High-purity adrenocorticotropic hormone, analogue and a large-scale preparation method thereof
11419919 · 2022-08-23
Assignee
Inventors
- Jundong Meng (Nanjing, CN)
- Kangning Rui (Nanjing, CN)
- Bin Liu (Nanjing, CN)
- Yuanyuan Han (Nanjing, CN)
- Song CHEN (Nanjing, CN)
- Haoning Zhang (Nanjing, CN)
Cpc classification
International classification
Abstract
The invention belongs to the technical field of polypeptide preparation methods, and in particular relates to a high-purity ACTH (human sequence) or analogue and large-scale preparation method thereof. The main steps include: amino acids are coupled from the C-terminal to the N-terminal by Fmoc solid-phase synthesis method to obtain the crude ACTH (human sequence) or analogue peptidyl-resin with protective groups, wherein the reaction temperature of C-15 peptide synthesis is 40-60° C. After cleavge and precipitation, the crude product of ACTH (human sequence) or analogue is obtained, and then the high-purity product is obtained by liquid chromatography. The chromatographic purity of ACTH (human sequence) or analogue prepared by the invention is more than 99%, the stability is good, and the yield of the target peptide is ≥63%.
Claims
1. A composition containing adrenocorticotropic hormone or analogue thereof and impurity, wherein the purity of the adrenocorticotropic hormone or analogue thereof is ≥99%, the content of the maximum single impurity is ≤0.5%, and the content of the total impurities is ≤1%, and wherein the sequence of the adrenocorticotropic hormone from the N-terminal to the C-terminal is TABLE-US-00016 (SEQ ID NO: 4) SYSMEHFRWGKPVGKKRRPVKVYPDGAEDESAEAFPLEF.
2. The composition according to claim 1, wherein the purity of the adrenocorticotropic hormone or analogue thereof is ≥99.5%, and the content of the total impurity is ≤0.5%.
3. A pharmaceutical composition comprising the composition of claim 1, and a medicinal carrier.
4. A method for preparing the composition according to claim 1, the method comprising coupling amino acids from C-terminal to N-terminal according to the amino acid sequence shown in SEQ ID NO: 4 by Fmoc solid-phase synthesis, thereby creating a synthesis product, and purifying the synthesis product to obtain the composition containing adrenocorticotropic hormone or adrenocorticotropic hormone analogue.
5. The method for preparing the composition according to claim 4, further comprising: 1) coupling amino acids from C- to N-terminal according to the amino acid sequence shown in SEQ ID NO: 4 by Fmoc solid-phase synthesis to obtain a peptidyl-resin of adrenocorticotropic hormone or analogue thereof with protective groups; 2) contacting the peptidyl-resin of adrenocorticotropic hormone or analogue thereof with protective groups with a cleavage cocktail thereby cleaving the peptide chain of adrenocorticotropic hormone or analogue thereof from the resin, and thereby removing the protective groups of the peptide chain, in order to obtain a solution containing adrenocorticotropic hormone or analogue thereof; 3) treating the solution containing adrenocorticotropic hormone or analogue thereof with a precipitation reagent to obtain a crude product of the adrenocorticotropic hormone or analogue thereof; and 4) purifying the crude product of the adrenocorticotropic hormone or analogue thereof by liquid chromatography to obtain the composition containing the adrenocorticotropic hormone or analogue thereof.
6. The method for preparing the composition according to claim 5, wherein in step 1), the amino acids are coupled stepwise or as fragments, with N-terminal protection by Fmoc group.
7. The method for preparing the composition according to claim 5, wherein in step 1), the resin used in the solid-phase synthesis method is Wang resin, 2-triphenylmethylchloromethane resin, Rink Amide AM Resin, Rink Amide MBHA Resin, Rink Amide Resin or resin connected with Fmoc-Phe-OH.
8. The method for preparing the composition according to claim 5, wherein step 1) comprises: i). swelling an Fmoc-AA.sub.n-resin by a first organic solvent, wherein AA.sub.n represents a polypeptide with n amino acid residues have been connected to the resin, wherein n is a natural number from 1 to 38; and wherein the structure of each amino acid in the polypeptide is the same or different, with the N-terminal amino acid being protected by Fmoc, Boc or Cbz group, and the side-chain of each amino acid having or without protective group; ii). deprotecting the Fmoc-AA.sub.n-resin with a second organic solvent until the Fmoc protecting group is removed, to obtain a deprotected H-AA.sub.n-resin by washing with a third organic solvent; iii). reacting Fmoc-AA.sub.m-OH with an activation reagent in the first organic solvent to obtain an activated Fmoc-AA.sub.m-OH derivative solution, wherein AA.sub.m is the (n+1)th amino acid from the C-terminus of SEQ ID NO: 4; wherein step iii) may be completed either before or after step i) or step ii); iv). mixing the activated Fmoc-AA.sub.m-OH derivative solution with the deprotected H-AA.sub.n-resin to obtain an Fmoc-AA.sub.(n+1)-resin through a coupling reaction, and washing the Fmoc-AA.sub.(n+1)-resin with the third organic solvent; and, v). repeating step ii) to step iv) to connect the remaining amino acid residues of SEQ ID NO: 4 to the Fmoc-AA.sub.(n+1)-resin to obtain the adrenocorticotropic hormone or analogue thereof peptidyl-resin with side-chain protective groups.
9. The method for preparing the composition according to claim 8, wherein the first organic solvent in step i) and step iii) is an aprotic solvent.
10. The method for preparing the composition according to claim 8, wherein the second organic solvent in step ii) is an aprotic solvent containing an organic base.
11. The method for preparing the composition according to claim 8, wherein the third organic solvent in step ii) is the first organic solvent or alcohol solvent.
12. The method for preparing the composition according to claim 8, wherein the activation reagent in step iii) is one of composition of DIC, HBTU and Oxyma Pure, composition of DIC and Oxyma Pure, composition of DIC and HOBt, composition of DIEA, TBTU and HOBt, composition of DIEA and PyBop.
13. The method for preparing the composition according to claim 8, wherein in step iv), the reaction temperature for coupling is 10-35° C. and the reaction time is 0.5-5 h besides the coupling at C-15 position; and the reaction temperature for coupling amino acid at C-15 position in step iv) is 40-60° C. and the reaction time is 0.5-16 h.
14. The method for preparing the composition according to claim 13, wherein urea or perchlorate is added to the reaction mixture in step iv) when coupling the amino acid at C-15 position.
15. The method for preparing the composition according to claim 14, wherein the mass ratio of urea or perchlorate to Fmoc-Phe-resin is 0.1:1-1:1.
16. The method for preparing the composition according to claim 5, wherein the peptidyl-resin of adrenocorticotropic hormone or analogue thereof with protective groups obtained in step 1) is deprotected with the second organic solvent, washed with the third organic solvent, and washed with the fourth organic solvent, wherein the fourth organic solvent comprises one or more of methanol, ethanol and DCM.
17. The method for preparing the composition according to claim 5, wherein the cleavage cocktail comprises trifluoroacetic acid and scavengers in step 2), and the scavengers is composed of one or more of phenol, benzyl sulfide, dimethyl sulfide, 1,2-ethanedithiol, triethylsilane, triisopropylsilane or water; the volume ratio of the scavengers and trifluoroacetic acid is 1:4-1:19.
18. The method for preparing the composition according to claim 5, wherein the precipitation reagent in step 3) is an ether solvent.
19. The method according to claim 7, wherein the resin is 2-triphenylmethylchloromethane resin.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DESCRIPTION OF THE EMBODIMENTS
(15) In order to facilitate those skilled in the art to understand the content of the present invention, the technical solutions of the invention will be further described below in conjunction with the examples, but the following contents should not limit the scope of the invention claimed by the appended claims in any way.
(16) The materials and reagents used in the following examples can be obtained from commercial channels without special instructions. Amino acids are L-type amino acids without special instructions
Example 1. Preparation of Fmoc-Phe-Resin
(17) 500 g of 2-triphenylmethylchloromethane resin was added to 5 L DCM and was swelled for 1 h, 4.25 mol DIPEA and 0.75 mol Fmoc-Phe-OH were added to react at room temperature for 4 h to obtain Fmoc-Phe-resin. 1.25 L DIPEA/methanol (volume ratio 1:9) solution was added to react for 0.5 h to cap the unreacted sites. The Fmoc-Phe-resin was filtrated and washed one time with 5 L DCM, then the resin was dried and washed three times with methanol, the amount of methanol used for each washing was 5 L. The Fmoc-Phe-resin was dried under vacuum to constant weight at room temperature, and the substitution was determined to be 0.7 mmol/g.
Example 2. Preparation of Fmoc-Glu(OtBu)-Phe-Resin
(18) 1) Resin swelling: 500 g of Fmoc-Phe-resin with substitution of 0.7 mmol/g prepared in example 1, namely, the molar amount of Fmoc-Phe was 0.35 mol, was added into the reactor, and 5 L DMF solution containing 50 g HOBt was added to fully swell Fmoc-Phe-resin.
(19) 2) Deprotection reaction: 5 L of deprotection reagent was added (20% piperidine/DMF solution, by volume ratio) to step 1), the mixture was stirred and reacted for 20 min, the resin was dried, and the deprotection reaction was repeated for 3 times until the deprotection reaction was complete. After the deprotection reaction, 5 L DMF solution once time was used to wash for 8 times in total to obtain Fmoc-Phe-resin.
(20) 3) Activation: 1.75 mol of Fmoc-Glu(OtBu)-OH was added into 5 L DMF solution, then 1.75 mol of Oxyma Pure and 1.75 mol of DIC were added respectively, and the mixture was reacted for 30 mins at room temperature to obtain the activated solution of Fmoc-Glu(OtBu)-OH;
(21) 4) Condensation reaction: the activated solution of Fmoc-Glu(OtBu)-OH was added to the Phe-resin obtained in step 2) to react for 3 h at 30° C., the resin was filtered and washed with DMF for 8 times to obtain Fmoc-Glu(OtBu)-Phe-resin. The reaction was monitored by the method of Ninhydrin Test. If the test result was negative, the next reaction was conducted. If the test result was positive, the above steps 3) to 4) condensation reaction was repeated until the test result was negative.
Example 3. Preparation of Fmoc-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-Resin
(22) Refer to the method described in step 2)-4) of example 2, that was, each coupling amino acid was subjected to deprotection reaction, activation reaction and condensation reaction to the Fmoc-Glu(OtBu)-Phe-resin prepared in example 2. The amino acids were coupled according to the amino acid sequence of ACTH (human sequence) from the C-terminal to the N-terminal in the order of 3-14, the sequence of coupling amino acids was as follows: Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH.
Example 4a. Preparation of Fmoc-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-Resin
(23) 1) 50 g of Fmoc-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-resin prepared in example 3 was taken, 500 mL of deprotection reagent (20% piperidine/DMF solution, by volume ratio) was added to the resin. The mixture was stirred and reacted for 20 min, the resin was dried, and the deprotection reaction was repeated for 3 times until the deprotection reaction was complete. After the deprotection reaction, 500 mL DMF solution each time was used to wash for 8 times in total. The reaction product Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-resin was evenly divided into 21 parts and respectively added into independent peptide reactor.
(24) 2) Preparation of Fmoc-Asn(Trt)-OH activated solution: several activated solutions were respectively prepared, 4.2 mmol Fmoc-Asn(Trt)-OH was added to 25 mL DMF solution for each solution, and then 4.2 mmol condensation reagent as shown in Table 1 was added to react at room temperature for 30 mins.
(25) 3) Each activated solution of Fmoc-Asn(Trt)-OH was added into the polypeptide reactor respectively for condensation reaction. The reaction temperature, type of condensation reagent and reaction time were shown in Table 1, wherein the amount of urea or sodium perchlorate was 0.1 Kg. After the reaction, the resin was filtered and washed with DMF for 8 times. During the reaction, the method of Ninhydrin Test was used to directly qualitatively detect whether the reaction was completed or not.Fmoc-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Ph e-Pro-Leu-Glu(OtBu)-Phe-resin was cut with 85% trifluoroacetic acid solution, the resulting solution was precipitated with ether, and the precipitate was dissolved with water. The chromatographic purity of 15 peptide, the residue of substrate 14 peptide and the racemic impurity of D-Asn-15 peptide were determined by HPLC, the details were shown in Table 1:
(26) TABLE-US-00011 TABLE 1 Condensation reactions under different reaction conditions Chroma- Racemic tographic impurity Temperature Time of Detection purity Residue of of of Type of condensation reaction/ of of 15 substrate 14 D-Asn-15 Appended # reaction reagent h ninhydrin peptides peptide peptide drawings 1. 20° C. DIC/Oxyma Pure 3 positive — — — — 2. DIC/Oxyma Pure 8 positive — — — — 3. DIC/Oxyma Pure 20 positive — — — — 4. DIC/Oxyma Pure/urea 3 positive — — — — 5. DIC/Oxyma Pure/urea 8 positive — — — — 6. DIC/Oxyma Pure/urea 20 positive 69.51% 5.19% not FIG. 2 detected 7. 30° C. DIC/HOBt 3 positive — — — — 8. DIC/HOBt 8 positive — — — — 9. DIC/HOBt 20 positive — — — — 10. DIC/HOBt/NaClO.sub.4 3 positive — — — — 11. DIC/HOBt/NaClO.sub.4 8 positive — — — — 12. DIC/HOBt/NaClO.sub.4 20 positive 69.81% 4.19% not FIG. 3 detected 13. 35° C. TBTU/DIPEA/HOBt 3 positive — — — — 14. TBTU/DIPEA/HOBt 8 positive — — — — 15. TBTU/DIPEA/HOBt 20 positive 68.75% 4.16% not FIG. 4 detected 16. 40° C. DIC/Oxyma Pure 3 — 69.45% 3.42% not FIG. 5 detected 17. 45° C. DIC/HOBt 3 — 69.80% 3.03% not FIG. 6 detected 18. 50° C. DIC/HOBt 3 — 71.06% 2.28% not FIG. 7 detected 19. DIC/HOBt/urea 3 — 68.00% 0.20% not FIG. 8 detected 20. DIC/HOBt/NaClO.sub.4 3 — 69.16% 1.03% not FIG 9 detected 21. 60° C. DIC/HOBt 3 — 65.75% 0.30% 1.11% FIG. 10
(27) In the above table, the 15 peptide was H-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH; The 14 peptide was H-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH; The racemic impurity of D-Asn-15 peptide was H-(D-Asn)-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH.
(28) The results showed that the residue of 14 substrate peptide was always more than 4% by using a variety of condensation reagents, repeating condensation times and prolonging condensation time when the reaction temperature was controlled at 20-35° C., which indicating that the reaction was not complete. When the reaction temperature was 40-60° C., the residue of 14 substrate peptide was controlled to be less than 4%. Especially when the reaction temperature was 50° C., the condensation rate could be significantly improved by adding appropriate amount of urea or NaClO.sub.4 to DIC/HOBt condensation reagent. Because the urea added was more helpful to the improvement of condensation rate, and the residue of 14 substrate peptide was better controlled at 0.20%.
Example 4b
(29) 950 g of Fmoc-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-resin prepared in example 3 was condensed under the optimal condition (#19) in Table 1, that was, the condensation reaction was conducted with Fmoc-Asn(Trt)-OH in the presence of the condensation reagent of DIC/HOBt/urea, and the obtained intermediate was used as the upstream raw material of example 5.
Example 5. Preparation of Fmoc-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala- Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-Resin
(30) Refer to the method described in steps 2)-4) of example 2, each coupling amino acid was subjected to deprotection reaction, activation reaction and condensation reaction in the polypeptide reactor of example 4b. The amino acids were coupled according to the amino acid sequence of ACTH (human sequence) from the C-terminal to the N-terminal in the order of 16-39, the sequence of coupling amino acids were as follows: Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH. At the end of the coupling, the peptidyl-resin of ACTH (human sequence) with protective groups was obtained.
Example 6. Preparation of H-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala- Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-Resin
(31) 5 L of deprotection reagent (20% piperidine/DMF solution) was added and stirred, the reaction was conducted for 20 min, the resin was dried, the deprotection reaction was repeated for 3 times until the deprotection reaction was complete. After deprotection reaction, the resin was washed 8 times with 5 L DMF solution each time. Then, 5 L methanol solution was added to wash for 8 times, and the resin was filtered and dried under vacuum at 40° C. to obtain 1.75 Kg of the peptidyl-resin of ACTH (human sequence) without the N-terminal protection by Fmoc.
Example 7. Preparation of Crude Peptide of ACTH(Human Sequence) H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH
(32) 1.75 Kg peptidyl-resin prepared in example 6 was added to 17.5 L cleavage cocktail which was precooled to −12° C. to react for 2 h (the cleavage cocktail was composed of 90% volume of trifluoroacetic acid and 10% volume of scavengers, in which the scavengers was composed of 1% phenol, 1% anisole, 1% dimethyl sulfide, 1% 1,2-ethanedithiol, 1% triethylsilane, 1% triisopropylsilane and 4% water in volume concentration), the temperature of the whole reaction was controlled not more than 40° C. After the reaction, the product was filtered and the filtrate was collected. The part of the cleavage cocktail was removed by reducing the pressure and concentrating the filtrate, and then the filtrate was slowly added to 17.5 L methyl tert-butyl ether which was precooled to −12° C. for precipitation. The wet solid was collected by centrifugation and washed with 87.5 L methyl tert-butyl ether. The solid was collected, dissolved with water and freeze-dried. 0.75 Kg solid crude peptide was obtained with purity of 68.96% and content of the target peptide of 48% (i.e. 0.36 Kg target peptide). As shown in
Example 8. Purification of Crude Peptide of ACTH (Human Sequence) H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH
(33) 1. Treatment of sample: 0.75 Kg of crude peptide of ACTH (human sequence) obtained in example 7 was taken to dissolve in 37.5 L acetonitrile water solution with the volume ratio of acetonitrile:water=30:70, the crude peptide was stirred to dissolve completely and filtered with 0.45 μm filter membrane, and then the filtrate was collected for use.
(34) 2. The first purification:
(35) Conditions of purification:
(36) Chromatographic column: DAC-20 dynamic axial compression column with octadecylsilane chemically bonded silica as stationary phase;
(37) Column diameter and packing length: 20*25 cm;
(38) Mobile phase A: Tris aqueous solution with molar concentration of 0.1 mol/L, pH was adjusted to 8.0 by ammonia water;
(39) Mobile phase B: acetonitrile;
(40) Flow rate: 80 mL/min;
(41) The detection wavelength: 280 nm.
(42) Gradient: B %: 30-60% (50 mins), the injection volume was 20 g.
(43) Process of purification: the chromatographic column was equilibrated with mobile phase A and then loaded with 5 L sample solution. Linear gradient elution was conducted for 50 mins and the target peptide solution with purity of more than 90% was collected. The yield of target peptide was 79%.
(44) 3. The second purification:
(45) Conditions of purification:
(46) Chromatographic column: DAC-20 dynamic axial compression column with octadecylsilane chemically bonded silica as stationary phase;
(47) Column diameter and packing length: 20*25 cm;
(48) Mobile phase A: Ammonium sulfate aqueous solution with molar concentration of 0.1 mol/L, pH was adjusted to 3.0 by sulfuric acid;
(49) Mobile phase B: acetonitrile;
(50) Flow rate: 80 mL/min;
(51) The detection wavelength: 280 nm.
(52) Gradient: B %: 30-60% (50 mins), the injection volume was 20 g.
(53) Process of purification: the chromatographic column was equilibrated with mobile phase A and then loaded with 1 L sample solution. Linear gradient elution was conducted for 50 mins and the target peptide solution with purity of more than 99% was collected. The yield of target peptide was 86%.
(54) 4. Desalting and ion control:
(55) Conditions of desalting:
(56) Chromatographic column: DAC-20 dynamic axial compression column with octadecylsilane chemically bonded silica as stationary phase;
(57) Column diameter and packing length: 20*25 cm;
(58) Mobile phase A: Acetic acid aqueous solution with concentration of 0.1%;
(59) Mobile phase B: acetonitrile;
(60) Flow rate: 80 mL/min;
(61) The detection wavelength: 280 nm.
(62) Gradient: B %: 30-60% (50 mins), the injection volume was 20 g.
(63) Process of desalting: the chromatographic column was equilibrated with mobile phase A and then loaded with 1 L sample solution. Linear gradient elution for was conducted 50 mins and the target peptide solution was collected. The yield of target peptide was 95%.
(64) 5. Freeze drying
(65) The target peptide solution obtained in the previous step was transferred to a stainless steel tray with appropriate size, and then detected by HPLC after freeze drying. The purity of ACTH acetate (human sequence, SEQ ID NO: 3) was 99.88% (as shown in
Example 9
(66) Referring to the method of example 4b-8, the amino acid of Asp(Trt) at position 15 was replaced with Asn(Trt) to obtain the ACTH (human sequence) analogue (SEQ ID NO: 4, H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asp-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH) with the purity of 99.58% (as shown in
Example 10 (Comparative Example)
(67) ACTH (human sequence) (SEQ ID NO: 3) was prepared strictly according to the specific steps from step 1 to step 14 in example 4 of U.S. Pat. No. 3,953,415. The total yield was 17%, and the purity of ACTH (human sequence) was only 75.72%, as shown in
Example 11 Study of Stability
(68) The samples prepared in example 8 were packed in double-layer medical low-density polyethylene bags and a layer of aluminum-plastic composite bags and stored at 2-8° C. for evaluation at 1, 2, 3 and 6 months respectively. The stability results of the samples after acceleration for 6 months were shown in Table 2:
(69) TABLE-US-00012 TABLE 2 Stability results of acceleration for 6 months Accelerated test Inspection Detection Start 1 month 2 months 3 months 6 months items method Result Conclusion Character Visual White White White White White Qualified powder powder powder powder powder Related HPLC 0.07% 0.17% 0.17% 0.16% 0.11% Qualified substances (maximum single impurity) Related HPLC 0.13% 0.29% 0.29% 0.38% 0.39% substances (total impurities) The purity HPLC 99.88% 99.71% 99.71% 99.62% 99.61% Qualified of chromatographic Moisture Karl 5.0% 7.3% 7.5% 8.4% 8.3% Qualified Fischer Polymer HPLC 0.03% 0.05% 0.03% 0.06% 0.04% Qualified
(70) The results showed that the character, related substances, purity of chromatographic, moisture and polymer of ACTH (human sequence SEQ ID NO: 3) had no obvious change trend at 2-8° C. It was suggested that the ACTH (human sequence SEQ ID NO: 3) was stable under this storage condition. The polymer refers to an impurity with a molecular weight greater than that of ACTH (human sequence SEQ ID NO: 3).
(71) The samples prepared in example 9 were packed in double-layer medical low-density polyethylene bags and a layer of aluminum-plastic composite bags and stored at 2-8° C. for evaluation at 1, 2, 3 and 6 months respectively. The stability results of the samples after acceleration for 6 months were shown in Table 3:
(72) TABLE-US-00013 TABLE 3 Stability results of acceleration for 6 months Accelerated test Inspection Detection Start 1 month 2 months 3 months 6 months items method Result Conclusion Character Visual White White White White White Qualified powder powder powder powder powder Related HPLC 0.16% 0.24% 0.17% 0.27% 0.10% Qualified substances (maximum single impurity) Related HPLC 0.42% 0.42% 0.43% 0.46% 0.49% substances (total impurities) The purity of HPLC 99.58% 99.58% 99.57% 99.54% 99.51% Qualified chromatographic Moisture Karl 5.2% 7.5% 7.6% 8.5% 8.6% Qualified Fischer Polymer HPLC 0.03% 0.04% 0.05% 0.06% 0.06% Qualified
(73) The results showed that the character, related substances, purity of chromatographic, moisture and polymer of ACTH analogue (human sequence SEQ ID NO: 4) had no obvious change trend at 2-8° C. It was suggested that the ACTH analogue (human sequence SEQ ID NO: 4) was stable under this storage condition. The polymer refers to an impurity with a molecular weight greater than that of ACTH analogue (human sequence SEQ ID NO: 4).
Example 12 Data of Security
(74) (1) Study of Activity In Vitro
(75) ACTH was a receptor agonist of melanocortin receptor 2 (MC2R). The combination of ACTH and MC2R could stimulate the signal pathway of the downstream and change the signal factors such as calcium ions and cAMP. The binding ability of ACTH and MC2R was determined by detecting calcium ion signals, and then the cell activity of ACTH in vitro was judged.
(76) The samples in example 8 and example 10 were taken for activity study in vitro. CHO-K1 cells expressing MC2R were cultured in 10 cm vessels and stored at 37° C. and 5% CO.sub.2.
(77) The results showed that high-purity ACTH (human sequence SEQ ID NO: 3, 99.88%) and low-purity ACTH (human sequence SEQ ID NO: 3, 75.72%) had binding ability to melanocortin receptor 2 (MC2R) and showed biological activity. The half inhibitory concentration (EC.sub.50) of high-purity ACTH was 180.2 nM, the half inhibitory concentration of low-purity ACTH was 287.9 nM. The lower the half inhibitory concentration, the higher the activity. Therefore, the activity of high-purity ACTH was better than that of low-purity ACTH, and the activity of the former was 1.6 times that of the latter. The specific activity test datas were shown in Table 4:
(78) TABLE-US-00014 TABLE 4 Activity test datas The source of sample Bottom Top HillSlope EC.sub.50 (nM) Example 8 −2.739 100.9 0.9745 180.2 Example 10 −1.846 94.88 1.249 287.9
(79) (2) Study of Toxicity In Vitro
(80) The cardiac metabolic toxicity of drugs was judged by detecting the affinity between drugs and heart receptor protein (hERG) in vitro. The higher the affinity between the drug and the hERG receptor, the lower the half inhibitory concentration, which indicated that the lower the dose required for its toxicity to the heart, and the stronger the metabolic toxicity to the heart.
(81) The samples in example 8, example 10 and positive control drug of dofetilide were taken for toxicity study in vitro. The results showed that the IC.sub.50 of high-purity ACTH and low-purity ACTH were more than 10000 nM, and the IC.sub.50 of positive control drug was 2.09 nM. Therefore, the toxic dose of high-purity ACTH and low-purity ACTH was much higher than that of the positive control drug, showing no cardiac metabolic toxicity. The specific metabolic toxicity datas were shown in Table 5:
(82) TABLE-US-00015 TABLE 5 Datas of cardiac metabolic toxicity MaxDose % Inh @ The source of sample IC.sub.50 (nM) (nM) MaxDose Exemple 8 >10000 10000 −1.28 Exemple 10 >10000 10000 12.47 positive control drug 2.090 10000 104.47