Capping of unprotected amino groups during peptide synthesis
11028123 · 2021-06-08
Assignee
Inventors
- Bernd Henkel (Frankfurt am Main, DE)
- Tobias Metzenthin (Frankfurt am Main, DE)
- Manfred Gerken (Frankfurt am Main, DE)
- Wolfgang Fiedler (Frankfurt am Main, DE)
Cpc classification
Y02P20/55
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
The present invention relates to a method for the synthesis of a polypeptide comprising a pre-determined amino acid sequence. The method according to the invention comprises coupling cycles of coupling an N-terminally protected amino acid building block C-terminally at an unprotected N-terminal amino group of an amino acid chain, wherein at least one coupling cycle comprises a coupling step (a), a capping step (b), and a de-protecting step (c).
Claims
1. A method for the solid-phase synthesis of lixisenatide SEQ ID NO:1), the method comprising coupling cycles of amino acid building blocks of lixisenatide to an amino acid chain, wherein said amino acid building blocks comprise an unprotected C-terminal carboxyl group and a protected N-terminal amino group comprising an Fmoc protecting group, wherein said amino acid chain comprises an unprotected N-terminal amino group, and wherein at least one coupling cycle comprises the steps: (a) coupling the amino acid building block C-terminally at the unprotected N-terminal amino group of the amino acid chain, so that an amide bond is formed between the amino acid chain and the amino acid building block, (b) contacting the product obtained in step (a) with a capping reagent comprising a capping compound, wherein the capping compound binds to an unprotected N-terminal amino group of the amino acid chain to which no building block has been coupled in step (a), and (c) de-protecting the N-terminal amino group of the amino acid building block, wherein the method comprises sufficient coupling cycles to produce lixisenatide (SEQ ID NO:1), and wherein step (b) is performed after coupling of the amino acid building block at positions Arg(20), Glu(17), Gln(13, Leu(10) or Gly(4) of the lixisenatide sequence.
2. The method of claim 1, wherein the capping compound is selected from the group consisting of acetic anhydride (CAS 108-24-7), homologues of acetic anhydride, benzoyl chloride (CAS 98-88-4), N-(benzyloxycarbonyloxy)succinimide (CAS 13139-17-8), benzyl chloroformate (CAS 501-53-1), esters of chloroformic acid, 1-acetylimidazole (CAS 2466-76-4), di-tert-butyl dicarbonate (CAS 24424-99-5) and N-(tert-butoxycarbonyloxy)succinimide (CAS 13139-12-3).
3. The method of claim 1, wherein the capping reagent comprises 0.5-5% v/v of acetic anhydride.
4. The method of claim 1, wherein the capping reagent comprises 0.2-2% v/v of diisopropylethylamine (DIPEA).
5. The method of claim 1, wherein the capping reagent comprises a solvent that comprises N,N-dimethylformamide (DMF).
6. The method of claim 1, wherein the step (b) is performed at a temperature of 15-25° C.
7. The method of claim 1, wherein the step (b) is performed for 5 to 15 min.
8. The method of claim 1, wherein the amino acid building block is a single amino acid selected from ARg, Glu, Gln, Leu, and Gly.
9. The method of claim 3, where the capping reagent comprises 1-3% v/v of acetic anhydride.
10. The method of claim 3, where the capping reagent comprises 2% v/v of acetic anhydride.
11. The method of claim 4, where the capping reagent comprises 0.5-2% v/v of diisopropylethylamine (DIPEA).
12. The method of claim 4, where the capping reagent comprises 1% v/v of diisopropylethylamine (DIPEA).
13. The method of claim 1, further comprising the step of cleaving lixisenatide polypeptide linked to the solid phase after synthesis of the lixisenatide amino acid chain is completed.
14. A method for the solid-phase synthesis of lixisenatide, the method comprising coupling cycles of amino acid building blocks to an amino acid chain, wherein said amino acid building blocks comprise an unprotected C-terminal carboxyl group and a protected N-terminal amino group comprising an Fmoc protecting group, wherein said amino acid chain comprises an unprotected N-terminal amino group, wherein at least one coupling cycle comprises the steps: (a) coupling the amino acid building block C-terminally at the unprotected N-terminal amino group of the amino acid chain, so that an amide bond is formed between the amino acid chain and the amino acid building block, (b) contacting the product obtained in step (a) with a capping reagent comprising a capping compound, wherein the capping compound binds to an unprotected N-terminal amino group of the amino acid chain to which no building block has been coupled in step (a), and (c) de-protecting the N-terminal amino group of the amino acid building block, and wherein the capping reagent comprises 0.5-5% v/v of acetic anhydride and 0.2-2% of diisopropylethylamine (DIPEA), and wherein step (b) is performed after coupling of the amino acid building block at positions Arg(20), Glu(17), Gln(13, Leu(10) or Gly(4) of the lixisenatide sequence.
15. The method of claim 14, wherein the capping reagent comprises 1-3% v/v of diisopropylethylamine (DIPEA) and 2% v/v of acetic anhydride.
16. The method of claim 14, wherein the capping reagent comprises N,N-dimethylformamide (DMF).
17. The method of claim 14, wherein the step (b) is performed at a temperature of 15-25° C.
18. The method of claim 14, wherein the step (b) is performed for 5 to 15 min.
19. The method of claim 14, where the capping reagent comprises 2% v/v of acetic anhydride and 1% v/v of diisopropylethylamine (DIPEA).
20. The method of claim 14, further comprising the step of cleaving lixisenatide polypeptide linked to the solid phase after synthesis of the lixisenatide amino acid chain is completed.
21. The method of claim 14, wherein the amino acid building block is a single amino acid selected from Arg, Glu, Gln, Leu and Gly.
Description
FIGURES
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EXAMPLE 1
(21) Synthesis of Lixisenatide
(22) The active substance Lixisenatide is a polypeptide amide composed of 44 amino acids; acetate functions as counterion.
(23) In the one-letter code, the amino acid sequence of Lixisenatide is as follows:
(24) TABLE-US-00001 H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W- L-K-N-G-G-P-S-S-G-A-P-P-S-K-K-K-K-K-K-NH.sub.2
(25) The peptide chain was constructed by means of linear solid-phase synthesis, starting from the C-terminus, Lys-44.
(26) The method of synthesis is Fmoc solid-phase peptide synthesis, in which a Rink amide resin was used in order to obtain a peptide amide. The reactions were carried out in DMF at room temperature. Between the reactions, washing was carried out repeatedly, mostly with DMF, with one of the middle washing steps being carried out with isopropanol.
(27) The synthesis of Lixisenatide on the polymeric support can be broken down into the following steps: Coupling of the first Fmoc-amino acid (Fmoc-Lys(Boc)-OH) to Rink resin Capping of the unreacted amino group Cleavage of the temporary protecting group Fmoc Coupling of the further Fmoc-amino acids or Fmoc-dipeptides Capping of the unreacted amino group Final Fmoc cleavage Cleavage of Lixisenatide from the resin and simultaneous removal of the side chain protecting groups
(28) The synthesis cycle is illustrated in
(29) 1.1 Coupling of the First Fmoc-Amino Acid (Fmoc-Lys(Boc)-OH) to Rink Resin
(30) Before the synthesis began, the Rink amide resin was swollen in DMF. The swelling was carried out for 2-15 h. Subsequently, the temporary protecting group Fmoc was cleaved from the Rink amide resin using 25% piperidine in DMF. This cleavage was undertaken twice; cleavage time of 5 minutes and 20 minutes. Following the Fmoc cleavage, the resin was washed repeatedly with DMF and once with isopropanol.
(31) The coupling of the first Fmoc-amino acid, Fmoc-Lys(Boc)-OH, was carried out in an excess of 2.4 eq, in order to load the resin. HOBt hydrate, HBTU and DIPEA served as coupling reagents. The coupling time was 60-120 min.
(32) In order to completely load the Rink resin with Fmoc-Lys(Boc)-OH, a further loading was carried out with the coupling reagents HOBt hydrate and DIC. The coupling time was 6-18 h. The mixture was stirred while step 1.1 was carried out. The capping was subsequently carried out.
(33) 1.2 Capping of the Unreacted Amino Group
(34) The consequence of incomplete loading of the resin is that as yet unreacted amino groups are found on the resin. These were inactivated, and hence made unavailable for further coupling, by adding a mixture of acetic anhydride/DIPEA/DMF (10:5:85). The capping mixture remained on the resin for 20 minutes while stirring. The remaining free amino group is acylated. Subsequently, the resin was washed repeatedly with DMF and once with isopropanol.
(35) A capping method according to the invention at least at 5 positions of a Lixisenatide synthesis is described in examples 4 and 5.
(36) 1.3. Cleavage of the Temporary Protecting Group Fmoc
(37) The temporary protecting group Fmoc was cleaved using 25% piperidine in DMF. This cleavage was undertaken twice; cleavage time of 5 minutes and 20 minutes. Following the Fmoc cleavage, the resin was washed repeatedly with DMF and once with isopropanol.
(38) 1.4 Coupling of the Further Fmoc-Amino Acids or Fmoc-Dipeptides
(39) The next Fmoc-amino acid was coupled to the deprotected amino group on the resin. The coupling was carried out in DMF at different equivalents. The coupling times were between 2 h and 18 h. HOBt/DIC, and also HBTU/DIPEA, were used as coupling reagents.
(40) The following derivatives were used as Fmoc-amino acids: Fmoc-Lys(Boc)-OH Fmoc-Ser(tBu)-OH Fmoc-Pro-OH Fmoc-Ala-OH×H.sub.2O Fmoc-Gly-OH Fmoc-Asn(Trt)-OH Fmoc-Leu-OH Fmoc-Trp(Boc)-OH Fmoc-Glu(OtBu)-OH×H.sub.2O Fmoc-Ile-OH Fmoc-Phe-OH Fmoc-Arg(Pbf)-OH Fmoc-Val-OH Fmoc-Met-OH Fmoc-Gln(Trt)-OH Fmoc-Asp(OtBu)-OH Fmoc-Thr(tBu)-OH Fmoc-His(Trt)-OH
(41) Alternatively, it was also possible to use Fmoc-dipeptides (method according to the invention): Fmoc-Pro-Pro-OH (CAS 129223-22-9) Fmoc-Ala-Pro-OH (CAS 186023-44-9) Fmoc-Ser(tBu)-Gly-OH (CAS 113247-80-6) Fmoc-Gly-Pro-OH (CAS 212651-48-4) Fmoc-Gly-Gly-OH (CAS 35665-38-4) Fmoc-Asn(Trt)-Gly-OH (from Bachem B-3630) Fmoc-Glu(OtBu)-Gly-OH (CAS 866044-63-5) Fmoc-His(Trt)-Gly-OH
(42) If the coupling was found to be incomplete according to the Kaiser test (E. Kaiser et al, Anal. Biochem. 34, 1970, 595), further coupling was possible. For this purpose, the Fmoc-amino acid was coupled again, together with HBTU/DIPEA/HOBt hydrate.
(43) 1.5 Capping of the Unreacted Amino Group
(44) See description under point 1.2.
(45) 1.6 Final Fmoc Cleavage
(46) The final Fmoc cleavage was carried out as described under point 1.3. The resin was finally washed again with diisopropyl ether and dried under reduced pressure.
(47) 1.7 Cleavage of Lixisenatide from the Resin and Simultaneous Removal of the Side Chain Protecting Groups
(48) The cleavage of Lixisenatide from the Rink resin was carried out as described in example 6.
(49) 1.8 Synthesis of Lixisenatide with Inventive Use of Dipeptides
(50) The coupling of the first Fmoc-Lys(Boc)-OH to the resin was carried out with HBTU/DIPEA/HOBt hydrate. After the coupling of the first amino acid Fmoc-Lys(Boc)-OH to the free amine of the Rink amide resin, the following process steps were conducted in an endlessly repeating cycle (see also steps 1.3 to 1.6): Fmoc cleavage Coupling Further coupling, if necessary Capping After coupling of the final amino acid unit, the N-terminal Fmoc group is cleaved.
(51) Standard Fmoc-protected amino acids were coupled with DIC/HOBt, with the excess of amino acids and coupling reagents being between 2 and 4 equivalents.
(52) At the positions Pro(36) and Pro(37), instead of two Fmoc-Pro-OH amino acid derivatives, the dipeptide Fmoc-Pro-Pro-OH was coupled with HBTU/DIPEA.
(53) At the position Pro(31), coupling was carried out with HBTU/DIPEA/HOBt hydrate.
(54) At the positions His(1) and Gly(2), instead of the amino acid derivatives Fmoc-His(Boc)-OH and Fmoc-Gly-OH, the dipeptide Fmoc-His(Trt)-Gly-OH was coupled.
(55) After the couplings, the capping was carried out in each case with Ac.sub.2O/DIPEA, as is described in examples 4 and 5.
(56) The Fmoc cleavage was performed with 25% piperidine in DMF, in each case successively first with 5 minutes of reaction time, then with 20-40 minutes of reaction time.
(57) The completeness of the coupling was checked by means of a Kaiser test.
(58) After the last coupling and last cleavage of the Fmoc group, the resin was washed, firstly repeatedly with DMF, then with isopropanol and finally with diisopropyl ether, and it was subsequently dried at 35° C. under reduced pressure.
(59) The cleavage of the raw peptide from the resin was carried out in trifluoroacetic acid with scavengers such as 1,2-ethanedithiol.
(60) The raw peptide was purified in a two-step HPLC process with C18 RP silica gel as solid phase. In the first purification step, a buffer system with acetonitrile/water with 0.1% TFA was used; in the second step, a buffer system with acetonitrile/water with AcOH was used. After concentration of the pooled solutions, the pure peptide was obtained by freeze-drying.
(61) Use of 3500 g of Rink amide resin with a loading of 0.3 mmol/g (i.e. a 1.05 mol batch) gave 9970 g of peptide on resin. 4636 g of raw peptide were obtained therefrom.
(62) After purification, 576 g of pure peptide were obtained therefrom. MS: 4855.5 (monoisotopic molar mass); found 4855.6. Amino acid sequencing: correct sequence found. Assay: 89.0% (as is).
(63) 1.9 Synthesis of Lixisenatide without Use of Dipeptides
(64) The peptide chain was constructed by means of linear solid-phase synthesis, starting from the C-terminus, Lys-44.
(65) Standard Fmoc-protected amino acids were coupled with DIC/HOBt, with the excess of amino acids and coupling reagents being between 2 and 4 equivalents.
(66) At the positions Pro(37), Pro(36), Pro(31), coupling was carried out with HBTU/DIPEA/HOBt hydrate.
(67) Each coupling was followed by capping with Ac.sub.2O/DIPEA. The Fmoc cleavage was performed with 25% piperidine in DMF, in each case successively first with 5 minutes of reaction time, then with 20 minutes of reaction time.
(68) The completeness of the coupling was checked by means of a Kaiser test. After the last coupling and last cleavage of the Fmoc group, the resin was washed, firstly repeatedly with DMF, then with isopropanol and finally with diisopropyl ether, and it was subsequently dried at 35° C. under reduced pressure.
(69) The cleavage of the raw peptide from the resin was carried out in trifluoroacetic acid with scavengers such as 1,2-ethanedithiol, thioanisole, phenol and water.
(70) The raw peptide was purified in a two-step HPLC process with C18 RP silica gel as solid phase. After concentration of the pooled solutions, the pure peptide was obtained by freeze-drying. Table 1 compares the contents of racemized D-His-Lixisenatide and the contents of some impurities in the pure peptide between the synthesis using the dipeptides and without the dipeptides.
(71) TABLE-US-00002 TABLE 1 Comparison of the Lixisenatide syntheses with and without use of the dipeptides. Content of Content of Content of Content desGly(2)- desPro(36)- diPro(36)- of D-His Lixisenatide Lixisenatide Lixisenatide Synthesis of 0.41% Not present Not present Not present lixisenatide with dipeptides Fmoc- His(Trt)-Gly-OH, Fmoc-Pro-Pro- OH according to the invention Comparative 4.1% 2.5% 1% 1% synthesis of lixisenatide without dipeptides
(72) The data show that the use of the dipeptide Fmoc-His(Trt)-Gly-OH gives a Lixisenatide which does not contain elevated values of D-His arising from racemization. Moreover, when using Fmoc-His(Trt)-Gly-OH, desGly(2)-Lixisenatide is no longer found. Furthermore, the N−1 and N+1 peptides in the vicinity of the chain position Pro(36) and Pro(37) (e.g. desPro(36)-Lixisenatide or diPro(36)Lixisenatide) did not occur.
EXAMPLE 2
(73) Synthesis, Purification and Characterization of Exendin-4 (According to the Invention)
(74) The active substance Exendin-4 is a polypeptide amide composed of 39 amino acids; acetate functions as counterion.
(75) In the one-letter code, the amino acid sequence is as follows:
(76) TABLE-US-00003 H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W- L-K-N-G-G-P-S-S-G-A-P-P-P-S-NH.sub.2
(77) MW 4186.66 g/mol; MW (monoisotopic)=4184.03 g/mol.
(78) The synthesis of Exendin-4 was carried out precisely as described in the synthesis of Lixisenatide, according to the abovementioned sequence. At positions 1 and 2, coupling was carried out in one cycle with Fmoc-His(Trt)-Gly-OH. At positions 37 and 38, coupling was carried out in one cycle with Fmoc-Pro-Pro-OH. At the other positions, coupling was carried out with Fmoc-amino acids (monoamino acid units).
(79) Use of 26.666 g of Rink amide resin with a loading of 0.42 mmol/g (i.e. a 11.2 mmol batch) gave 74 g of peptide on resin. From this, 65 g of peptide on resin were cleaved, and 28 g of raw peptide were obtained. For the purification, from this, 21.3 g of raw peptide were used, and 4.01 g of pure peptide were obtained. MS: 4184.03 (monoisotopic molar mass): found 4185.1 [M+H]. Purity 98.25 Fl %.
(80) The use of the dipeptides confirms the results which were obtained for Lixisenatide. The use of the dipeptide Fmoc-His(Trt)-Gly-OH gives an Exendin-4 which does not contain elevated values of D-His arising from racemization. Moreover, when using Fmoc-His(Trt)-Gly-OH, desGly(2)-Exendin-4 is no longer found. Furthermore, the N−1 and N+1 peptides in the vicinity of the chain position Pro(36) and Pro(37) (e.g. desPro(36)-Exendin-4 or diPro(36)Exendin-4) did not occur.
EXAMPLE 3
(81) Synthesis of Fmoc-His(Trt)-Gly-OH
(82) 3.1 Fmoc-His(Trt)-Gly-OBzl
(83) ##STR00001##
(84) 40 g of Fmoc-His(Trt)-OH were dissolved together with 32.7 g of H-Gly-OBzl tosylate and 29.37 g of HBTU in 400 ml of ethyl acetate. Thereafter, 33.32 ml of N-ethylmorpholine were added. The reaction was stirred for 4 h at 30° C. Thereafter, extraction was carried out three times with 256 g of an 8% sodium bicarbonate solution each time, and then washing was carried out once with 250 ml of water. Half of the resulting ethyl acetate solution was evaporated and processed further in the next step.
(85) 3.2 Fmoc-His(Trt)-Gly-OH
(86) ##STR00002##
(87) THF and methanol were added to the ethyl acetate phase, such that a 5:2:2 (w/w/w) THF/ethyl acetate/MeOH mixture was formed. Subsequently, 10 g of palladium on carbon catalyst (5%) were added, and this mixture was hydrogenated at 30° C. and a hydrogen pressure of 1.1 bar for 2.5 h. Thereafter, the catalyst was filtered off and the resulting solution was evaporated until a precipitate began to form. Subsequent stirring was carried out for 1 h and the solution was left to stand at room temperature for 4 days. The product was filtered off and subsequently extracted by stirring in 2-butanone at 80° C. for 4 h. Yield: 32.9 g of Fmoc-His(Trt)-Gly-OH (75%).
EXAMPLE 4
(88) Acetylated Erroneous Sequences During the Synthesis of Lixisenatide
(89) 4.1 Determining the Content of Acetylated Erroneous Sequences During the Synthesis of Lixisenatide
(90) Some acetylated erroneous sequences can be seen in the HPLC profile of the crude Lixisenatide product. These usually arise from unreacted amino groups on the resin being capped. What is achieved by the capping is that no (N−1) impurities can occur, which differ only slightly from the desired product and are hence difficult to remove by purification.
(91) The completeness and also the coupling kinetics at selected positions were monitored by Edman degradation. A resin sample was taken from the synthesis of Lixisenatide and the Fmoc group was cleaved therefrom. This resin sample was then subjected to Edman degradation and in this way it was possible to determine the ratio of coupled amino acid to the (N−1) amino acid, from which the coupling yield could be directly inferred. The results of the Edman degradation (table 2) show high coupling values. These values are so high that they cannot account for the amounts of acetylated erroneous sequences (HPLC data in table 2). This means that there must be an alternative way of forming these by-products. The elucidation of this situation will be described in the following sections.
(92) TABLE-US-00004 TABLE 2 Coupling yields and contents of acetylated fragments during synthesis of Lixisenatide. The percentage contents of acetylated erroneous sequences from HPLC data and Edman results (coelution of Ac(6-44), Ac(5-44) and Ac(4-44)) are compared to one another. Amino acid to be Coupling yield Impurities content Impurity coupled (Edman data) (HPLC) Ac(36-44) Ala(35) 99.4-99.5% 4.7% (SEQ ID NO: 26) Ac(23-44) Phe(22) >98.4% 0.9% (SEQ ID NO: 23) Ac(20-44) Val(19) 99.7% 2.0% (SEQ ID NO: 20) Ac(13-44) Lys(12) 98.7-99.5% 2.1% (SEQ ID NO: 15) Ac(6-44) Thr(5) 98.4-99.5% Approx. 4.3% (SEQ ID NO: 8) Ac(5-44) Gly(4) 99.1-99.8% (SEQ ID NO: 7) Ac(4-44) Glu(3) 98.2-99.4% (SEQ ID NO: 6)
(93) 4.2 Formation of Acetylated Erroneous Sequences
(94) In order to investigate the points in the synthesis cycle at which the acetylated erroneous sequences are formed, resin samples were taken over a coupling cycle, and the peptide was cleaved and investigated using LC-MS. These investigations were carried out at the positions of coupling of Fmoc-Arg(20)-OH and coupling of Fmoc-Gln(13)-OH.
(95) In the coupling of Fmoc-Arg(20)-OH to the solid-phase-bonded peptide of the Lixisenatide partial sequence H(22-24), samples were taken after coupling times of 1 h, 2 h, 4 h, 8 h and 24 h and also after capping, the subsequent Fmoc cleavage and the coupling of valine(19). As can be seen in
(96) The same experiment was conducted for the coupling of Fmoc-Gln(13)-OH during the Lixisenatide synthesis (
(97) The experiment shows that it is necessary to search for capping conditions, under which the undesired formation of the acetylated erroneous sequence of the Nth amino acid (the last one coupled) is prevented, without the capping ability of the mixture used being reduced to such a significant extent that a potential (N−1) impurity is no longer capped.
(98) 4.3 Variation in the Capping Conditions
(99) The couplings of Fmoc-Arg(20)-OH, Fmoc-Leu(10)-OH, Fmoc-Gly(4)-OH and Fmoc-Thr(5)-OH were investigated. Various capping conditions were compared to one another.
(100) The capping conditions were varied in a laboratory synthesis of Lixisenatide. Particular attention was paid to the contents of undesired Ac(N-44) and desired Ac([N−1]-44). The conditions tested are as follows: 10% acetic anhydride/5% DIPEA in DMF for 20 minutes 10% acetic anhydride/5% DIPEA in DMF for 10 minutes 2% acetic anhydride/1% DIPEA in DMF for 20 minutes 2% acetic anhydride/1% DIPEA in DMF for 10 minutes
(101) The investigations were carried out at the positions Arg(20), Leu(10), Thr(5) and Gly(4). The results are compiled in tables 3-6.
(102) The data were also compared with the result of a GMP synthesis of Lixisenatide (“GMP capping” in tables 3-6). The capping conditions corresponded to the conditions 10% acetic anhydride/5% DIPEA in DMF. The contact time of the resin with the capping mixture in the GMP batch was 7-8 minutes longer, and was therefore 27-28 minutes. This arose from the longer time taken to pump the capping mixture away.
(103) 4.3.1 Coupling at Position Arg(20)
(104) Fmoc-Arg(Pbf)-OH was coupled to Leu(21). On those chains on which no coupling took place (product H(21-44)), the product Ac(21-44)(SEQ ID NO:21) was formed by the subsequent capping. Both products Ac(20-44)(SEQ ID NO:20) and H(20-44) are formed when, during capping, the Fmoc group is undesirably cleaved (formation of H(20-44)) and acetylation occurs (formation of Ac(2044)).
(105) It can be clearly seen in table 3 that the degree of formation of the undesired products H(20-44) and Ac(20-44)(SEQ ID NO:20) is dependent both on the capping time and on the amount of acetic anhydride and DIPEA (see Ac(20-44)(SEQ ID NO:20)% column). The highest percentage value can be seen in the GMP capping. The lowest content of Ac(20-44)(SEQ ID NO:20) is found under the conditions “2% acetic anhydride/1% DIPEA in DMF for 10 minutes”.
(106) The capping power of the various capping mixtures (and hence the original intended use) is approximately the same (see column Ac(21-44)(SEQ ID NO:21)), i.e. all capping mixtures convert H(21-44)). The mixture “2% acetic anhydride/1% DIPEA in DMF for 10 minutes” also fulfils the desired purpose of avoiding (N−1) impurities.
(107) TABLE-US-00005 TABLE 3 Results of the coupling of Fmoc-Arg(Pbf)-OH at position 20. The table shows the content of acetylated and non-acetylated fragments depending on the capping conditions. The results were obtained by means of LC-MS. The data were compared with the results from a GMP synthesis (“GMP capping”). Ac(20-44) Ac(21-44) (SEQ ID (SEQ ID Capping conditions NO: 20) % Fmoc(20-44) % H(20-44) % H(21-44) % NO: 21) % 10 min/2% acetic 0.75 96.48 0.08 0.66 2.03 anhydride, 1% DIPEA 10 min/10% acetic 0.92 95.87 0.55 0.69 1.96 anhydride, 5% DIPEA 20 min/2% acetic 1.63 95.83 0.14 0.55 1.85 anhydride, 1% DIPEA 20 min/10% acetic 2.26 95.32 0.06 0.60 1.77 anhydride, 5% DIPEA GMP capping 2.64 94.47 0.03 0.68 2.18
(108) 4.3.2 Coupling at the Positions Leu(10), Gly(4) and Thr(5)
(109) The results for Leu(10) are given in table 4 and confirm the results which were obtained for position Arg(20). The content of undesired products Ac(10-44)(SEQ ID NO:12) and H(10-44), which are formed during the capping of the free amino groups of the product H(11-44), is lowest under the conditions “2% acetic anhydride, 1% DIPEA for 10 minutes”. The capping power is comparable in the different capping mixtures.
(110) TABLE-US-00006 TABLE 4 Results of the coupling of Fmoc-Leu-OH at position 10. The table shows the content of acetylated and non-acetylated fragments depending on the capping conditions. The results were obtained by means of LC-MS. Ac(10-44) Ac(11-44) (SEQ ID (SEQ ID Capping conditions NO: 12) % Fmoc(10-44) % H(10-44) % H(11-44) % NO: 13) % 10 min/2% acetic 0.06 98.90 0.42 0.18 0.43 anhydride, 1% DIPEA 10 min/10% acetic 0.20 98.57 0.61 0.16 0.46 anhydride, 5% DIPEA 20 min/2% acetic 0.13 98.24 0.90 0.18 0.56 anhydride, 1% DIPEA 20 min/10% acetic 0.45 98.44 0.52 0.15 0.44 anhydride, 5% DIPEA
(111) For the coupling of Gly(4) as well, the contents of the undesired products Ac(4-44)(SEQ ID NO:6) are dependent on the capping mixture and the reaction time. The capping power is the same in the different mixtures (table 5).
(112) TABLE-US-00007 TABLE 5 Results of the coupling of Fmoc-Gly-OH at position 4. The table shows the content of acetylated and non-acetylated fragments depending on the capping conditions. The results were obtained by means of LC-MS. Ac(4-44) Ac(5-44) (SEQ ID (SEQ ID Capping conditions NO: 6) % Fmoc(4-44) % H(4-44) % H(6-44) % NO: 7) % 10 min/2% acetic 0.09 98.21 0.55 0.56 0.61 anhydride, 1% DIPEA 10 min/10% acetic 0.26 98.42 0.39 0.41 0.52 anhydride, 5% DIPEA 20 min/2% acetic 0.10 98.40 0.47 0.36 0.67 anhydride, 1% DIPEA 20 min/10% acetic 0.39 98.02 0.43 0.39 0.77 anhydride, 5% DIPEA GMP capping 0.92 97.54 0.51 0.40 0.63
(113) In addition to the positions Arg(20), Leu(10) and Gly(4), the position Thr(5) was also investigated. In contrast to the three former positions, the contents of the undesired product Ac(N−44) (Ac(5-44)(SEQ ID NO:7) at position 5) are approximately the same under the various capping conditions. However, the capping power of the different mixtures is also comparable here (table 6).
(114) TABLE-US-00008 TABLE 6 Results of the coupling of Fmoc-Thr(tBu)-OH at position 5. The table shows the content of acetylated and non-acetylated fragments depending on the capping conditions. The results were obtained by means of LC-MS. Ac(5-44) Ac(6-44) (SEQ ID (SEQ ID Capping conditions NO: 7) % Fmoc(5-44) % H(5-44) % H(6-44) % NO: 8) % 10 min/2% acetic 0.04 97.80 0.33 0.24 1.58 anhydride, 1% DIPEA 10 min/10% acetic 0.07 97.93 0.15 0.24 1.61 anhydride, 5% DIPEA 20 min/2% acetic 0.03 97.69 0.36 0.23 1.70 anhydride, 1% DIPEA 20 min/10% acetic 0.03 97.70 0.42 0.29 1.55 anhydride, 5% DIPEA GMP capping 0.07 97.77 0.25 0.24 1.67
(115) 4.3.3 Summary
(116) At the positions Arg(20), Leu(10) and Gly(4), the mild capping mixture (2% acetic anhydride/1% DIPEA in DMF for 10 minutes) is sufficient in order to maintain the desired effect of avoiding (N−1) impurities by acylation. However, in these three cases, the respective formation of Ac(20-44)(SEQ ID NO:20), Ac(10-44)(SEQ ID NO:12) and Ac(4-44)(SEQ ID NO:6) is dependent on the capping time and also on the capping mixture. This does not apply to the position Thr(5).
EXAMPLE 5
(117) Synthesis of Lixisenatide
(118) The example relates to the synthesis of Lixisenatide (cf. SEQ ID NO:1). At the start of the synthesis, the solid-phase-bonded linker bears an Fmoc protecting group. The individual amino acid units were coupled starting from the C-terminus (position 44) towards the N-terminus in coupling cycles, which consist of the steps of Fmoc cleavage Coupling of the Fmoc-protected amino acid unit and Capping.
(119) At the positions Arg(20), Glu(17), Gln(13), Leu(10) and Gly(4), the capping method according to the invention (2% acetic anhydride/1% DIPEA in DMF for 10 minutes) was used. For these positions, the instructions for a coupling cycle are described below. At the other positions, capping was carried out with 10% acetic anhydride/5% DIPEA in DMF for 20 minutes. This capping is described, by way of example, at the position Thr(5). The capping method according to the invention comprises milder conditions.
(120) The batch size was 1050 mmol of Rink resin.
(121) 5.1. Coupling of Fmoc-Arg(Pbf)-OH at Position 20
(122) 5.1.1 Fmoc Cleavage
(123) 7 l of DMF were added to the reactor, followed by a mixture of 7.9 l of piperidine in 16.6 l of DMF. This mixture was stirred for 5 minutes, then filtered with suction. This process was repeated and stirring was carried out for 30 minutes; then filtering with suction was carried out again. After the Fmoc cleavage, the resin was washed 7 times in the following sequence: DMF (31.1 l), DMF (31.1 l), isopropanol (31.1 l), DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF (31.1 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(124) 5.1.2 Coupling of Fmoc-Arg(Pbf)-OH
(125) 21 l of DMF were added to the reactor. Thereafter, 2.125 kg of FmocArg(Pbf)-OH were weighed in and 5.3 l of DMF were added. After complete dissolution, this solution was emptied into the reactor, followed by a solution of 502 g hydroxybenzotriazole hydrate (HOBt hydrate) in 2.2 l of DMF. Finally, 413 g of N,N-diisopropylcarbodiimide (DIC) were added to the reactor. The coupling time was 6-18 h. After coupling, the solvent was filtered off from the resin by suction and the capping was immediately continued.
(126) 5.1.3 Capping (According to the Invention)
(127) The reactor was filled with 26.3 l of DMF. At the same time, 1.2 l of DMF, 0.53 l of acetic anhydride and 0.26 l of diisopropylethylamine (DIPEA) were mixed in a 2 l Schott bottle and added to the resin in the reactor. The reactor was stirred for 10 minutes, then filtering with suction was carried out. After the capping, the resin was washed 5 times in the following sequence: DMF (24 l), isopropanol (31.1 l), DMF (8 l), DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(128) 5.2. Coupling of Fmoc-Glu(OtBu)-OH Hydrate at Position 17
(129) 5.2.1 Fmoc Cleavage
(130) 7 l of DMF were added to the reactor, followed by a mixture of 7.9 l of piperidine in 16.6 l of DMF. This mixture was stirred for 5 minutes, then filtered with suction. This process was repeated and stirring was carried out for 30 min; then filtering with suction was carried out again. After the Fmoc cleavage, the resin was washed 7 times in the following sequence: DMF (31.1 l), DMF (31.1 l), isopropanol (31.1 l), DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF (31.1 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(131) 5.2.2 Coupling of Fmoc-Glu(OtBu)-OH Hydrate
(132) 21 l of DMF were added to the reactor. Thereafter, 1.453 kg of FmocGlu(OtBu)-OH hydrate were weighed in and 5.3 l of DMF were added. After complete dissolution, this solution was emptied into the reactor, followed by a solution of 502 g hydroxybenzotriazole hydrate (HOBt hydrate) in 2.2 l of DMF. Finally, 413 g of N,N-diisopropylcarbodiimide (DIC) were added to the reactor. The coupling time was 6-18 h. After coupling, the solvent was filtered off from the resin by suction and the capping was immediately continued.
(133) 5.2.3 Capping (According to the Invention)
(134) The reactor was filled with 26.3 l of DMF. At the same time, 1.2 l of DMF, 0.53 l of acetic anhydride and 0.26 l of diisopropylethylamine (DIPEA) were mixed in a 2 l Schott bottle and added to the resin in the reactor. The reactor was stirred for 10 minutes, then filtering with suction was carried out. After the capping, the resin was washed 5 times in the following sequence: DMF (24 l), isopropanol (31.1 l), DMF (8 l), DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(135) 5.3 Coupling of Fmoc-Gln(Trt)-OH at Position 13
(136) 5.3.1 Fmoc Cleavage
(137) 7 l of DMF were added to the reactor, followed by a mixture of 7.9 l of piperidine in 16.6 l of DMF. This mixture was stirred for 5 minutes, then filtered with suction. This process was repeated and stirring was carried out for 35 minutes; then filtering with suction was carried out again. After the Fmoc cleavage, the resin was washed 7 times in the following sequence: DMF (31.1 l), DMF (31.1 l), isopropanol (31.1 l), DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF (31.1 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(138) 5.3.2 Coupling of Fmoc-Gln(Trt)-OH
(139) 21 l of DMF were added to the reactor. Thereafter, 2.001 kg of FmocGln(Trt)-OH were weighed in and 5.3 l of DMF were added. After complete dissolution, this solution was emptied into the reactor, followed by a solution of 502 g of hydroxybenzotriazole hydrate (HOBt hydrate) in 2.2 l of DMF. Finally, 413 g of N,N-diisopropylcarbodiimide (DIC) were added to the reactor. The coupling time was 6-18 h. After coupling, the solvent was filtered off from the resin by suction and the capping was immediately continued.
(140) 5.3.3 Capping (According to the Invention)
(141) The reactor was filled with 26.3 l of DMF. At the same time, 1.2 l of DMF, 0.53 l of acetic anhydride and 0.26 l of diisopropylethylamine (DIPEA) were mixed in a 2 l Schott bottle and added to the resin in the reactor. The reactor was stirred for 10 minutes, then filtering with suction was carried out. After the capping, the resin was washed 5 times in the following sequence: DMF (24 l), isopropanol (31.1 l), DMF (8 l), DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(142) 5.4 Coupling of Fmoc-Leu-OH at Position 10
(143) 5.4.1 Fmoc Cleavage
(144) 7 l of DMF were added to the reactor, followed by a mixture of 7.9 l of piperidine in 16.6 l of DMF. This mixture was stirred for 5 minutes, then filtered with suction. This process was repeated and stirring was carried out for 35 minutes; then filtering with suction was carried out again. After the Fmoc cleavage, the resin was washed 7 times in the following sequence: DMF (31.1 l), DMF (31.1 l), isopropanol (31.1 l), DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF (31.1 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(145) 5.4.2 Coupling of Fmoc-Leu-OH
(146) 21 l of DMF were added to the reactor. Thereafter, 1.158 kg of Fmoc-Leu-OH were weighed in and 5.3 l of DMF were added. After complete dissolution, this solution was emptied into the reactor, followed by a solution of 502 g hydroxybenzotriazole hydrate (HOBt hydrate) in 2.2 l of DMF. Finally, 413 g of N,N-diisopropylcarbodiimide (DIC) were added to the reactor. The coupling time was 6-18 h. After coupling, the solvent was filtered off from the resin by suction and the capping was immediately continued.
(147) 5.4.3 Capping (According to the Invention)
(148) The reactor was filled with 26.3 l of DMF. At the same time, 1.2 l of DMF, 0.53 l of acetic anhydride and 0.26 l of diisopropylethylamine (DIPEA) were mixed in a 2 l Schott bottle and added to the resin in the reactor. The reactor was stirred for 10 minutes, then filtering with suction was carried out. After the capping, the resin was washed 5 times in the following sequence: DMF (24 l), isopropanol (31.1 l), DMF (8 l), DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(149) 5.5 Coupling of Fmoc-Gly-OH at Position 4
(150) 5.5.1 Fmoc Cleavage
(151) 7 l of DMF were added to the reactor, followed by a mixture of 7.9 l of piperidine in 16.6 l of DMF. This mixture was stirred for 5 minutes, then filtered with suction. This process was repeated and stirring was carried out for 35 minutes; then filtering with suction was carried out again. After the Fmoc cleavage, the resin was washed 7 times in the following sequence: DMF (31.1 l), DMF (31.1 l), isopropanol (31.1 l), DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF (31.1 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(152) 5.5.2 Coupling of Fmoc-Gly-OH
(153) 21 l of DMF were added to the reactor. Thereafter, 1.217 kg of Fmoc-Gly-OH were weighed in and 5.3 l of DMF were added. After complete dissolution, this solution was emptied into the reactor, followed by a solution of 627 g of hydroxybenzotriazole hydrate (HOBt hydrate) in 2.2 l of DMF. Finally, 517 g of N,N-diisopropylcarbodiimide (DIC) were added to the reactor. The coupling time was 6-18 h. After coupling, the solvent was filtered off from the resin by suction and the capping was immediately continued.
(154) 5.5.3 Capping (According to the Invention)
(155) The reactor was filled with 26.3 l of DMF. At the same time, 1.2 l of DMF, 0.53 l of acetic anhydride and 0.26 l of diisopropylethylamine (DIPEA) were mixed in a 2 l Schott bottle and added to the resin in the reactor. The reactor was stirred for 10 minutes, then filtering with suction was carried out. After the capping, the resin was washed 5 times in the following sequence: DMF (24 l), isopropanol (31.1 l), DMF (8 l), DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(156) 5.6 Coupling of Fmoc-Thr(tBu)-OH at Position 5
(157) 5.6.1 Fmoc Cleavage
(158) 7 l of DMF were added to the reactor, followed by a mixture of 7.9 l of piperidine in 16.6 l of DMF. This mixture was stirred for 5 minutes, then filtered with suction. This process was repeated and stirring was carried out for 35 minutes; then filtering with suction was carried out again. After the Fmoc cleavage, the resin was washed 7 times in the following sequence: DMF (31.1 l), DMF (31.1 l), isopropanol (31.1 l), DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF (31.1 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(159) 5.6.2 Coupling of Fmoc-Thr(tBu)-OH
(160) 21 l of DMF were added to the reactor. Thereafter, 1.628 kg of FmocThr(tBu)-OH were weighed in and 5.3 l of DMF were added. After complete dissolution, this solution was emptied into the reactor, followed by a solution of 627 g of hydroxybenzotriazole hydrate (HOBt hydrate) in 2.2 l of DMF. Finally, 517 g of N,N-diisopropylcarbodiimide (DIC) were added to the reactor. The coupling time was 6-18 h. After coupling, the solvent was filtered off from the resin by suction and the capping was immediately continued.
(161) 5.6.3 Capping
(162) The reactor was filled with 10.5 l of DMF. At the same time, 15.8 l of DMF, 3.2 l of acetic anhydride and 1.6 l of diisopropylethylamine (DIPEA) were mixed in a mixing vessel and added to the resin in the reactor. The reactor was stirred for 20 minutes, then filtering with suction was carried out. After the capping, the resin was washed 5 times in the following sequence: DMF (24 l), isopropanol (31.1 l), DMT (8 l), DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time with the respective washing solvent, then stirring was carried out for 3 minutes and filtering with suction was carried out again.
(163) 5.7 Results
(164) The HPLC chromatogram of the crude product of the Lixisenatide synthesis with the capping method according to the invention at the positions Arg(20), Glu(17), Gln(13), Leu(10) and Gly(4), and capping in the other couplings as described under 5.6.3, is shown in
(165) 5.8 Comparison
(166) The capping steps of all couplings, as described under 5.6.3, were carried out, leading to increased formation of the undesired erroneous sequences Ac(20-44)(SEQ ID NO:20), Ac(17-44)(SEQ ID NO:17), Ac(13-44)(SEQ ID NO:15), Ac(10-44)(SEQ ID NO:12) and Ac(4-44)(SEQ ID NO:6)/Ac(6-44)(SEQ ID NO:8). The HPLC chromatogram of a crude Lixisenatide from this test is shown in
(167)
(168)
(169) By using a milder capping mixture (2% acetic anhydride/1% DIPEA in DMF for 10 minutes), it was possible to reduce the level of acetylated erroneous sequences of Ac(20-44)(SEQ ID NO:20), Ac(17-44)(SEQ ID NO:17), Ac(13-44)(SEQ ID NO:15), Ac(10-44)(SEQ ID NO:12) and Ac(4-44)(SEQ ID NO:6) in the crude product of Lixisenatide or eliminate them therefrom. Since a Lixisenatide crude product which was prepared by the capping according to the invention included the acetylated by-products Ac(17-44)(SEQ ID NO:17), Ac(13-44)(SEQ ID NO:15) and Ac(10-44)(SEQ ID NO:12) in particular in considerably reduced amounts, the purification of Lixisenatide was simplified. As a result, pooling of the fractions after the first preparative chromatography run of Lixisenatide gave more fractions which met the specification criteria and thus did not have to be discarded. This led to an improved yield.
EXAMPLE 6
(170) Capping at 9 Specific Positions in the Synthesis of Lixisenatide
(171) As discussed in Example 5, the use of “mild” capping conditions in the synthesis of lixisenatide at positions Arg(20), Glu(17), Gln(13), Leu(10) or/and Gly(4) could improve the profile of undesired by-products.
(172) This Example describes the influence of capping conditions upon the formation of acetylated and non-acetylated by-products. Variations in the temperature (15° C., room temperature [20° C.-23° C.], 30° C.), capping duration and the ingredients of the capping composition were performed: no capping, mild capping conditions: 10 min capping with 2% acetic anhydride and 1% of DIPEA (diisopropylethylamine) “normal” capping conditions: 20 min capping with 10% acetic anhydride and 5% of DIPEA 40 min capping with 10% acetic anhydride and 5% of DIPEA
(173) Capping conditions of the present invention are the “mild conditions”. These conditions were used in Example 5. These conditions were found to be advantageous.
(174) At the 9 positions selected in this Example, acetylated sequences are obtained at capping of the (N−1) position (
(175) 6.1 Capping at Position 36/35, after Coupling of the Dipeptide Building Block Pro-Pro, (36-44)
(176) Peptide Fmoc-(36-44)-AVE0010 was produced by solid phase synthesis. The resin was dried in divided into 4 portions. Each portion underwent one of the four capping procedures described above at room temperature (20° C.-23° C.). Samples were dried, and the peptide was cleaved from the resin. This procedure was repeated, wherein capping was performed at 15° C. or 30° C.
(177) In a total 12 peptide samples were obtained. The 12 peptide samples were analyzed with LCMS. Molecular weights were determined from the TIC (total ion current). The molecular weights of the following compounds were determined:
(178) TABLE-US-00009 TABLE 7 Ac(36-44) can be formed by Fmoc cleavage during capping and (SEQ ID NO: 26) subsequent acetylation (undesired by-product) Fmoc(36-44) desired product (main product) of solid phase synthesis (36-44) can be formed by Fmoc cleavage during capping, but no acetylation takes place (undesired by-product) (38-44) may be still present if coupling of the Fmoc-dipeptide building block was incomplete, but no acetylation takes place during the capping step (undesired by-product) Ac(38-44) desired capping product, may be formed by capping if coupling of the Fmoc-dipeptide building block was incomplete.
(179) Table 8 shows the content of products obtained after Fmoc-ProPro coupling and subsequence capping (% of total peptide content).
(180) TABLE-US-00010 Ac(36-44) (SEQ ID NO: 26) Fmoc(36-44) (36-44) (38-44) Ac(38-44) Position 36/35 Pro-Pro, 15° C. without capping 0.02 99.96 0 0.02 0 10 min, 2% Ac2O, 1% DIPEA 0.37 99.6 0 0.03 0 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.09 99.73 0 0.18 0 (normal) 40 min, 10% Ac2O, 5% DIPEA 0.4 99.57 0 0.02 0 Position 36/35 Pro-Pro, RT without capping 0.09 99.84 0 0 0.06 10 min, 2% Ac2O, 1% DIPEA 0.47 99.47 0 0 0.06 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.9 99.04 0 0 0.06 (normal) 40 min, 10% Ac2O, 5% DIPEA 1.52 98.42 0 0 0.05 Position 36/35 Pro-Pro, 30° C. without capping 0 100 0 0 0 10 min, 2% Ac2O, 1% DIPEA 0.21 99.79 0 0 0 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.66 99.34 0 0 0 (normal) 40 min, 10% Ac2O, 5% DIPEA 2.39 97.61 0 0 0
(181) The results are described in
(182) 6.2 Capping at Position 23, after Coupling of the Building Ile, (23-44)
(183) The synthesis of Fmoc(23-44) was performed as described in section 6.1. Experiments at 15° C./30° C. and at room temperature were performed with different batches.
(184) Table 9 shows the content of products obtained after Fmoc-Ile coupling and subsequence capping (% of total peptide content)
(185) TABLE-US-00011 Ac(23-44) (SEQ ID NO: 23) Fmoc(23-44) (23-44) (24-44) Ac(24-44) Position 23 Ile, 15° C. without capping 0 99.7 0 0.16 0.14 10 min, 2% Ac2O, 1% DIPEA 0 99.56 0.18 0.11 0.15 (mild) 20 min, 10% Ac2O, 5% DIPEA 0 99.57 0.2 0.11 0.13 (normal) 40 min, 10% Ac2O, 5% DIPEA 0 99.59 0.16 0.1 0.15 Position 23 Ile, RT without capping 0 99.81 0 0.19 0 10 min, 2% Ac2O, 1% DIPEA 0.13 99.7 0 0.16 0 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.26 99.54 0 0.2 0 (normal) 40 min, 10% Ac2O, 5% DIPEA 0.61 99.21 0 0.18 0 Position 23 Ile, 30° C. without capping 0 99.66 0 0.18 0.16 10 min, 2% Ac2O, 1% DIPEA 0.1 99.38 0.19 0.16 0.17 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.77 98.65 0.25 0.15 0.17 (normal) 40 min, 10% Ac2O, 5% DIPEA 1.43 98.15 0.16 0.12 0.14
(186) The results are described in
(187) Formation of the desired product Ac(24-44) is independent from the capping composition.
(188) 6.3 Capping at Position 21, after Coupling of the Building Block Leu, (21-44)
(189) The synthesis of Fmoc(21-44) was performed as described in section 6.1. Experiments at 15° C./30° C. and at room temperature were performed with different batches.
(190) Table 10 shows the content of products obtained after Fmoc-Leu coupling and subsequence capping (% of total peptide content)
(191) TABLE-US-00012 Ac(21-44) Ac(22-44) (SEQ ID (SEQ ID NO: 21) Fmoc(21-44) (21-44) (22-44) NO: 22) Position 21 Leu, 15° C. without capping 0 99.91 0 0 0.09 10 min, 2% Ac2O, 1% DIPEA 0.03 99.78 0.07 0 0.12 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.09 99.74 0.05 0 0.12 (normal) 40 min, 10% Ac2O, 5% DIPEA 0.36 99.48 0.03 0 0.13 Position 21 Leu, RT without capping 0 99.77 0 0.07 0.16 10 min, 2% Ac2O, 1% DIPEA 0.06 99.64 0 0.14 0.16 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.2 99.62 0 0.04 0.14 (normal) 40 min, 10% Ac2O, 5% DIPEA 0.46 99.34 0 0.04 0.16 Position 21 Leu, 30° C. without capping 0 99.86 0.04 0 0.11 10 min, 2% Ac2O, 1% DIPEA 0.1 99.67 0.11 0 0.12 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.86 98.95 0.06 0 0.13 (normal) 40 min, 10% Ac2O, 5% DIPEA 2.57 97.22 0.05 0 0.16
(192) The results are described in
(193) Formation of the desired compound Ac(22-44)(SEQ ID NO:22) is independent from the capping composition. Even without capping, this compound is formed.
(194) 6.4 Capping at Position 19, after Coupling of the Building Block Val, (19-44)
(195) The synthesis of Fmoc(19-44) was performed as described in section 6.1. Experiments at 15° C./30° C. and at room temperature were performed with different batches.
(196) Table 1 l shows the content of products obtained after Fmoc-Val coupling and subsequence capping (% of total peptide content)
(197) TABLE-US-00013 Ac(19-44) Ac(20-44) (SEQ ID (SEQ ID NO: 19) Fmoc(19-44) (19-44) (20-44) NO: 20) Position 19 Val, 15° C. without capping 0 98.98 0.13 0.46 0.44 10 min, 2% Ac2O, 1% DIPEA 0.11 98.79 0.44 0.25 0.4 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.56 98.68 0.14 0.25 0.37 (normal) 40 min, 10% Ac2O, 5% DIPEA 1 98.17 0.09 0.23 0.51 Position 19 Val, RT without capping 0 99.61 0 0.23 0.16 10 min, 2% Ac2O, 1% DIPEA 0.14 99.52 0 0.15 0.2 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.43 99.23 0 0.17 0.17 (normal) 40 min, 10% Ac2O, 5% DIPEA 0.9 98.9 0 0 0.2 Position 19 Val, 30° C. without capping 0 99.16 0.08 0.4 0.36 10 min, 2% Ac2O, 1% DIPEA 0.41 98.59 0.4 0.27 0.33 (mild) 20 min, 10% Ac2O, 5% DIPEA 2.3 96.89 0.14 0.22 0.45 (normal) 40 min, 10% Ac2O, 5% DIPEA 5.09 94.1 0.11 0.22 0.48
(198) The results are described in
(199) Formation of the desired compound Ac(20-44)(SEQ ID NO:20) increases with the strength of the capping composition. The content of undesired (20-44) decreases with increasing strength of the capping composition.
(200) 6.5 Capping at Position 18, after Coupling of the Building Block Ala, (18-44)
(201) The synthesis of Fmoc(18-44) was performed as described in section 6.1. Experiments at 15° C./30° C. and at room temperature were performed with different batches.
(202) Table 12 shows the content of products obtained after Fmoc-Ala coupling and subsequence capping (% of total peptide content)
(203) TABLE-US-00014 Ac(18-44) Ac(19-44) (SEQ ID (SEQ ID NO: 18) Fmoc(18-44) (18-44) (19-44) NO: 19) Position 18 Ala, 15° C. without capping 0 98.77 0.48 0.33 0.42 10 min, 2% Ac2O, 1% DIPEA 0.48 98.24 0.53 0.26 0.49 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.76 98.18 0.27 0.2 0.59 (normal) 40 min, 10% Ac2O, 5% DIPEA 1.12 97.91 0.23 0.22 0.52 Position 18 Ala, RT without capping 0 99.63 0 0 0.37 10 min, 2% Ac2O, 1% DIPEA 0.1 99.43 0.1 0 0.36 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.77 98.69 0.18 0 0.36 (normal) 40 min, 10% Ac2O, 5% DIPEA 0.38 99.28 0 0 0.38 Position 18 Ala, 30° C. without capping 0 98.76 0.53 0.2 0.5 10 min, 2% Ac2O, 1% DIPEA 0.92 98.07 0.32 0.11 0.58 (mild) 20 min, 10% Ac2O, 5% DIPEA 2.44 96.67 0.09 0.14 0.65 (normal) 40 min, 10% Ac2O, 5% DIPEA 6.33 92.73 0.05 0.14 0.73
(204) The results are described in
(205) Formation of the desired compound Ac(19-44)(SEQ ID NO:19) increases at 15° C. and 30° C. with the strength of the capping composition.
(206) 6.6 Capping at Position 15, after Coupling of the Building Block Glu (15-44)
(207) The synthesis of Fmoc(15-44) was performed as described in section 6.1. Experiments at 15° C./30° C. and at room temperature were performed with different batches.
(208) Table 13 shows the content of products obtained after Fmoc-Glu coupling and subsequence capping (% of total peptide content)
(209) TABLE-US-00015 Ac(15-44) Fmoc(15-44) (15-44) (16-44) Ac(16-44) Position 15 Glu, 15° C. without 0 99.28 0 0.59 0.13 capping 10 min, 2% 0.05 99.08 0.15 0.57 0.15 Ac2O, 1% DIPEA (mild) 20 min, 10% 0.19 99.08 0 0.58 0.15 Ac2O, 5% DIPEA (normal) 40 min, 10% 0.39 98.82 0 0.63 0.16 Ac2O, 5% DIPEA Position 15 Glu, RT without 0 99.72 0.12 0 0.17 capping 10 min, 2% 0.1 99.4 0.36 0 0.16 Ac2O, 1% DIPEA (mild) 20 min, 10% 0.42 99.13 0.2 0.04 0.21 Ac2O, 5% DIPEA (normal) 40 min, 10% 0.89 98.65 0.22 0.05 0.19 Ac2O, 5% DIPEA Position 15 Glu, 30° C. without 0 98.93 0 0.91 0.16 capping 10 min, 2% 0.17 98.7 0 0.95 0.18 Ac2O, 1% DIPEA (mild) 20 min, 10% 1.62 97.3 0 0.88 0.2 Ac2O, 5% DIPEA (normal) 40 min, 10% 3.24 95.63 0 0.94 0.19 Ac2O, 5% DIPEA
(210) The results are described in
(211) Formation of the desired compound Ac(16-44) is independent from the capping composition. Even without capping, this compound is formed.
(212) 6.7 Capping at Position 12, after Coupling of the Building Block Lys (12-44)
(213) The synthesis of Fmoc(12-44) was performed as described in section 6.1. Experiments at 15° C./30° C. and at room temperature were performed with different batches.
(214) Table 14 shows the content of products obtained after Fmoc-Lys coupling and subsequence capping (% of total peptide content)
(215) TABLE-US-00016 Ac(12-44) Ac(13-44) (SEQ ID (SEQ ID NO: 14) Fmoc(12-44) (12-44) (13-44) NO: 15) Position 12 Lys, 15° C. without capping 0 99.43 0.13 0 0.44 10 min, 2% Ac2O, 1% DIPEA 0.15 99.25 0.17 0 0.43 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.3 99.03 0.17 0 0.49 (normal) 40 min, 10% Ac2O, 5% DIPEA 0.55 98.88 0.16 0 0.41 Position 12 Lys, RT without capping 0 99.12 0 0.17 0.71 10 min, 2% Ac2O, 1% DIPEA 0 99.29 0 0 0.71 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.5 98.76 0 0 0.74 (normal) 40 min, 10% Ac2O, 5% DIPEA 1.12 98.15 0 0 0.73 Position 12 Lys, 30° C. without capping 0 99.41 0.15 0 0.44 10 min, 2% Ac2O, 1% DIPEA 0.35 99.02 0.16 0 0.47 (mild) 20 min, 10% Ac2O, 5% DIPEA 1.55 97.89 0.14 0 0.41 (normal) 40 min, 10% Ac2O, 5% DIPEA 3.53 95.87 0.16 0 0.44
(216) The results are described in
(217) Formation of the desired compound Ac(13-44)(SEQ ID NO:15) is independent from the capping composition. Even without capping, this compound is formed.
(218) 6.8 Capping at Position 8, after Coupling of the Building Block Ser (8-44)
(219) The synthesis of Fmoc(8-44) was performed as described in section 6.1. Experiments at 15° C., RT and 30° C. were performed with the same batch.
(220) Table 15 shows the content of products obtained after Fmoc-Ser coupling and subsequence capping (% of total peptide content)
(221) TABLE-US-00017 Ac(8-44) Ac(9-44) (SEQ ID (SEQ ID NO: 10) Fmoc(8-44) (8-44) (9-44) NO: 11) Position 8 Ser, 15° C. without capping 0 100 0 0 0 10 min, 2% Ac2O, 1% DIPEA 0 99.79 0 0 0.21 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.29 99.53 0 0 0.18 (normal) 40 min, 10% Ac2O, 5% DIPEA 1.08 98.72 0 0 0.21 Position 8 Ser, RT without capping 0 99.67 0 0.18 0.16 10 min, 2% Ac2O, 1% DIPEA 0.22 99.78 0 0 0 (mild) 20 min, 10% Ac2O, 5% DIPEA 1.12 98.88 0 0 0 (normal) 40 min, 10% Ac2O, 5% DIPEA 2.1 97.9 0 0 0 Position 8 Ser, 30° C. without capping 0 100 0 0 0 10 min, 2% Ac2O, 1% DIPEA 0.29 99.27 0.27 0 0.18 (mild) 20 min, 10% Ac2O, 5% DIPEA 2.1 97.8 0 0 0.1 (normal) 40 min, 10% Ac2O, 5% DIPEA 5.02 94.74 0 0 0.24
(222) The results are described in
(223) 6.9 Capping at Position 6, after Coupling of the Building Block Phe (6-44)
(224) The synthesis of Fmoc(86-44) was performed as described in section 6.1. Experiments at 15° C., RT and 30° C. were performed with the same batch.
(225) Table 16 shows the content of products obtained after Fmoc-Phe coupling and subsequence capping (% of total peptide content)
(226) TABLE-US-00018 Ac(6-44) Ac(7-44) (SEQ ID (SEQ ID NO: 8) Fmoc(6-44) (6-44) (7-44) NO: 9) Position 6 Phe, 15° C. without capping 0 99.21 0 0.38 0.41 10 min, 2% Ac2O, 1% DIPEA 0 99 0.39 0.28 0.34 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.35 98.73 0.32 0.26 0.33 (normal) 40 min, 10% Ac2O, 5% DIPEA 0.62 98.6 0.3 0.18 0.3 Position 6 Phe, RT without capping 0 99.24 0 0.39 0.37 10 min, 2% Ac2O, 1% DIPEA 0.2 98.68 0.6 0.25 0.28 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.57 98.49 0.31 0.25 0.38 (normal) 40 min, 10% Ac2O, 5% DIPEA 1.32 97.9 0.33 0.2 0.24 Position 6 Phe, 30° C. without capping 0 99.24 0 0.43 0.33 10 min, 2% Ac2O, 1% DIPEA 0.33 98.36 0.55 0.29 0.46 (mild) 20 min, 10% Ac2O, 5% DIPEA 1.54 97.42 0.37 0.3 0.37 (normal) 40 min, 10% Ac2O, 5% DIPEA 3.73 95.91 0 0 0.36
(227) The results are described in
(228) Formation of the desired compound Ac(7-44)(SEQ ID NO:9) is independent from the capping composition. Even without capping, this compound is formed.
(229) Temperature has only slight influence on formation of the desired compound Ac(7-44)(SEQ ID NO:9). The content of undesired (7-44) decreases with increasing strength of the capping composition.
(230) 6.10 Summary
(231) Undesired formation of Ac(X-44)-compound strongly depends upon the capping duration, the capping composition and the capping temperature. With increasing capping duration, increasing capping temperature, and increased content of acetic anhydride and DIPEA in the capping composition the content of undesired Ac(X-44) compound increases.
(232) 6.1 l Capping Under “Normal” Conditions, Depending Upon Temperature.
(233)
(234)
(235) The formation of undesired product Ac(X-44) is 0.5% in 5 of 9 positions, in 3 positions between 0.5% and 1%, and in only one position >1%. A large increase is observed at 30° C., while at 15° C., formation of Ac(X-44) slightly decreases.
(236) This means that GMP capping “20 min, 10% Ac2O, 5% DIPEA” can be performed at different positions between 15° C. and room temperature, which can be 20-23° C.
(237)
(238) Regarding the desired formation of the Ac[(X-1)-44] compounds at RT, the deviation at 15° C. is between +0.23 und −0.25%. At 30° C., the deviation is between +0.29 und −0.33%. Formation of the desired capping product Ac[(X-1)-44] is thus less dependent upon the temperature than the undesired formation of Ac(X-44).
(239) At 15° C. and 30° C., negative deviations of the content of desired compound Ac[(X-1)-44] are observed in view of capping at room temperature. This means that capping with “normal” conditions should be performed at room temperature.
(240) 6.12 Capping with Different Capping Compositions at Room Temperature.
(241)
(242)
(243) Formation of undesired product Ac(X-44) under “20 min, 10% Ac2O, 5% DIPEA” and “40 min, 10% Ac2O, 5% DIPEA” is largest. Formation of Ac(X-44) under “normal” conditions (40 min, 10% Ac2O, 5% DIPEA) is between 0.2% and 1.12%. A strong decrease is observed at mild capping conditions.
(244)
(245) The formation of the desired product Ac[(X-1)-44] at the conditions “no capping”, “mild” and “40 min” is within −0.14% and +0.16% in view of the “normal” conditions.
(246) In particular, under “mild” conditions (10 min, 2% Ac2O, 1% DIPEA) of the invention, sufficient capping can be achieved in the synthesis of lixisenatide.
(247) In summary, mild capping conditions, in particular capping for 10 min with 2% Ac2O and 1% DIPEA in a solvent, are advantageous in the solid phase synthesis of lixisenatide, as described herein.
(248) If capping is omitted after coupling at certain amino acid positions, undesired by-products comprising an incomplete amino acid sequence and being present in small amount, may be difficult to remove during the purification process.
EXAMPLE 7
(249) Cleavage of Lixisenatide from the Solid Phase
(250) This example relates to the cleavage according to the invention of Lixisenatide from a solid phase. A solid phase (Rink resin) was provided, to which the peptide Lixisenatide was bonded. The peptide was synthesized on the resin by stepwise coupling of amino acid units.
(251) As comparative test, a cleavage according to the prior art (King et al., Int. J. Peptide Protein Res. 1990, 36: 255-266) was carried out.
(252) The cleavage method according to the invention is distinguished from the method of the prior art by the following changes: Reaction temperature from 20° C. to 26° C. Number of components in the cleavage mixture reduced from 5 to 2 constituents, combined with increase in the ratio of resin to cleavage mixture used.
(253) TABLE-US-00019 TABLE 17 Comparison of the cleavage method according to the invention and the cleavage according to the prior art. The differences are indicated in bold/underlined. Comparative process (prior art) Method according to the invention Cleavage mixture [g or ml/g “peptide on resin”]: Cleavage mixture [ml/g “peptide on resin”]: a) 0.5 g phenol a) 0.25 ml 1,2-ethanedithiol b) 0.5 ml thioanisole b) 8.25 ml TFA c) 0.25 ml 1,2-ethanedithiol d) 0.5 ml water e) 8.25 ml TFA (“King's cocktail”) Cleavage mixture is cooled to 5-10° C. and Cleavage mixture is cooled to 5-10° C. and added to Lixisenatide-resin(1-44) added to Lixisenatide-resin(1-44) (SEQ ID NO: 1) (SEQ ID NO: 1) Reaction mixture heated to 20° C. and Reaction mixture heated to 26° C. and stirred for 4 h stirred for 4 h Reaction mixture filtered Reaction mixture filtered Subsequent cleavage Subsequent cleavage The resin filtered off is added to TFA The resin filtered off is added to TFA (10 ml per g of resin), stirred for 1 h and (10 ml per g of resin), stirred for 1 h and the resin is filtered off the resin is filtered off Filtrate purified and solution concentrated Filtrate purified and solution concentrated by distillation under reduced pressure at by distillation under reduced pressure at 35-40° C. to at least 1/16th of the original 35-40° C. to at least 1/16th of the original volume. volume. Crude Lixisenatide precipitated by addition Crude Lixisenatide precipitated by addition of the concentrate to 6 times the volume of of the concentrate to 6 times the volume of DIPE DIPE The precipitate is resuspended twice in The precipitate is resuspended twice in ethyl acetate and filtered off. ethyl acetate and filtered off. The precipitate is dried and the crude The precipitate is dried and the crude Lixisenatide is isolated. Lixisenatide is isolated.
(254) By using the cleavage according to the invention, compared to the cleavage according to the prior art, it was possible to increase the yields of the crude Lixisenatide by approximately 5% (from 20% to 25%), while the impurities profile was only slightly altered.
(255) The method of this example is suitable for scale-up to the pilot-plant and production scale.
(256) Table 18 summarizes the results obtained in the comparative process (see Table 17). Three different batches 2E002, 26008 and 26006 of lixisenatide-resin (1-44)(SEQ ID NO:1) were used. Means and standard deviation are calculated for each batch separately. Comparison between different cleavage conditions should be made in tests using the same batch of lixisenatide-resin (1-44)(SEQ ID NO:1). In different batches, the solid phase synthesis may have an impact on the yield. If not otherwise indicated, 10 g of lixisenatide-resin(1-44)(SEQ ID NO:1) were used as starting material.
(257) TABLE-US-00020 TABLE 18 Content of lixisenatide in the resin, and yield of lixisenatide after cleavage of lixisenatide from the resin under standard conditions (comparative process, see Table 17). Batch of lixisenatide- resin(1-44) Output Number of (SEQ ID weight Content Yield experiment NO: 1) [g] [%] [%] Batch 2E002 71002-002 2E002 2.60 22.9 10.2 71002-003 2E002 3.36 23.1 13.4 71002-012 2E002 1.88 20.3 6.6 separate subsequent 0.77 25.0 3.3 cleavage 70609-068 2E002 3.01 21.5 11.1 71002-035 2E002 3.08 16.3 8.6 71002-036 2E002 3.07 16.3 8.6 70586-043 2E002 2.40 24.3 10.1 71003-003 2E002 2.50 24.0 10.3 Mean ± standard 10.3 ± 1.5 deviation Batch 2B008 70586-052 2B008 2.84 26.9 14.7 71001-006 2B008 3.03 20.0 11.7 71002-048 2B008 2.89 22.8 12.7 71001-016 2B008 3.41 21.6 14.2 Mean ± standard 13.4 ± 1.4 deviation Batch 2B006 70586-056 2B006 3.02 25.0 14.3
(258) 7.1 Cleavage Yield Depending Upon the Cleavage Temperature Between 20° C. and 35° C.
(259) Cleavage from the lixisenatide-resin(1-44)(SEQ ID NO:1) was performed under standard conditions (comparative process, see Table 17) for 4 h.
(260) TABLE-US-00021 TABLE 19 Cleavage yield depending upon temperature Batch of lixisenatide- resin(1-44) Number of (SEQ ID Temperature Duration Yield experiment NO: 1) [° C.] [h] [%] Standard 2E002 20 4 10.3 ± 1.5 70586-050 2E002 23 4 11.7 70586-044 2E002 26 4 14.8 70586-046 2E002 30 4 14.2 70586-049 2E002 35 4 12.4
(261) Results: The yield of lixisenatide after cleavage under standard conditions increases with increasing temperature until the optimum of about 26° C. Surprisingly, an increase of temperature from 23° C. to 26° C. results in a significant increase in yield.
(262) 7.2 Cleavage Yield Depending Upon the Cleavage Duration
(263) Cleavage from the lixisenatide-resin(1-44)(SEQ ID NO:1) was performed under standard conditions (comparative process, see Table 17) at 20° C.
(264) TABLE-US-00022 TABLE 20 Cleavage yield depending upon the cleavage duration Batch of lixisenatide- resin(1-44) Number of (SEQ ID Temperature Duration Yield experiment NO: 1) [° C.] [h] [%] Standard 2E002 20 4 10.3 ± 1.5 71002-037 2E002 20 6 11.3 71002-038 2E002 20 8 13.4 70586-037, 2E002 20 12 13.1 ± 0.9 71003-002, 71003-004
(265) Results: The yield of lixisenatide increases with increased cleavage duration. A maximum yield is reached after about 8 h cleavage.
(266) 7.3 Cleavage Yield Depending Upon the Temperature at Cleavage Duration of 12 h
(267) Cleavage from the lixisenatide-resin(1-44)(SEQ ID NO:1) was performed under standard conditions (comparative process, see Table 17) for 4 h.
(268) TABLE-US-00023 TABLE 21 Cleavage yield depending upon the temperature at cleavage duration of 12 h Batch of lixisenatide- resin(1-44) Number of (SEQ ID Temperature Duration Yield experiment NO: 1) [° C.] [h] [%] 70586-040 2E002 17 12 10.7 70586-037 2E002 20 12 14.1 70586-039 2E002 23 12 13.0 70586-045 2E002 26 12 14.0 70586-047 2E002 30 12 12.1
(269) Results: The yield increases at a cleavage duration of 12 h if reaction temperature is increased. A maximum yield is obtained at 26° C., as described in Example 7.1 for 4 h cleavage. Tests 70586-044 (4 h, 26° C., Example 7) and 70586-045 (12 h, 26° C.) resulted in similar yields (14.8% vs. 14.0%).
(270) 7.4 Cleavage Yield Depending Upon the Cleavage Temperature Up to 20° C.
(271) Cleavage from the lixisenatide-resin(1-44)(SEQ ID NO:1) was performed under standard conditions (comparative process, see Table 17) for 4 h.
(272) TABLE-US-00024 TABLE 22 Cleavage yield depending upon the cleavage temperature up to 20° C. Batch of lixisenatide- resin(1-44) Number of (SEQ ID Temperature Duration Yield experiment NO: 1) [° C.] [h] [%] 71002-028 2E002 0-5° C. 21.5 6.0 71002-029 2E002 8-13° C. 28 8.7 71002-030 2E002 8-13° C. 40.8 11.2 70586-040 2E002 17° C. 12 10.7 Standard 2E002 20° C. 4 10.3 ± 1.5 70586-037, 2E002 20° C. 12 13.1 ± 0.9 71003-002, 71003-004
(273) Results: The cleavage at a temperature below 20° C. requires longer cleavage durations, as expected, to reach the yield obtained by cleavage at 20° C. for 4 h (standard conditions, comparative process, Table 17).
(274) 7.5 Modified Cleavage Cocktail
(275) The standard process uses a cleavage cocktail containing five components: phenol, thioanisole, 1,2-ethandithiole, water and TFA. Subject of the example are simplified cleavage cocktails, omitting one to three of thioanisole, phenol and water. The yield of lixisenatide cleavage from lixisenatide-resin(1-44)(SEQ ID NO:1) is determined. The “no modification” cocktail is described in Table 17, “Comparative process”.
(276) TABLE-US-00025 TABLE 23 Modified cleavage cocktail Batch of lixisenatide- resin(1-44) Modification of Number of (SEQ ID cleavage Yield experiment NO: 1) composition [%] Standard 2E002 no modification 10.3 ± 1.5 71002-010 2E002 without thioanisole 10.7 71002-009 2E002 without phenol 12.1 71002-006 2E002 without water 13.2 71002-008 2E002 without phenol and 13.3 water 71003-008 2E002 water content is 13.3 reduced to 2.5% w/w 71002-042 2E002 without thioanisole, 12.7 phenol and water, i.e. only TFA and 1,2-ethanedithiol
(277) Results: Omission of one or more components results in an increased yield, except test 71002-010 (omission of thioanisole).
(278) A simplified cleavage mixture (cleavage cocktail) has several advantages:
(279) (a) simplification of analytics and quality control,
(280) (b) reduced costs,
(281) (c) facilitated handling in the production process.
(282) 7.6 TFA and 1,2-Ethanedithiol Content in the Cleavage Cocktail
(283) Starting from test 71002-042, the influence of the TFA:1,2-ethaneditihiol ratio upon cleavage yield was investigated:
(284) TABLE-US-00026 TABLE 24 Different TFA and 1,2-ethanedithiol ratio in the cleavage cocktail Batch of Lixisenatide- Volume in mL of resin (1-44) TFA and 1,2- Number of (SEQ ID ethanedithiol per g Yield Experiment NO: 1) .sub.“peptide on resin” [%] Standard 2E002 10.3 ± 1.5 71002-045 2E002 8:2 6.5 71002-043 2E002 9:1 9.3 71002-044 2E002 .sup. 9:0.5 11.0 71002-042 2E002 8.25:0.25 12.7 Standard 2B008 13.4 ± 1.4 71002-046 2B008 8.25:0.25 15.6 71002-047 2B008 8.25:0.25 14.3
(285) Results: An increase in the 1,2-ethanedithiol content results in a significant decrease of lixisenatide yield. The TFA:1,2-ethanedithiol ratio of 8.25:0.25 was found to be the ratio with largest yield (batch 2E002). This finding was confirmed by to experiments using batch 2B008.
(286) 7.7 Volume of the Cleavage Cocktail
(287) The influence of volume (and thus concentration) of the cleavage cocktail was investigated
(288) TABLE-US-00027 TABLE 25 Cleavage yield, depending upon volume of the cleavage cocktail. Batch of Lixisenatide- resin (1-44) Reduction Number of (SEQ ID of volume Yield Experiment NO: 1) [%] [%] Standard 2E002 0% 10.3 ± 1.5 71002-026 2E002 −10% 13.3 71002-040 2E002 −15% 10.3 71002-025 2E002 −25% 11.0 70609-069 2E002 −30% 10.5 71002-031 2E002 −50% 7.9
(289) Results: The reduction of up to 30% has no influence upon cleavage yield. Larger volume reductions lead to a decreased yield.
(290) 7.8 Swelling of the “Peptide on Resin” with a Co-Solvent (Toluol or CH.sub.2Cl.sub.2) Before Cleavage
(291) The rationale behind this experiment is the finding that cleavage of lixisenatide from the resin may result in an increase in temperature of up to 5-8° C., which may lead to formation of undesired by-products and potentially has a negative impact upon stability and thus the cleavage yield. Swelling of the “peptide on resin” in an organic solvent may reduce the exotherm and thus may increase the yield.
(292) TABLE-US-00028 TABLE 26 Cleavage yield, depending upon the presence of a co-solvent. Batch of Lixisenatide- resin (1-44) Swelling Increase of Number of (SEQ ID with organic Duration temperature Yield Experiment NO: 1) solvent [h] [° C.] [%] Standard 2E002 without 5-8° C. 10.3 ± 1.5 71002-016 2E002 30 ml toluol* 4 h 1-2° C. 9.8 71002-019 2E002 30 ml toluol 6 h 1-2° C. 6.1 71002-021 2E002 30 ml toluol 17 h 1-2° C. 9.3 71002-017 2E002 50 ml toluol 28 h 1-2° C. 4.7 71002-024 2E002 30 ml CH.sub.2Cl.sub.2 24 h 1-2° C. 7.3 *The total volume of TFA and toluol/CH.sub.2Cl.sub.2 is kept constant.
(293) Results: Swelling with an organic co-solvent does not increase the cleavage yield.
(294) 7.9 Concentration in the Presence of a Co-Solvent
(295) The presence of a co-solvent, having a higher boiling point than TFA, and in which lixisenatide is insoluble, may increase the yield after cleavage from the resin, because during distillation of TFA from the filtrate, the presence of the co-solvent may lead to precipitation of lixisenatide, and therefore can prevent the degradation of lixisenatide during cleavage in King's cocktail.
(296) TABLE-US-00029 TABLE 27 Cleavage yield, depending upon the presence of a co-solvent during TFA distillation. Batch of lixisenatide- resin(1-44) Number of (SEQ ID Yield Experiment NO: 1) Solvent [%] Standard 2E002 Ohne 10.3 ± 1.5 71002-004 2E002 Toluol 12.0 71002-014 2E002 n-Heptan 11.1
(297) Results: The presence of toluol in the distillation of the filtrate after cleavage of lixisenatide from the resin leads to a slightly increased yield.
(298) 7.10 Optimized Cleavage Procedure of the Invention
(299) Based upon the above-described results obtained in this Example, optimized cleavage conditions as follows were selected and tested: (a) reaction temperature of 26° C., (b) cleavage cocktail consists of TFA and 1,2-ethanedithiol. The cocktail contained about 97% of TFA and about 3% of 1,2-ethanedithiol. An amount of 8.25 ml/g “peptide on resin” of TFA and 0.25 ml/g “peptide on resin” of 1,2-ethanedithiol was used.
(300) The cleavage yield of this cocktail, compared with the standard comparative cocktail, was tested in batches 2E002 and 26008.
(301) TABLE-US-00030 TABLE 28 Optimized cleavage procedure of the invention Batch of Lixisenatide- resin (1-44) Modification Number of (SEQ ID of cleavage Yield experiment NO: 1) composition [%] Standard 2E002 no 10.3 ± 1.5 modification 70586-051 2E002 26° C., only 14.9 TFA and EDT 71001-012 2E002 26° C., only 15.8 TFA and EDT Mean ± standard 15.4 ± 0.6 deviation Standard 2B008 no 13.4 ± 1.4 modification 70586-051 2B008 26° C., only 18.5 TFA and EDT 71001-013 2B008 26° C., only 19.7 TFA and EDT Mean ± standard 19.4 ± 0.4 deviation
(302) Results: In both batches, the yield increased by about 5%, indicating a significant improvement of the peptide cleavage from the solid phase by the method of the invention.
(303) 7.11 Second (Subsequent) Cleavage
(304) After the cleavage, using the comparative cocktail (King's cocktail) or the cleavage cocktail of the invention, a second (subsequent) cleavage, was performed (see Table 17).
(305) The first cleavage was performed, a filtrate was obtained. TFA was added to the TFA-wet resin. After 1 h stirring, the resin was filtrated. The filtrates were combined and concentrated.
(306) The effect of the second, subsequent cleavage upon lixisenatide yield was investigated.
(307) TABLE-US-00031 TABLE 29 Influence of a second (subsequent) cleavage of lixisenatide yield. Batch of Lixisenatide- resin (1-44) Modification Subsequent Number of (SEQ ID of cleavage cleavage Yield experiment NO: 1) composition (TFA only) [%] Standard 2B008 26° C., only yes 13.4 ± 1.4 TFA and EDT 70586-051 2B008 26° C., only yes 18.5 TFA and EDT 71001-013 2B008 26° C., only yes 19.7 TFA and EDT Mean ± 19.1 ± 0.4 standard deviation 70001-018 2B008 26° C., only no 17.9 TFA and EDT 70001-019 2B008 26° C., only no 18.1 TFA and EDT 70609-078 2B008 26° C., only no 18.1 TFA and EDT 70001-020 2B008 26° C., only no 20.1 TFA and EDT Mean ± 18.4 ± 1.1 standard deviation EDT: 1.2-ethanedithiol.
(308) Results: Subsequent cleavage results in an increase of the yield of only about 0.7%. This increase is associated with a significant increase in costs for starting materials (TFA), and additional efforts to remove the TFA from the peptide preparation. It must be considered that by combination of the filtrates of the first and second cleavage step, the amount of TFA significantly increases.
(309) It is concluded that in view of the small increase in yield, omission of the second cleavage leads to a cost reduction, and handling during the production process is facilitated. The amounts of TFA are reduced, so that removal of TFA is facilitated.
(310) 7.12 Analytics
(311) Two batches, 71001-016 (comparative batch, cleavage with King's cocktail according to the standard method), and 71001-013 (lixisenatide cleavage according to the invention) were prepared.
(312) TABLE-US-00032 TABLE 30 analytics Output Content against weight external standard Purity Yield [g] [%] [Fl.-%] [%] 71001-016 3.41 23.0 35.6 14.2 (comparative) 71001-013 5.20 20.5 35.9 19.7 (invention)
(313) Results: The batches showed almost identical purity. The content in the batch produced according to the invention is slightly decreased. In the batch of the invention, the output weight is increased, resulting in an increased yield.
(314) 7.13 Summary
(315) The cleavage method of the invention has the following advantages: (a) increase of lixisenatide yield by about 5%, resulting in a cost reduction and an increase of production capacity. (b) only two components are present in the cleavage cocktail (in view of five components in the comparative King's cocktail), thus analytic quality control is improved and costs are reduced, (c) omission of the second cleavage leads to a cost reduction, and handling during the production process is facilitated. The amounts of TFA are reduced, so that removal of TFA is facilitated.
(316) The following aspects are also subject of the invention: 1. A method for the synthesis of a polypeptide comprising a pre-determined amino acid sequence, the method comprising coupling cycles of amino acid building blocks to an amino acid chain, wherein said amino acid building blocks comprise an unprotected C-terminal carboxyl group and a protected N-terminal amino group, and wherein said amino acid chain comprises an unprotected N-terminal amino group, wherein at least one coupling cycle comprises the steps: (a) coupling the amino acid building block C-terminally at the unprotected N-terminal amino group of the amino acid chain, so that an amide bond is formed between the amino acid chain and the amino acid building block, (b) contacting the product obtained in step (a) with a capping reagent comprising a capping compound, wherein the capping compound binds to an unprotected N-terminal amino group of the amino acid chain to which no building block has been coupled in step (a), and (c) de-protecting the N-terminal amino group of the amino acid building block. 2. The method of item 1, wherein the capping compound is selected from the group consisting of acetic anhydride (CAS 108-24-7), homologues of acetic anhydride, benzoyl chloride (CAS 98-88-4), N-(benzyloxycarbonyloxy)succinimide (CAS 13139-17-8), benzyl chloroformate (CAS 501-53-1), esters of chloroformic acid, 1-acetylimidazole (CAS 2466-76-4), di-tert-butyl dicarbonate (CAS 24424-99-5) and N-(tert-butoxycarbonyloxy)succinimide (CAS 13139-12-3). 3. The method of item 1 or 2, wherein the capping reagent comprises 0.5-5% v/v of acetic anhydride. 4. The method of any one of the preceding items, wherein the capping reagent comprises 1-3% v/v of acetic anhydride. 5. The method of any one of the preceding items, wherein the capping reagent comprises 2% v/v of acetic anhydride. 6. The method of any one of the preceding items, wherein the capping reagent comprises 0.2-2% v/v of diisopropylethylamine. 7. The method of any one of the preceding items, wherein the capping reagent comprises 0.5-2% v/v of diisopropylethylamine. 8. The method of any one of the preceding items, wherein the capping reagent comprises 1% v/v of diisopropylethylamine. 9. The method of any one of the preceding items, wherein the capping reagent comprises 1% v/v of diisopropylethylamine and 2% v/v of acetic anhydride. 10. The method of any one of the preceding items, wherein the capping reagent comprises DMF. 11. The method of any one of the preceding items, wherein the step (b) is performed at a temperature of 15-25° C. 12. The method of any one of the preceding items, wherein the step (b) is performed for 5 to 15 min. 13. The method of any one of the preceding items, wherein the amino acid building block comprises an α-amino acid. 14. The method of any one of the preceding items, wherein the amino acid building block is selected from Ser, Thr, Trp, Lys, Ala, Asn, Asp, Val, Met, Phe, Ile, Pro, Arg, Glu, Gln, Leu and Gly. 15. The method of any one of the preceding items, wherein the amino acid building block is selected from Arg, Glu, Gln, Leu and Gly. 16. The method of any one of the preceding items, wherein the side chain of the amino acid building block comprises a protecting group which is orthogonal to the N-terminal protecting group of the amino acid building block. 17. The method of any one of the preceding items, comprising a solid phase synthesis. 18. The method of any one of the preceding items, wherein the N-terminal amino group at the amino acid building block is protected by a base-labile protecting group. 19. The method of any one of the preceding items, wherein the N-terminal amino group at the amino acid building block is protected by Fmoc. 20. The method of any one of the preceding items, wherein the polypeptide is a GLP-1 agonist. 21. The method of any one of the preceding items, wherein the polypeptide is selected from GLP-1, analogs and derivatives thereof, exendin-3, analogs and derivatives thereof, and exendin-4, analogs and derivatives thereof. 22. The method of any one of the items 1 to 21, wherein the polypeptide is selected from exendin-4 and lixisenatide. 23. The method of any one of the items 1 to 22, wherein the polypeptide is lixisenatide. 24. The method of any one of the items 1 to 21, wherein the polypeptide is selected from albiglutide, dulaglutide and semaglutide. 25. The method of in item 1, wherein the polypeptide is lixisenatide, or exendin-4, wherein after coupling of the amino acid building block Arg(20), Glu (17), Gln(13), Leu(10) or/and Gly(4), step (b) is performed for about 10 min with a capping reagent comprising 2% v/v acetic anhydride and 1% v/v diisopropylethylamine. 26. Composition, characterized in that it comprises 0.5-5% v/v of acetic anhydride and 0.2-2% v/v of diisopropylethylamine in DMF. 27. The composition of item 26, comprising 1% v/v of diisopropylethylamine and 2% v/v of acetic anhydride. 28. Use of the composition of item 26 or 27 for acetylation of an unprotected amino group in polypeptide synthesis. 29. Use of the composition of item 28, wherein the polypeptide is lixisenatide.