Manufacture of glucagon peptides
The present invention refers to a method of purifying a glucagon peptide, the method comprising a two dimensional reversed phase high performance liquid chromatography protocol, wherein the first step is carried out at using a mobile phase comprising a triethylammonium phosphate (TEAP) buffer and acetonitrile, and the second step is carried out at using a mobile phase comprising aqueous acetic acid and acetonitrile.
1 . A method for the preparation of purified glucagon peptide, comprising:
a) providing a liquid composition C comprising a chemically synthesized glucagon peptide and at least one peptidic impurity selected from a glucagon derivative, a glucagon truncation variant, a derivative of a glucagon truncation variant, a glucagon deletion variant, a derivative of a glucagon deletion variant, and a combination thereof;
b) subjecting the composition C to a first reversed phase high performance liquid chromatography (RP-HPLC) purification, wherein a hydrocarbon bonded silica is used as a stationary phase, an aqueous mobile phase of pH 6.5 to 7.5 comprising triethylammonium phosphate and acetonitrile is used, and elution is effected by gradually increasing the acetonitrile concentration within the mobile phase while collecting fractions containing the glucagon peptide; and
c) subjecting the pooled glucagon peptide-containing fractions obtained in step b) to a second reversed phase HPLC purification, wherein a hydrocarbon bonded silica is used as a stationary phase, a mobile phase comprising acetic acid and acetonitrile is used, wherein the concentration of acetic acid in the mobile phase of step c) is between 0.5 and 5% v/v, and elution is effected by gradually increasing the acetonitrile concentration within the mobile phase while collecting and pooling fractions containing the purified glucagon peptide,
wherein the same stationary phase is used in steps b) and c).
2 . The method according to claim 1 ,
wherein elution in step b) is effected by a gradient of increasing the acetonitrile content from 2 to 4% v/v acetonitrile to 30 to 50% v/v acetonitrile, and
wherein elution in step c) is effected by a gradient of increasing the acetonitrile content from 2 to 4% v/v acetonitrile to 40 to 60% v/v acetonitrile.
3 . The method according to claim 1 , wherein C18 bonded silica is used as a stationary phase in step b) and step c).
4 . The method according to claim 1 , further comprising a step of rinsing the stationary phase with an aqueous solution comprising TFA and acetonitrile prior to loading with the liquid composition C, prior to loading with the pooled glucagon peptide containing fractions obtained in step b), or prior to loading with the liquid composition C and prior to loading with the pooled glucagon peptide containing fractions obtained in step b).
5 . The method according to claim 1 , further comprising the step of:
d) performing an anion exchange.
6 . The method according to claim 1 , wherein step a) comprises:
(a-i) providing a glucagon peptide conjugated to a solid phase, wherein at least the side chains of Glu, Asp, and Lys carry protecting groups; and
(a-ii) cleaving the glucagon peptide from the solid phase and removing the protection groups from the glucagon peptide.
7 . The method according to claim 6 , wherein the glucagon peptide conjugated to a solid phase comprises two pseudoproline dipeptides.
8 . The method according to claim 6 , wherein step (a-i) comprises performing Fmoc-based Solid Phase Peptide Synthesis of a glucagon peptide using suitably protected amino acid derivatives or dipeptide derivatives, wherein said protected amino acid derivatives or dipeptide derivatives are activated by means of one or more coupling reagent mixtures comprising reagents selected for each step independently from the group consisting of:
(E) (benzotriazolyl)tetramethyluronium tetrafluoroborate (TBTU) plus diisopropylethylamine (DIPEA);
(F) diisopropylcarbodiimide (DIC) plus cyano-hydroxyimino-acetic acid ethyl ester (Oxyma);
(G) 3-(diethoxy-phosphoryloxy)-3H-benzo[d][1,2,3]triazin-4-one (DEPBT) plus DIPEA; and
(H) DIC plus hydroxybenzotriazole (HOBt).
9 . The method according to claim 6 , wherein an N-terminal histidine moiety is introduced into the glucagon peptide conjugated to the solid phase using an amino acid derivative selected from the group consisting of Boc-His(Boc)-OH, Boc-His(Trt)-OH, and Fmoc-His(Trt)-OH and a coupling reagent mixture comprising DEPBT plus DIPEA or DIC plus Oxyma.
10 . The method according to claim 1 , wherein the glucagon peptide is a peptide of SEQ ID NO: 1.
11 . The method according to claim 1 , wherein step a) comprises subjecting a sample comprising a glucagon peptide to a reversed phase high performance liquid chromatography (RP-HPLC) purification, wherein a hydrocarbon bonded silica is used as a stationary phase, an aqueous mobile phase comprising trifluoroacetic acid and acetonitrile is used, and elution is effected by gradually increasing the acetonitrile concentration within the mobile phase while collecting fractions containing the glucagon peptide.
12 . The method according to claim 6 , wherein the step of cleaving the glucagon peptide from the solid phase and removing the protection groups from the glucagon peptide is achieved by incubation with a cleavage composition comprising at least 50% TFA and one or more scavenger(s).
13 . A composition GC comprising a glucagon peptide obtained from a method according to claim 1 , characterized in that said composition contains a glucagon peptide at a purity above 99.3%, and does contain detectable levels, but not more than 0.5% of one or more selected from the groups consisting of Glu 24 -glucagon, des-Ser 2 -glucagon, and des-Thr 5 -glucagon, wherein said concentrations are determined as the relative peak area observed in analytical RP-HPLC with UV detection at 220 nm.
14 . The method according to claim 1 , further comprising the step of:
d) Performing an anion exchange, wherein acetate is at least partially replaced by chloride, wherein the anion exchange is achieved by lyophilization, ultrafiltration, dialysis, solid phase extraction, reversed-phase chromatography or by ion exchange chromatography, or wherein acetate is at least partially replaced by chloride and wherein the anion exchange is achieved by lyophilization, ultrafiltration, dialysis, solid phase extraction, reversed-phase chromatography or by ion exchange chromatography.
15 . The method according to claim 6 , wherein the glucagon peptide conjugated to a solid phase comprises two pseudoproline dipeptides, wherein the two pseudoproline dipeptides are introduced at positions corresponding to or identical with the positions selected from the group consisting of: Gly 4 -Thr 5 and Phe 6 -Thr 7 , Gly 4 -Thr 5 and Thr 7 -Ser 8 , Gly 4 -Thr 5 and Tyr 10 -Ser 11 , Gly 4 -Thr 5 and Asp 15 -Ser 16 , Phe 6 -Thr 7 and Tyr 10 -Ser 11 , Phe 6 -Thr 7 and Asp 15 -Ser 16 , Thr 7 -Ser 8 and Tyr 10 -Ser 11 , Thr 7 -Ser 8 and Asp 15 -Ser 16 , or Tyr 10 -Ser 11 and Asp 15 -Ser 16 of the peptide of SEQ ID NO: 1.
16 . The method according to claim 6 , wherein one or more agents selected from the group consisting of iodide salts, dimethyl sulfide, 1,4-dithiothreitol, trimethylsilylbromide, and ascorbic acid is or are added to a suspension comprising the solid phase.
17 . The composition GC according to claim 13 , characterized in that said composition does contain detectable levels, but not more than 0.3% of one or more selected from the groups consisting of Glu24-glucagon, des-Ser 2 -glucagon, and des-Thr 5 -glucagon, wherein said concentrations are determined as the relative peak area observed in analytical RP-HPLC with UV detection at 220 nm.
18 . The composition GC according to claim 13 , characterized in that said composition does contain detectable levels, but not more than 0.2% of one or more selected from the groups consisting of Glu24-glucagon, des-Ser 2 -glucagon, and des-Thr 5 -glucagon, wherein said concentrations are determined as the relative peak area observed in analytical RP-HPLC with UV detection at 220 nm.
19 . The method of claim 1 , wherein the at least one peptidic impurity results from omission or addition of at least one amino acid during glucagon peptide synthesis.
20 . The method of claim 1 , wherein the at least one peptidic impurity results from incomplete removal of protecting groups, from side reactions occurring during peptide cleavage from a solid support, from racemization, and/or from deamidation.
21 . The method of claim 1 , wherein the collected and pooled glucagon peptide comprises no more than 0.5% of any individual peptidic impurity as assessed by UV detection at a wavelength between 205 and 230 nm.