WO2014077801A1

WO2014077801A1 - Purification process for preparing highly pure taspoglutide

Info

Publication number: WO2014077801A1
Application number: PCT/US2012/064843
Authority: WO
Inventors: Philip SHANAHAN; Patrick Foley; Floriana STOMEO; Zhengxin Dong; Jundong Zhang; Xiaojun ZOU
Original assignee: Ipsen Pharma S.A.S.
Priority date: 2012-11-13
Filing date: 2012-11-13
Publication date: 2014-05-22

Abstract

The present invention relates to a purification process of (Aib^8,35)GLP-1(7-36)NH₂applying reversed phase HPLC, comprising a first and a second chromatography step, to obtain a peptide product with a high purity. The first step is performed with TFA in an water solution as eluent A and TFA in an acetonitrile solution as eluent B of the mobile phases, with a gradient increase of eluent B. The second step is performed in an acidic buffer and corresponding to a salt exchange step followed by a gradient elution. The resulting high purity peptide is suitable for clinical applications.

Description

Purification process for preparing highly pure Taspoglutide

The present invention relates to a process for the purification of analogues of human glucagon-like peptide-1 (GLP-1 ), particularly to a process for the purification of the GLP-1 analogue (Aib^8,35)GLP-1 (7-36)NH₂ with the amino acid sequence:

His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-lle-Ala-Trp-Leu-Val-Lys-Aib-Arg-NH₂.

The synthesis of GLP-1 analogues can follow a hybrid approach encompassing both solid phase peptide synthesis (SPPS) and fragment couplings in solution. The peptide (Aib^8,35)GLP-1 (7-36)NH₂ designates an analogue formally derived from natural human GLP-1 (7-36)NH₂ by substituting the naturally occurring amino acid residues in positions 8 (Ala) and 35 (Gly) with a-aminoisobutyric acid (Aib).

This peptide and its therapeutical use as well as its preparation by SPPS are known from the PCT patent application WO 2000/34331 . However, the process of this peptide as described in WO 2000/34331 is not suitable for commercial scale production.

The PCT Publication WO 2007/147816 describes a preparation of this particular analogue of GLP-1 by preparing three fragments and coupling these fragments in solution whereas the PCT patent application WO 2010/033254 describes also a preparation comprising a stepwise solid-phase Fmoc-chemistry. However, whatever the preparation process used, the purity is in general less than 95 %.

Purification for human glucagon-like peptide-1 using chromatography has been widely described in the art.

For instance, according to the PCT Publication WO 2007/147816, the GLP-1 analogue is subjected to a two-step RP-HPLC process using tetrahydrofuran. However, the use of this eluent is detrimental for performing RP-HPLC on a large scale as it can form peroxides.

The PCT patent application WO 201 1 /161007 proposes to employ an acidic RP-HPLC step followed by a RP-H PLC performed at high pH of the mobile phase. However, the high pH conditions make the peptide less stable and generate new impurities, resulting in inconsistancy of purities between batches. There is thus a real need to develop a process to obtain a highly pure (Aib^8,35)GLP-1 (7- 36)NH₂ on a commercial scale, aiming to reduce impurities, and thus side effects, and to increase the tolerance of the treatment. The Applicant has now discovered that by two specific chromatography steps for the purification of (Aib^8,35)GLP-1 (7-36)NH₂, it was possible to obtain a higher purity of this product at levels of production suitable for commercial scale production.

Indeed, the purity of the final product is in one preferred embodiment of this invention required to be higher than 99.3 % with an individual impurity not larger than 0.3 %. For example, the purity of the final product is about 99.3 %, about 99.4 %, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, and the individual impurity is about 0.3 %, about 0.2 %, about 0.1 %. In another preferred embodiment, the purity of the final product is at least 99.9 % and the individual impurity is less than 0.1 %. The availability of highly pure (Aib^8,35)GLP-1 (7-36)NH₂ is critical for its clinical evaluation.

Moreover, the purification process according to the invention leads to a reduction of acetate content in the final product. In one preferred embodiment of the invention, the acetate content is not larger than 0.3%.

One subject of the present invention is thus a purification process of (Aib^8,35)GLP-1 (7-36)NH₂ applying reversed phase HPLC comprising a first and a second chromatography step,

- the first step being performed with TFA (trifluoroacetic acid) in a water solution as eluent A and TFA in an acetonitrile solution as eluent B of the mobile phases, with a gradient increase of eluent B, and - the second step being performed in an acidic buffer and corresponding to a salt exchange step followed by a gradient elution.

In the text herein below, unless otherwise indicated, the limits of a range of values are included in that range, especially in the expression "ranging from".

An acidic buffer is an acidic solution containing a buffer agent which prevents a change in the pH value.

The term "an individual impurity" defines herein a peptide that is not (Aib⁸'³⁵)GLP-1 (7-36)NH₂.

Unless otherwise indicated, the following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein. A water solution is a solution containing at least 50 % (w/w) of water, preferably at least 60 % (w/w) of water, more preferably at least 70 % (w/w) of water. In a preferred embodiment, the water solution contains at least 80 % (w/w) of water, preferably at least 90 % (w/w) of water, more preferably at least 95 % (w/w) of water. A water solution may contain water and another solvent selected from acetonitrile and alcohol, in particular methanol, ethanol and propyl alcohol. In a preferred embodiment, a water solution is a solution containing only water or water and acetonitrile. In a more preferred embodiment, a water solution is a solution containing only water.

An acetonitrile solution is a solution containing 50 % (w/w) of acetonitrile, preferably at least 60 % (w/w) of acetonitrile, more preferably at least 70 % (w/w) of acetonitrile. In a preferred embodiment, the acetonitrile solution contains at least 80 % (w/w) of acetonitrile, preferably at least 90 % (w/w) of acetonitrile, more preferably at least 95 % (w/w) of acetonitrile. An acetonitrile solution may contain acetonitrile and another solvent selected from water and alcohol, in particular methanol, ethanol and propyl alcohol. In a preferred embodiment, an acetonitrile solution is a solution containing acetonitrile alone or acetonitrile and water. In a more preferred embodiment, an acetonitrile solution is a solution containing only acetonitrile.

Another subject of the present invention is thus a purification process of (Aib^8,35)GLP-1 (7- 36)NH₂ applying reversed phase H PLC comprising a first and a second chromatography step,

- the first step being performed with TFA in water as eluent A and TFA in acetonitrile as eluent B of the mobile phases, with a gradient increase of eluent B, and

- the second step being performed in an acidic buffer and corresponding to a salt exchange step followed by a gradient elution.

Preferably, the gradient of the first step is ranging from 100 to 0 % (v/v) of eluent A and 0 to 100 % (v/v) of eluent B. In another preferred embodiment, the gradient of the first step is ranging from 90 to 5 % (v/v) of eluent A and from 10 to 95 % (v/v) of eluent B.

In a preferred embodiment, the first step is performed at a pH ranging from 1 .0 to 4.0. More preferably, the first step is performed at a pH ranging from 1 .5 to 2.5, even more preferentially from 1 .8 to 2.2 and is about 2 (2 ± 0.1 ).

In a particular embodiment, TFA used in the mobile phase of the first step is present in a concentration ranging from 0.005 to 0.5 % by volume relative to the total volume of the water solution. In another preferred embodiment, TFA used in the mobile phase of the first step is present in a concentration ranging from 0.01 to 0.2 % by volume, more preferably from 0.05 to 0.15 % by volume and even more preferably the concentration is about 0.1 % (0.1 ± 0.05 %) by volume relative to the total volume of the water solution.

In another particular embodiment, TFA used in the mobile phase of the first step is present in a concentration ranging from 0.005 to 0.5 % by volume relative to the total volume of the acetonitrile solution. In another preferred embodiment, TFA used in the mobile phase of the first step is present in a concentration ranging from 0.01 to 0.2 % by volume, more preferably from 0.05 to 0.15 % by volume and even more preferably the concentration is about 0.1 % (0.1 ± 0.05 %) by volume relative to the total volume of the acetonitrile solution. After the first chromatography step, the purity of the product is higher than 95 %, and more preferably higher than 96 %.

The product not having the required purity of 95 % is re-purified under the same conditions as previously for the first step. If the product does not have the required purity of 95 %, it may be repeatedly re-purified under the same conditions as used in the first step until the resulting product has the required purity of 95 %. For example, the product not having the required purity of 95 % may be re-purified under the same conditions as used in the first step at least one, at least twice, at least three times, etc.

The product having the required purity of 95 % is then purified by the second chromatography step. According to the process of the invention, the second step is performed in an acidic buffer and corresponds to a salt exchange step followed by a gradient elution.

In a preferred embodiment, the second step is performed with an acidic buffer at a pH ranging from 1 .0 to 4.0. More preferably, the second step is performed with an acidic buffer at a pH ranging from 2.0 to 3.0, even more preferentially from 2.5 to 2.9, and in particular around 2.75 (2.75 ± 0.05).

According to the present invention, in step b), the gradient elution may be performed with one or more acidic eluent(s), and more particularly with two acidic eluents, eluent x and eluent y. In such a case, the total amount of eluent x and eluent y is always 100 %. Eluents x and y may be an acidic aqueous or organic solution. Such solution may be a solution of acid, such as acetic acid in water, or in an organic solvent, such as acetonitrile or alcohol, in particular methanol, ethanol and propyl alcohol.

In a preferred embodiment, the salt exchange step is performed with ammonium acetate as acidic buffer. Preferably, ammonium acetate is used in the salt exchange step in a concentration ranging from 0.005 to 0.5 N. More preferably, ammonium acetate is used in the salt exchange step in a concentration ranging from 0.01 to 0.2 N, even more preferably from 0.05 to 0.15 N and more particularly in a concentration of about 0.1 N (0.1 ± 0.05 N). In another particular embodiment, the acidic buffer used in the salt exchange step additionally comprises acetonitrile.

In a particular embodiment, the gradient elution of the second step is performed with acetic acid in water as eluent D and with acetic acid in acetonitrile as eluent E.

Advantageously, the gradient of the second step is ranging from 0 to 70 % (v/v) of eluent E and from 100 to 30 % (v/v) of eluent D, and more preferably the gradient of the second step is ranging from 0 to 60 % (v/v) of eluent E and from 100 to 40 % (v/v) of eluent D.

In another particular embodiment, the gradient elution of the second step is performed with acetic acid in water as eluent D and with acetonitrile as eluent F.

Advantageously, the gradient of the second step is ranging from 100 to 0 % (v/v) of eluent D and from 0 to 100 % (v/v) of eluent F, and more preferably, the gradient of the second step is ranging from 90 to 0 % (v/v) of eluent D and from 10 to 90 % (v/v) of eluent F.

In another preferred embodiment, the concentration of acetic acid in eluents D and E used as mobile phases in the second step is ranging from 0.1 to 0.5 N. More preferably, the concentration of acetic acid in water and in acetonitrile used as mobile phase in the second step is ranging from 0.2 to 0.3 N.

Advantageously, the GLP-1 analogue (Aib⁸'³⁵)GLP-1 (7-36)NH₂ thus obtained by the process according to the present invention contains less than 10 % (w/w) of acetic acid. In a preferred embodiment, the GLP-1 analogue (Aib^8,35)GLP-1 (7-36)NH₂ product obtained contains less than 7 % (w/w) of acetic acid, more preferentially less than 6.5 % (w/w) of acetic acid, and even more preferably, less than 6 % (w/w) of acetic acid. By way of example, the GLP-1 analogue (Aib^{8 35})GLP-1 (7-36)NH₂ obtained by the process of the present invention contains 6 % ± 1 % (w/w) acetic acid, 5 % ± 1 % (w/w) acetic acid, 4 % ± 1 % (w/w) acetic acid, 3 % ± 1 % (w/w) acetic acid, 2 % ± 1 % (w/w) acetic acid, 1 % ± 0.5 % (w/w) acetic acid, or 0.5 % ± 0.1 % (w/w) acetic acid.

The RP-HPLC is expediently performed using a silica gel sorbent as stationary phase. Suitable silica gel types used in RP-HPLC may be selected from, but are not limited to the following silica gel sorbents : Kromasil® 100-16-C18, Kromasil^® 100-10-C18, Kromasif 100-16-C8, Kromasif 100-16-C4, Kromasif 100-10-Phenyl, Kromasif Eternity- 5-C18, Kromasil® 100-5-C4, Chromatorex^® C18 SMB 100-15 HE, Chromatorex® C8 SMB 100-15 HE, Chromatorex® C4 SMB 100-15 HE, Daisogel® SP-120-15-ODS-AP, Daisogel® SP-120-10-C4-Bio, Daisogel® SP-200-10-C4-Bio, Zeosphere® 100 C18, Zeosphere® 100 C8, Zeosphere® 100 C4, SepTech® ST 150-10-C18, Luna® 100-10-C18, Gemini 1 10-10-

C18, YMC-Triart 120-5-C18 and YMC-Triart 200-10-C8. The Kromasil^® silica gel types as listed above are particularly suitable.

Alternatively the RP-HPLC may be performed by using polymeric based stationary phases. Suitable polymeric phases may be selected from PLRP-S 100-10 or Amberchrom'™' Profile XT20.

After the first and the second chromatography steps, the purity of the peptide product is higher than 99 %. For example, the purity of the final product is about 99.3 %, about 99.4 %, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%. More preferably, the purity of the peptide product is higher than 99.3 %. Most preferably, the purity of the final product is at least 99.9 %. Individual impurities are not larger than 0.5 %.

For example, the individual impurity is about 0.3 %, about 0.2 %, about 0.1 %, about 0.05%, or about 0.01 %. More preferably, individual impurities are not larger than 0.3 %. Most preferably, the individual impurity is less than 0.1 %.

In order to obtain a dry final product which is suitable for the drug formulation, the solution may be subjected to precipitation, lyophilization or spray-drying techniques.

Preferably, the purification process as described above is followed by a lyophilisation step and a relyophilisation step.

The following examples are presented to illustrate the above procedures without limiting the scope of the invention. Experimental part

Example 1 : Preparation of the GLP-1 compound

The crude peptide (Aib^8,35)GLP-1 (7-36)NH₂ can be prepared according to the methods described in WO 2007/147816 and WO 2009/074483 by producing three fragments and coupling these fragments in solution. The purification involves a first pass chromatographic purification at a pH of 2, followed by a second pass at a pH of 2.75. Example 2 : Purification process 1 a) First chromatography step Specifications of the process used for the first chromatography step are given in Table 1 .

Table 1

200 mg of crude (Aib ' )GLP-1 (7-36)NH₂ being a mass concentration of 10 mg/mL was dissolved in 0.1 % TFA based water at a pH of 2 and loaded onto a RP-HPLC column. The peptide was eluted by gradient increase of eluent B.

Parameters and purification program of the above-mentioned chromatography step are respectively shown in Tables 2 and 3:

Table 2

Table 3

Duration (min) Flow rate (mL/min) Eluent A (% (v/v)) Eluent B (% (v/v))

5 10 100 0

15 10 100 — > 70 0— > 30

45 10 70— > 55 30 — > 45

10 10 55— > 0 45 — -> 100

1 10 0 > 100 100 > 0

9 10 100 0 Fractions were then collected and the ones with a purity above 95 % were combined and lyophilized. Fractions not having the required purity were pooled, evaporated under reduced pressure, and re-purified using the method performed in the first step.

The lyophilized product had purity of about 97 %.

The pooled fractions are further purified by a second chromatographic purification. b) Second chromatography step Specifications of the process used for the RP-HPLC step are given in Table 4.

Table 4

The pooled fractions from the first chromatography step are loaded onto the HPLC column and the purification program is continued by a salt exchange followed by a gradient elution.

Parameters and purification program of the above-mentioned step-two RP-HPLC are respectively shown in Tables 5 and 6:

Table 5

Table 6

Duration (min) Flow rate (mL/min) Eluent D (% (v/v)) Eluent E (% (v/v)) Eluent C (% (v/v))

45 10 0 0 100 5 10 100 0 0

5 10 100— > 80 0—> 20 0

60 10 80 > 70 20— 30 0

10 10 70— >· 40 30—> 60 0

1 10 40 —M OO 60— > 0 0

9 10 100 0 0

Fractions were collected. Only fractions having purity above 99.3 % were pooled and lyophilized. Fractions not having the required purity were pooled, evaporated under reduced pressure and re-purified under the same conditions as performed in the gradient elution. Fractions were stored at -40° C right after collection. The purity of the peptide product was 99.9 %. This peptide product purity corresponds to a high level for purification process standard.

The peptide product contains 6.17 % of acetic acid.

Example 3: Purification process 2 a) First chromatography step

Specifications of the process used for the first chromatography step are given in Table 7.

Table 7

Crude (Aib ' )GLP-1 (7-36)NH₂ at a concentration of approximately 10-15 g/L was dissolved in a mixture of acetic acid and acetonitrile, filtered and loaded onto a RP-HPLC column at up to 15 g of peptide per kg of column packing. The peptide was eluted by gradient increase of eluent B.

Parameters and purification program of the above-mentioned chromatography step are respectively shown in Tables 8 and 9:

Table 8

Parameter Description

Eluent A 0.1 % TFA in purified water

Eluent B 0.1 % TFA in acetonitrile

Table 9

Fractions were then collected and the ones which are purity above 98-99 % were combined. Fractions not having the required purity were diluted to reduce acetonitrile content and combined for further purification using the method performed in the first step.

The pooled fractions are further purified by a second chromatographic purification. b) Second chromatography step

Specifications of the process used for the second RP-HPLC step are described in Table 7.

The loading may be increased relative to the first step by up to a factor of 2, i.e. up to 30 g peptide per kg column packing.

Parameters and purification program of the above-mentioned step-two RP-HPLC are respectively shown in Tables 10 and 1 1 : Table 10

Table 1 1

Fractions were collected. Only fractions having purity equal to or above 99.3 % were pooled, evaporated to reduce acetonitrile content and lyophilized. Fractions not having the required purity were diluted to reduce acetonitrile content and combined for further purification under the same conditions performed by either repeating the second step or if necessary by repeating both first and second steps.

The purity of the peptide after performing the first and second steps was higher than 99.5 % with no impurity larger than 0.3 %. This peptide product purity corresponds to a high level for a purification process on large scale. After lyophilisation steps performed after the purification process, the peptide product contains less than or equal to 3 % (w/w) of acetic acid.

Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all patents and patent applications and patent publications, are specifically and entirely hereby incorporated herein by reference. It is intended that the specification and examples be considered exemplary only, with the true scope and spirit of the invention indicated by the following claims.

Claims

1. Purification process of (Aib^8,35)GLP-1 (7-36)NH₂ applying reversed phase HPLC comprising a first and a second chromatography step,

- the first step being performed with TFA in a water solution as eluent A and TFA in an acetonitrile solution as eluent B of the mobile phases, with a gradient increase of eluent B, and

2. Process according to claim 1 , wherein the water solution is water and the acetonitrile solution is acetonitrile.

3. Process according to claim 1 , wherein the gradient of the first step is ranging from 100 to 0 % (v/v) of eluent A and from 0 to 100 % (v/v) of eluent B.

4. Process according to claim 1 , wherein the gradient of the first step is ranging from 90 to 5 % (v/v) of eluent A and from 10 to 95 % (v/v) of eluent B.

5. Process according to any one of the preceding claims, wherein the first step is performed at a pH ranging from 1.0 to 4.0, more preferably at a pH from 1.5 to 2.5.

6. Process according to any one of the preceding claims, wherein TFA used in the mobile phase of the first step is present in a concentration ranging from 0.005 to 0.5 % by volume, more preferably from 0.01 to 0.2 % by volume relative to the total volume of the water solution.

7. Process according to any one of claims 1 to 5, wherein TFA used in the mobile phase of the first step is present in a concentration ranging from 0.005 to 0.5 % by volume, more preferably from 0.01 to 0.2 % by volume relative to the total volume of the acetonitrile solution.

8. Process according to any one of the preceding claims, wherein the second step is performed with an acidic buffer at a pH ranging from 1 .0 to 4.0 and more preferably at a pH ranging from 2.0 to 3.0.

9. Process according to any one of the preceding claims, wherein the salt exchange step is performed with ammonium acetate as acidic buffer.

10. Process according to claim 9, wherein the concentration of ammonium acetate used as acid buffer in the salt exchange step is ranging from 0.005 to 0.5 N, more preferably from 0.01 to 0.2 N .

11. Process according to claim 9 or 10, wherein the acidic buffer additionally comprises acetonitrile.

12. Process according to any one of claims 1 to 10, wherein the gradient elution of the second step is performed with acetic acid in water as eluent D and with acetic acid in acetonitrile as eluent E.

13. Process according to claim 1 1 , wherein the gradient of the second step is ranging from 0 to 70 % (v/v) of eluent E and from 100 to 30 % (v/v) of as eluent D, more preferably from

0 to 60 % (v/v) of eluent E and from 100 to 40 % (v/v) of eluent D.

14. Process according to any one claims 1 to 10, wherein the gradient elution of the second step is performed with acetic acid in water as eluent D and with acetonitrile as eluent F.

15. Process according to claim 14, wherein the gradient of the second step is ranging from 100 to 0 % (v/v) of eluent D and from 0 to 100 % (v/v) of eluent F, more preferably from 90 to 10 % (v/v) of eluent D and from 10 to 90 % (v/v) of eluent F.

16. Process according to any one of the preceding claims, wherein the concentration of acetic acid in eluents D and E used as mobile phases in the second step is ranging from 0.1 to 0.5 N, more preferably from 0.2 to 0.3 N.

17. Process according to any one of the preceding claims, wherein the GLP-1 analogue (Aib^8,35)GLP-1 (7-36)NH₂ obtained contains less than 10 % (w/w) of acetic acid, more preferably less than 7 % (w/w) of acetic acid.

18. Process according to any of the preceding claims, wherein the GLP-1 analogue (Aib^8,35)GLP-1 (7-36)NH₂ obtained from the first and second steps has a purity of at least

99 % with an individual impurity less than 0.5 %.

19. Process according to any of the preceding claims, wherein the GLP-1 analogue (Aib^8,35)GLP-1 (7-36)NH₂ obtained from the first and second steps has a purity of at least 99.3 % with an individual impurity less than 0.3 %.

20. Process according to any of the preceding claims, wherein the GLP-1 analogue (Aib^8,35)GLP-1 (7-36)NH₂ obtained from the first and second steps has a purity of at least 99.9 % with an individual impurity less than 0.1 %.