 1, wherein is a C2-C10 unbranched alkyl.

5. R １ and R ２ 2. The silica particles of claim 1, wherein is a C 4 -C 8 unbranched alkyl.

6. The silica particles according to claim 1, wherein the silica particles have an average particle size of 0.1 to 100 μm.

7. 1. A method for preparing silica particles, comprising: (i) providing silica particles having a micropore size of 80 to 250 angstroms (Å); and (ii) modifying silica particles with a compound of the formula: 【Chemistry 2】 In the formula, R １ and R ２ are each independently a C 2 -C 20 branched or unbranched alkyl; and X １ and X ２ are each independently a halogen or an alkoxy group; The pore size is measured using ISO 9277:2010.

8. The method of claim 7, wherein the pore size is 80 to 150 angstroms (Å).

9. R １ and R ２ The method of claim 7 , wherein:

10. R １ and R ２ The method of claim 7, wherein is a C 2 -C 10 unbranched alkyl.

11. R １ and R ２ The method of claim 7, wherein is a C 4 -C 8 unbranched alkyl.

12. X １ and X ２ The method of claim 7, wherein is chlorine.

13. A separation column for chromatography, comprising silica particles according to any one of claims 1 to 6.

14. Use of silica particles according to any one of claims 1 to 6 as a stationary phase for purifying modified conjugated peptides.

15. The use according to claim 14, wherein the modified conjugated peptide is semaglutide or liraglutide.

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