NOVEL STATIONARY PHASE FOR LARGE SCALE REVERSE-PHASE HPLC PURIFICATION

Abstract

Silica particles have a fine pore size of 1 to 250 Angstrom (?) and comprise a silane group which comprises two groups which are each independently chosen from alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl groups. The silica particles are prepared by a method. The silica particles can be used as a stationary phase for purifying a modified conjugated peptide, such as a GLP-1 agonist or a GLP-2 analog.

Claims

1. A silica particle having a fine pore size of from about 1 to about 250 Angstrom (?) and comprising a silane group according to the following formula: ##STR00006## wherein R.sup.1 and R.sup.2 are each independently chosen from alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl groups; R is hydrogen or silica; and SiO.sub.2 is silica.

2. The silica particle of claim 1, wherein the fine pore size is from about 50 to about 150 Angstrom (?).

3. The silica particle of claim 1, wherein R.sup.1 and R.sup.2 are the same.

4. The silica particle of claim 1, wherein each of R.sup.1 and R.sup.2 is independently a C.sub.2-C.sub.20 branched or unbranched alkyl group.

5. The silica particle of claim 1, wherein each of R.sup.1 and R.sup.2 is independently a C.sub.4-C.sub.8 unbranched alkyl group.

6. The silica particle of claim 1, wherein the silica particle has an average particle diameter of from about 0.1 to about 100 ?m.

7. The silica particle of claim 1, wherein the silica particle has an average particle diameter of from about 1 to about 50 ?m.

8. A method for preparing a silica particle, said method comprising the steps of: (i) providing a silica particle with a fine pore size of from about 1 to about 250 Angstrom (?); and (ii) modifying the silica particle with a compound of the following formula: ##STR00007## wherein R.sup.1 and R.sup.2 are each independently chosen from alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl groups; and X.sup.1 and X.sup.2 are each independently a halogen or an alkoxy group.

9. The method of claim 8, wherein the fine pore size is from about 50 to about 150 Angstrom (?).

10. The method of claim 8, wherein R.sup.1 and R.sup.2 are the same.

11. The method of claim 8, wherein R.sup.1 and R.sup.2 are each C.sub.2-C.sub.10 unbranched alkyl groups.

12. The method of claim 8, wherein R.sup.1 and R.sup.2 are each C.sub.4-C.sub.8 unbranched alkyl group.

13. The method of claim 8, wherein X.sup.1 and X.sup.2 are both chlorine.

14. A separation column for chromatography comprising the silica particle of claim 1.

15. A method of purifying a modified conjugated peptide wherein the method comprises the step of exposing the silica particle of claim 1 to the modified conjugated peptide.

16. The method of claim 15 wherein the modified conjugated peptide is a GLP-1 agonist or a GLP-2 analog.

17. The method of claim 16 wherein the modified conjugated peptide is a semaglutide or liraglutide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present disclosure will hereinafter be described in conjunction with the following drawing figs., wherein like numerals denote like elements, and:

[0014] FIG. 1A is a chromatogram obtained for the first purification step of example 2a);

[0015] FIG. 1B is a chromatogram obtained for the second purification step of example 2a);

[0016] FIG. 2A is a chromatogram obtained for the first purification step of example 2b);

[0017] FIG. 2B is a chromatogram obtained for the second purification step of example 2b);

[0018] FIG. 3A is a chromatogram obtained for the first purification step of example 3; and

[0019] FIG. 3B is a chromatogram obtained for the second purification step of example 3.

DETAILED DESCRIPTION

[0020] The following detailed description is merely exemplary in nature and is not intended to limit the disclosure or the application and uses of the subject matter as described herein. Furthermore, there is no intention to be bound by any theory presented in the preceding background or the following detailed description. Moreover, it is contemplated that, in various non-limiting embodiments, it is to be appreciated that all numerical values as provided herein, save for the actual examples, are approximate values with endpoints or particular values intended to be read as about or approximately the value as recited.

[0021] The present disclosure is, therefore, directed to finding such improved silica particles and methods for producing the same. It has been found that the above problems can be solved by improved silica particles having a fine pore size of 1 to 250 Angstrom (?) and comprising a silane group which comprises two groups which are each independently chosen from alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl.

[0022] In a first aspect, the present disclosure is directed to silica particles having a fine pore size of 1 to 250 Angstrom (?) and comprising a silane group according to the following formula:

##STR00002##

wherein R.sup.1 and R.sup.2 are each independently chosen from alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl groups; R is hydrogen or silica; and SiO.sub.2 is silica.

[0023] In a second aspect, the present disclosure is further directed to a method for preparing a silica particle comprising the following steps: [0024] (i) providing a silica particle with a fine pore size of 1 to 250 Angstrom (?); and [0025] (ii) modifying the silica particle with a compound of the following formula

##STR00003##

wherein R.sup.1 and R.sup.2 are each independently chosen from alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl; and X.sup.1 and X.sup.2 are independently a halogen or an alkoxy group.

[0026] The present disclosure is even further directed to the use of the silica particle according to the first aspect or a silica particle obtained by the method according to the second aspect as stationary phase for purifying a modified conjugated peptide, typically a GLP-1 agonist or a GLP-2 analog.

[0027] In a first aspect, the present disclosure discloses novel stationary phases for large scale reversed phase HPLC purification, in particular for purification of modified conjugated peptides, such as GLP-1 receptor agonists and GLP-2 analogs. In particular, the present disclosure provides a silica particle which can be used as a stationary phase for large scale reversed phase HPLC purification.

[0028] The novel stationary phases according to the present disclosure comprise a silica particle having a fine pore size of 1 to 250 Angstrom (?) and comprising a silane group according to the following formula:

##STR00004##

wherein R.sup.1 and R.sup.2 are each independently chosen from alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl; R is hydrogen or silica; and SiO.sub.2 is silica.

[0029] Surprisingly, it has been found that such modified silica particle can be used for a stationary phase in HPLC application and achieves improved separation characteristics in particular for modified conjugated peptides, such as modified conjugated peptides, for instance GLP-1 receptor agonists and GLP-2 analogs, and in particular for liraglutide and semaglutide. With these new modified silica particles in stationary phase for HPLC applications, specific difficult impurities in crude active pharmaceutical ingredients, such as in liraglutide and semaglutide, can be successfully removed already in a first chromatographic step, for instance under alkaline conditions. This allows to deliver an already more purified active pharmaceutical ingredient per cycle and hour to the second chromatographic step which in turn increases the possibility to meet the pharmaceutical industry's purity demands of >99.5% and single impurity amount of <0.1% already in a second HPLC purification step with, at the same time, commercially acceptable overall yields. In other words, the modified silica particle of the present disclosure increases the selectivity as well as the recovery significantly, resulting in an overall significantly improved separation productivity.

[0030] In the above Formula I, each of R.sup.1 and R.sup.2 are each independently chosen from alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl groups.

[0031] The term alkyl refers to a straight, branched chain and/or cyclic (cycloalkyl) hydrocarbon having from 1 to 30 (e.g. 1 to 20, or 1 to 4) carbon atoms. Alkyl moieties having from 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 may be monocyclic or multicyclic, and examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and adamantyl. Additional examples of alkyl moieties have linear, branched and/or cyclic portions (e.g. 1-ethyl-4-methyl-cyclohexyl). The term alkyl as used herein includes saturated as well as unsaturated hydrocarbons, typically saturated hydrocarbons.

[0032] The term aryl refers to an aromatic ring or an aromatic or partially aromatic ring system composed of carbon and hydrogen atoms. An aryl moiety may comprise multiple rings bound 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.

[0033] The term alkylaryl refers to a moiety comprising at least one alkyl group and at least one aryl group, such as an alkyl moiety bound to an aryl moiety, wherein the terms alkyl and aryl are as defined above.

[0034] The term heteroalkyl refers to an alkyl group which also include at least one heteroatom, such as a nitrogen, oxygen, phosphor, sulfur and/or boron atom, wherein the term alkyl is as defined above.

[0035] The term heteroaryl refers to an aryl group which also contains at least one heteroatom, such as a nitrogen, oxygen, phosphor, sulfur and/or boron atom, wherein the term aryl is as defined above. For instance, the term heteroaryl typically refers to an aryl group wherein at least one of its carbon atoms has been replaced with nitrogen, oxygen, or sulfur. Examples of heteroaryl moieties include acridinyl, benzimidazolyl, benzofuranyl, benzoisothiazolyl, benzoisoxazolyl, benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furyl, imidazolyl, indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, phthalazinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolinyl, tetrazolyl, thiazolyl, and triazinyl.

[0036] The term heteroalkylaryl refers to a moiety comprising at least one alkyl group, at least one aryl group, and at least one heteroatom, such as a nitrogen, oxygen, phosphor, sulfur and/or boron atom, wherein the terms alkyl and aryl are as defined above.\

[0037] Any of the above groups alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl may be unsubstituted or may be further substituted.

[0038] The term substituted, when used to describe a chemical structure or moiety, refers to a derivative of that structure or moiety wherein one or more of its hydrogen atoms is substituted with an atom, chemical moiety or functional moiety such as, but not limited to, alcohol, aldehylde, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl), alkynyl, alkylcarbonyloxy (OC(O)alkyl), amide (C(O)NH-alkyl- or -alkyl-NHC(O)alkyl), amidinyl (C(NH)NH-alkyl or C(NR)NH2), amine (primary, secondary and tertiary such as alkylamino, arylamino, arylalkylamino), aroyl, aryl, aryloxy, azo, carbamoyl (NHC(O)O-alkyl- or OC(O)NH-alkyl), carbamyl (e.g. CONH.sub.2, as well as 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. CCl.sub.3, CF.sub.3, C(CF.sub.3).sub.3), heteroalkyl, hemiacetal, imine (primary and secondary), ketone, nitrile, nitro, oxygen (i.e., to provide an oxo moiety), phosphodiester, sulfide, sulfonamido (e.g. SO.sub.2NH.sub.2), sulfone, sulfonyl (including alkylsulfonyl, arylsulfonyl and arylalkylsulfonyl), sulfoxide, thiol (e.g. sulfhydryl, thioether) and urea (NHCONH-alkyl-).

[0039] In some embodiments, both R.sup.1 and R.sup.2 independently include 2 to 20 carbon atoms.

[0040] In some embodiments, R.sup.1 and R.sup.2 are typically both independently chosen from alkyl and heteroalkyl. In some embodiments, R.sup.1 and R.sup.2 are both independently alkyl, such as C.sub.2-C.sub.20 branched or unbranched alkyl, typically C.sub.2-C.sub.10-branched or unbranched alkyl, such as C.sub.3-C.sub.10-branched or unbranched alkyl or C.sub.3-C.sub.8-branched or unbranched alkyl, and even more typically C.sub.4-C.sub.8-branched or unbranched alkyl, such as C.sub.4-branched or unbranched alkyl, C.sub.5-branched or unbranched alkyl, C.sub.6-branched or unbranched alkyl, C.sub.7-branched or unbranched alkyl, or C.sub.8-branched or unbranched alkyl, or for instance C.sub.4-C.sub.6-branched or unbranched alkyl. Particular typical are unbranched alkyls, such as C.sub.2-C.sub.20-unbranched alkyl, C.sub.2-C.sub.10-unbranched alkyl, C.sub.3-C.sub.10-unbranched alkyl, C.sub.3-C.sub.8-unbranched alkyl, or even C.sub.4-C.sub.8-unbranched alkyl, such as C.sub.4-unbranched alkyl, C.sub.5-unbranched alkyl, C.sub.6-unbranched alkyl, C.sub.7-unbranched alkyl, or C.sub.8-unbranched alkyl, or for instance C.sub.4-C.sub.6-unbranched alkyl. In a particularly typical embodiment, R.sup.1 and R.sup.2 are both independently C.sub.4-, C.sub.6- and/or C.sub.8-branched or unbranched alkyl, and even more typical C.sub.4-, C.sub.6- and/or C.sub.8-unbranched alkyl. In another typical embodiment, R.sup.1 and R.sup.2 are both the same. For instance, in some embodiments R.sup.1 and R.sup.2 are both C.sub.4-, C.sub.6- or C.sub.8-branched or unbranched alkyl, such as C.sub.4- or C.sub.8-branched or unbranched alkyl, and even more typical C.sub.4-, C.sub.6- or C.sub.8-unbranched alkyl, such as C.sub.4- or C.sub.8-unbranched alkyl.

[0041] SiO.sub.2 in the above Formula I means silica. This means the silane group of Formula I is bond to a silica structure, i.e. a silica particle, via a OSi-bond.

[0042] The silica or particle of silica may be obtained from e.g. precipitated silica, micro silica (silica fume), pyrogenic silica (fumed silica), silica sols or silica gels, and mixtures thereof. The particle of silica may be in the form of a porous particle.

[0043] Suitably, the particle of silica has a specific surface area from 20 to 1500, typically from 50 to 900, and most typically from 70 to 800 m.sup.2/g. The specific surface area can be measured by titration with sodium hydroxide as described by Sears in Analytical Chemistry 28(1956), 12, 1981-1983 and in U.S. Pat. No. 5,176,891. The given area thus represents the average specific surface area of the particles.

[0044] The rest R in the above formula is either hydrogen or silica. Silica means in this context that the silane group of Formula I is bond to the silica or silica particle via a further OSi-bond.

[0045] The silica particle of the present disclosure has a fine pore size of 1 to 250 Angstrom (?), such as for instance from 10 to 200 ?, or from 50 to 250 ?, or from 60 to 200 ?, or even from 70 to 180 ?. Particularly typical are fine pore sizes of 80 to 150 ?, such as from 100 to 130 ?, and in particular a fine pore size of around 100 ? or around 120 ?. It has been found that fine pore sizes above 250 ? tend to significantly impair the overall separation productivity of the silica particle in a HPLC column, i.e. the overall yield of purified components is low. All references to a fine pore size as used herein refer to the average fine pore size. Methods for determining the fine pore size of a silica particle are commonly known by the skilled person and include for instance the Brunauer-Emmett-Teller (BET)-method by for instance using the device TriStar II Plus from Micromeritics.

[0046] The particle size of the silica particle of the present disclosure is not particularly limited. For instance, the silica particle of the present disclosure may have an average particle diameter of 0.1 to 100 ?m, and typically from 1 to 50 ?m. In some embodiments, it may even have an average particle diameter ranging from 1.8 to 25 ?m. Methods for determining the average particle diameter of a silica particle are commonly known by the skilled person. For instance, the particle diameter may be calculated from the formula relating to specific surface area and particle diameter in ller (The Chemistry of Silica, Wiley, 1979). As conventional in silica chemistry, the particle size refers to the average size of the primary particles. The particle size may also be measured via the coulter principle on a Multisizer 4e Coulter Counter from Beckman Coulter.

[0047] The pore volume of the particle of modified silica is suitably from 0.1 to 4 ml/g, typically from 0.2 to 2 ml/g, most typically from 0.3 to 1.2 ml/g.

[0048] The specific surface area (BET method) of the particle of modified silica is suitably from 1 to 1000 m.sup.2/g, typically from 25 to 700 m.sup.2/g, most typically from 50 to 500 m.sup.2/g.

[0049] A suitable way of measuring surface area, pore volume and average fine pore size (from the surface area and pore volume) is by the Brunauer-Emmett-Teller (BET) method, also based on nitrogen adsorption/desorption, the surface area typically being calculated from the linear part of the isotherm. Examples of such methods are given in ISO9277:2010 (for BET) and ISO 15901-2:2006 (for gas adsorption/desorption). Typically, surface area, pore volume and average fine pore size are measured using ISO9277:2010.

[0050] In another aspect, the present disclosure describes a method for preparing a silica particle comprising the following steps: [0051] (i) providing a silica particle with a fine pore size of 1 to 250 Angstrom (?); and [0052] (ii) modifying the silica particle with a compound of the following formula

##STR00005## [0053] wherein R.sup.1 and R.sup.2 are each independently chosen from alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl groups; and [0054] X.sup.1 and X.sup.2 are each independently a halogen or an alkoxy group.

[0055] With this method it is possible to obtain a silica particle according to the first aspect of the present disclosure as described above. Hence, the present disclosure is also directed to silica particles obtained by the method as described above according to this aspect of the present disclosure.

[0056] The silica particle provided in step (i) of the method according to the second aspect of the present disclosure may be as described above and may have the same typical fine pore sizes as described above in the context of the first aspect of the present disclosure.

[0057] The compound according to Formula II which is used in step (i)i of the method according to the second aspect of the present disclosure to modify the silica particle is a silane which comprises two groups R.sup.1 and R.sup.2 which are each independently chosen from alkyl, aryl, alkylaryl, heteroalkyl, heteroaryl, and heteroalkylaryl, and two further groups X.sup.1 and X.sup.2 which are each independently a halogen or an alkoxy group.

[0058] Groups R.sup.1 and R.sup.2 may be the same as described above in the context of the first aspect of the present disclosure.

[0059] Groups X.sup.1 and X.sup.2 which are each independently a halogen or an alkoxy group, typically a halogen.

[0060] Halogens include fluorine, chlorine, bromine and iodine, of which chlorine is particularly typical.

[0061] The term alkoxy group refers to an O-alkyl moiety, wherein the alkyl group can be as defines above in the context of the first aspect of the present disclosure. Typical are alkoxy groups wherein the alkyl moiety has 1-8 carbon atoms, such as 1-4 carbon atoms. Examples of alkoxy moieties include OCH.sub.3, OCH.sub.2CH.sub.3, O(CH.sub.2).sub.2CH.sub.3, O(CH.sub.2).sub.3CH.sub.3, O(CH.sub.2).sub.4CH.sub.3, and O(CH.sub.2).sub.5CH.sub.3.

[0062] The modification reaction as such is known by the skilled person and can be adjusted according to needs. For instance, the reaction of the compound of Formula II with the silica particle is performed at a temperature suitable for performing the reaction, typically from 40 to 180, more typically from 60 to 160, and most typically from 80 to 140? C.

[0063] Typically, the compound of Formula II may be added to the silica particle under agitation and at a controlled rate, until a suitable amount of compound of Formula II has been added. The reaction time may be from 1 to 24 hours.

[0064] The reaction of the compound of Formula II and the silica particle may be carried out in an organic solvent, typically under stirring. Such an organic solvent is typically an aprotic solvent. Examples of such an aprotic solvent may be acetonitrile, acetone, xylene or toluene, typically toluene, or mixtures thereof.

[0065] The silica particle may be added to the solvent, followed by an optional step of evaporating any water present. The compound of Formula II may then be added to a solvent, and thereafter added to the dispersion of silica particle and solvent.

[0066] The proportion of the compound of Formula II may be from 4 to 8 ?moles per m.sup.2 particle of silica surface. The amount of solvent is typically selected in such a way that the amount of silica particle in the dispersion is from 5 to 20 wt %.

[0067] The silanol surface moieties on the silica particle react with the compound of Formula II to form a covalent link between the silica and the compound of Formula II.

[0068] When the dispersion of modified silica particles has been formed, the dispersion may be cooled and purified, e.g. by ultra filtration, or by washing, e.g. by using toluene, ethanol or formic acid. The dispersion may then be dried, for example at 40 to 100? C., typically at 60 to 90? C., for 2 to 30 hours, typically from 10 to 25 hours.

[0069] According to an even further aspect, the present disclosure relates to a separation column for chromatography comprising the silica particle of the present disclosure, i.e. the silica particle according to the first aspect of the present disclosure or a silica particle obtained by the method according to the second aspect of the present disclosure. In this aspect, the silica particles of the disclosure have been packed into the separation column. Hence, in this aspect the silica particles of the disclosure are used a stationary phase for chromatography. The silica particles may thus be used in a separation column for any type of chromatographic separation methods, such as HPLC, supercritical fluid chromatography (SFC), and simulating moving bed (SMB).

[0070] According to an even further aspect, the present disclosure relates to the use of the silica particle according to the first aspect of the present disclosure or a silica particle obtained by the method according to the second aspect of the present disclosure as stationary phase for purifying a modified conjugated peptide, typically a GLP-1 agonist or a GLP-2 analog. It is particularly typical that the silica particle according to the first aspect of the present disclosure or a silica particle obtained by the method according to the second aspect of the present disclosure is used as stationary phase for purifying semaglutide or liraglutide.

EXAMPLES

Example 1: Preparation of Modified Silica Particles

[0071] The following commercially available Kromasil? particles (from Nouryon B.V.) were used as the starting material to prepare different modified silica particles.

[0072] Silica particles having an average particle size of 5 ?m, an average pore size of 100 ? and a specific surface area of 315 m.sup.2/g (Kromasil? KR-100-5 SIL of Nouryon B.V.).

[0073] Silica particles having an average particle size of 10 ?m, an average pore size of 100 ? and a specific surface area of 320 m.sup.2/g (Kromasil? KR-100-10 SIL of Nouryon B.V.).

[0074] Silica particles having an average particle size of 5 ?m, an average pore size of 200 ? and a specific surface area of 195 m.sup.2/g (Kromasil? KR-200-5 SIL of Nouryon B.V.).

[0075] Silica particles having an average particle size of 10 ?m, an average pore size of 300 ? and a specific surface area of 105 m.sup.2/g (Kromasil? KR-300-10 SIL of Nouryon B.V.).

Example 1 a)

[0076] 20 g of KR-100-5 SIL and 10.3 g imidazole was added to 175 g 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 dispersion of toluene, silica, and imidazole under stirring. The temperature was raised, and the dispersion was refluxed overnight. The reaction was cooled to 70? C. and 32 g ethanol was added to deactivate the silane. The dispersion was washed by 87 g toluene and 2?79 g ethanol. The silica was dried at 90? C. in a lab oven overnight. The resulting powder was white and is further referred to herein as Kromasil? 100-5-diC4. Elemental analysis of carbon gave 8.3 wt % C.

Example 1 b)

[0077] KR-100-10 SIL was modified in the same manner with dibutyldichlorosilane as described above in Example 1 a) for KR-100-5 SIL. The resulting powder was white and is further referred to herein as Kromasil? 100-10-diC4. Elemental analysis of carbon gave 8.4 wt % C.

Example 1 c)

[0078] KR-100-10 SIL was modified in the same manner as described above in Example 1 a) except of that 5.7 g butyldimethylchlorosilane and 4.1 g trimethylchlorosilane were added instead of dibutyldichlorosilane as surface modification. The resulting powder was white and is further referred to herein as Kromasil? 100-10-C4. Elemental analysis of carbon gave 7.7 wt % C.

[0079] Example 1 d)

[0080] KR-200-5 SIL was modified in the same manner with 5.0 g dibutyldichlorosilane, as described above in Example 1 a) for KR-100-5 SIL. The resulting powder was white and is further referred to herein as Kromasil? 200-5-diC4. Elemental analysis of carbon gave 5.2 wt % C.

Example 1 e)

[0081] KR-100-5 SIL was modified in the same manner as described above in Example 1 a) except of that instead of dibutyldichlorosilane, 12.4 g dioctyldichlorosilane was used. The resulting powder was white and is further referred to herein as Kromasil? 100-5-diC8. Elemental analysis of carbon gave 14.8 wt % C.

Example 1 f)

[0082] KR-100-5 SIL was modified in the same manner as described above in Example 1 a) except of that instead of dibutyldichlorosilane, 10.2 g dihexyldichlorosilane was used. The resulting powder was white and is further referred to herein as Kromasil? 100-5-diC6. Elemental analysis of carbon gave 11.9 wt % C.

Example 1 g)

[0083] KR-300-10 SIL was modified with 2.7 g dibutyldichlorosilane in the same manner as described above in Example la) for KR-100-5 SIL. The resulting powder was white and is further referred to herein as Kromasil? 300-10-diC4. Elemental analysis of carbon gave 3.1 wt % C.

Example 2: Two Step Purification of Liraglutide

[0084] In all following examples, the samples to be purified was a mixture including 5.0 mg mL.sup.?1 of crude liraglutide (purity ?10%) which was prepared by dissolving 80 mg of crude liraglutide in 16 mL of NH.sub.4HCO.sub.3 buffer (0.1 mol L.sup.?1, pH 7.0)/Acetonitrile (95:5), filtered through a 0.45 ?m syringe filter and which was injected directly in the column.

Example 2 a): Two Step Purification of Liraglutide on Kromasil@ 100-10-diC4

[0085] The first purification step was performed at pH 7 with ammonium hydrogen carbonate, and the second purification was performed at pH 8 with ammonium acetate as buffer, and in both systems, acetonitrile was used as the organic modifier. The purification was performed on an Agilent 1260 system equipped with an automated fraction collector. Both purification steps were made on a 4.6?250 mm column packed with Kromasil@ 100-10-diC4 from Example 1 b).

[0086] First purification step: A crude liraglutide sample (purity ?10%), was loaded onto the column, 32 mg/g stationary phase, where the mobile phase included 0.1 M ammonium hydrogen carbonate, pH 7, and acetonitrile. The purification was performed under gradient elution, according to the following sequence. A loading step with 5% acetonitrile for 5 minutes, between 5 minutes and 138 minutes, the acetonitrile concentration was increased linearly from 29.4%to 39.8%. After 128 minutes the acetonitrile concentration was increased to 80% for 11 minutes to elute strongly adsorbed components and to wash the column, before the acetonitrile concentration was decreased to 5% to equilibrate the column. Liraglutide was collected between 105 minutes and 115 minutes, and the pooled fractions showed a purity of 97.1%, with a recovery of 81%. The obtained chromatogram is shown in FIG. 1a.

[0087] Second purification step: The pooled fractions from the first purification were diluted to about 10% acetonitrile and loaded onto the column including Kromasil@ 100-10-diC4, where the mobile phase included 0.1 M ammonium acetate buffer at pH 8, and acetonitrile as organic modifier. The purification was performed under gradient elution, according to the following sequence A loading step with 5% acetonitrile for 5 minutes, between 5 minutes and 95 minutes, the acetonitrile concentration was increased linearly from 30.5% to 41.3%. After 95 minutes the acetonitrile concentration was increased to 80% for 11 minutes to elute strongly adsorbed components and to wash the column before the acetonitrile concentration was decreased to 5% to equilibrate the column. Liraglutide was collected between 79 minutes and 86 minutes, and the pooled fractions showed a purity of ?99.5%, with recovery of 98.2%. Hence, the total recovery over both purification steps was 79.6%. The obtained chromatogram is shown in FIG. 1b.

Example 2 b): Two Step Purification of Liraglutide on Kromasil@ 100-5-diC8

[0088] The first purification step was performed at pH 7 with ammonium carbonate, and the second purification was performed at pH 8 with ammonium acetate as buffer, and in both systems, acetonitrile was used as the organic modifier. The purification was performed on an Agilent 1260 system equipped with an automated fraction collector. both purification steps were made on a 4.6?150 mm column packed with Kromasil@ 100-5-diC8 from Example 1 c) above.

[0089] Frist purification step: A crude liraglutide sample (purity ?10%), was loaded onto the column, 12 mg/g stationary phase, where the mobile phase included 0.1 M ammonium hydrogen carbonate, pH 7, and acetonitrile. The purification was performed under gradient elution, according to the following sequence. A loading step with 5% acetonitrile for 5 minutes, between 5 minutes and 45 minutes, the acetonitrile concentration was increased linearly from 29.8% to 40.2%. After 45 minutes the acetonitrile concentration was increased to 70% for 11 minutes to elute strongly adsorbed components and to wash the column before the acetonitrile concentration was decreased to 5% to equilibrate the column. Liraglutide was collected between 37 minutes and 40 minutes, and the pooled fractions showed a purity of 96.4%, with a recovery of 96%. The obtained chromatogram is shown in FIG. 2a.

[0090] Second purification step: The pooled fractions from the first purification was diluted to about 10% acetonitrile, and loaded on to the column including Kromasil@ 100-5-diC8, where the mobile phase included 0.1 M ammonium acetate buffer at pH 8, and acetonitrile as organic modifier. The purification was performed under gradient elution, according to the following sequence. A loading step with 5% acetonitrile for 5 minutes, between 5 minutes and 45 minutes, the acetonitrile concentration was increased linearly from 29.8% to 40.2%. After 45 minutes the acetonitrile concentration was increased to 70% for 11 minutes to elute strongly adsorbed components and to wash the column before the acetonitrile concentration was decreased to 5% to equilibrate the column. Liraglutide was collected between 43 minutes and 45 minutes, and the pooled fractions showed a purity of 99.6%, with a total recovery over both purification steps of 79%. The obtained chromatogram is shown in FIG. 2b.

Example 2 c): Two Step Purification of Liraglutide on Kromasil? 100-10-C4 (Comparative Example)

[0091] A two-step purification of a liraglutide sample on Kromasil? 100-10-C4 from Example 1 c) above was performed in the same manner as described above in Example 2 a) for the two step purification of liraglutide on Kromasil@ 100-10-diC4 using the same solvent gradients and equipment parameters.

[0092] In the first purification step, the fractions were collected every minute between 100 and 125 minutes. Fractions No. 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%.

[0093] In the second purification step, the fractions were collected every 30 seconds, between 77 and 87 minutes. Pooled fractions No. 12 to 16 presented a purity of 99.5%, with a recovery of 65.6%. Hence, the total recovery over both purification steps was merely 57.3%. This is considerably lower than the total recovery achieved for instance by using Kromasil@ 100-10-diC4 (see above Example 2 a)) for which a total recovery of 79.6% was achieved.

Example 2 d): Two Step Purification of Liraglutide on Kromasil? 300-10-diC4 (Comparative Example)

[0094] A two-step purification of a liraglutide sample on Kromasil? 300-10-diC4 from Example 1 g) above was performed in the same manner as described above in Example 2 a) for the two-step purification of liraglutide on Kromasil@ 100-10-diC4 using the same solvent gradients and equipment parameters.

[0095] In the first purification step, the fractions were collected every minute between 92 and 109 minutes. Fractions No. 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%.

[0096] In the second purification step, the fractions were collected every 30 seconds, between 71 and 86 minutes. Only fraction No. 16 presented an acceptable purity of 99.5%, with a recovery of 4.7%. Hence, the total recovery over both purification steps was merely 3.4%. This is considerably lower than the total recovery achieved for instance by using Kromasil@ 100-10-diC4 (see above Example 2 a)) for which a total recovery of 79.6% was achieved.

Example 3: Two Step Purification of Semaglutide on Kromasil@ 100-10-diC4

[0097] The first purification step was performed at pH 7 with ammonium hydrogen carbonate, and the second purification was performed at pH 8 with ammonium acetate as buffer, and in both systems, acetonitrile was used as the organic modifier. The purification performed on an Agilent 1260 system equipped with an automated fraction collector. both purification steps were made on a 4.6?250 mm column packed with Kromasil@ 100-10-diC4 from Example 1 c) above. The semaglutide sample to be purified was a mixture including 5.0 mg mL.sup.?1 of crude semaglutide (purity ?60%) which was prepared by dissolving 80 mg of semaglutide in 16 mL of NH.sub.4HCO.sub.3 buffer (0.1 mol L.sup.?1, pH 7.0)/Acetonitrile (95:5), filtered through a 0.45 ?m syringe filter and which was injected directly in the column.

[0098] First purification step: A crude semaglutide sample (purity ?60%), was loaded on to the column, 34 mg/g stationary phase, where the mobile phase included 0.1 M ammonium hydrogen carbonate, pH 7, and acetonitrile. The purification was performed under gradient elution, according to the following sequence. A loading step with 5% acetonitrile for 5 minutes, between 5 minutes and 138 minutes, the acetonitrile concentration was increased linearly from 30% to 40.5%. After 128 minutes the acetonitrile concentration was increased to 80% for 11 minutes to elute strongly adsorbed components and to wash the column, before the acetonitrile concentration was decreased to 5% to equilibrate the column. Semaglutide was collected between 50 minutes and 70 minutes, and the pooled fractions showed a purity of 98.1%, with a recovery of 95%. The obtained chromatogram is shown in FIG. 3a.

[0099] Second purification step: The pooled fractions from the first purification was diluted to about 10% acetonitrile, and loaded on to the column including Kromasil@ 100-10-diC4, where the mobile phase included 0.1 M ammonium acetate buffer at pH 8, and acetonitrile as organic modifier. The purification was performed under gradient elution, according to the following sequence. A loading step with 5% acetonitrile for 5 minutes, between 5 minutes and 95 minutes, the acetonitrile concentration was increased linearly from 30.5% to 41.3%. After 95 minutes the acetonitrile concentration was increased to 80% for 11 minutes to elute strongly adsorbed components and to wash the column before the acetonitrile concentration was decreased to 5% to equilibrate the column. Liraglutide was collected between 40 minutes and 65 minutes, and the pooled fractions showed a purity of 99.6%, with total a recovery over both purification steps of 76%. The obtained chromatogram is shown in FIG. 3b.

Example 4: Further One Step Purification Tests for Liraglutide

[0100] Further experiments were made to confirm that various silica particles of the present disclosure are suitable to achieve already in a first purification step purity levels for liraglutide of at least 95% with a commercially acceptable yield for a first purification step, such as at least 70%, typically at least 80%, and more typically at least 90%. The liraglutide sample to be purified, the solvent gradient and the equipment parameters were the same as described above for Example 2 a). Merely the silica particles used in the HPLC column as stationary phase have been changed. In Example 4 a) Kromasil? 100-5-diC4 from Example 1 a) above was used. In Example 4 b) Kromasil? 200-5-diC4 from Example 1 d) above was used. In Example 4 c) Kromasil? 100-5-diC6 from Example 1 f) above was used.

[0101] Table 1 summarizes the results and also incorporates the results of Examples 2 and 3 above.

TABLE-US-00001 TABLE 1 Liraglutide purification using different silica phases Second purification step (%) First purification step (%) overall Example Silica phase Purity Recovery Purity recovery * 2 a) 100-10-diC4 97.1 81 ?99.5 79.6 2 b) 100-5-diC8 96.4 96 99.6 79 2 c) .sup.+ 100-10-C4 96.5 87.4 99.5 57.3 2 d) .sup.+ 300-10-diC4 94.5 73.8 99.5 3.4 3 ** 100-10-diC4 98.1 95 99.6 76 4 a) 100-5-diC4 97.8 90.1 4 b) 200-5-diC4 96 70.5 4 c) 100-5-diC6 95.6 96 * = overall recovery over both purification steps .sup.+ = comparative example ** = semaglutide was purified rather than liraglutide = not performed

[0102] It is apparent from above Table 1 that the silica particles of the present disclosure are particularly suitable to achieve already in a first purification step purity rates of above 95% with commercially acceptable yields of at least 70%. Moreover, the silica particles of the present disclosure also achieve over all recovery rates (i.e. yields) of well above 75% at the pharmaceutically relevant purification rate of at least 99.5%.

[0103] While at least one exemplary embodiment has been presented in the foregoing detailed description, it should be appreciated that a vast number of variations exist. It should also be appreciated that the exemplary embodiment or exemplary embodiments are only examples, and are not intended to limit the scope, applicability, or configuration in any way. Rather, the foregoing detailed description will provide those skilled in the art with a convenient road map for implementing an exemplary embodiment. It being understood that various changes may be made in the function and arrangement of elements described in an exemplary embodiment without departing from the scope as set forth in the appended claims.