Cracking process of G-type natriuretic peptide

Abstract

The invention provides a cracking process of G-type natriuretic peptide, which is used for cracking synthesized G-type natriuretic peptide resin to obtain a linear peptide crude product, and comprises the following steps: S41, preparing a cracking reagent; s42, performing concentration; s43, carrying out precipitation; s44, filtering is carried out; and S45, drying. The cracking reagent comprises the following components: TFA, H2O, EDTA, TIS, ArOH and ArSCH3, wherein the ratio of TFA to H2O to EDTA to TIS to ArOH to ArSCH3 is 81.5: 5: 2.5: 1: 5: 5 (V / V / V / V / W / V). The dosage ratio of the cracking reagent to the G-type natriuretic peptide resin is 12-15ml / g. And carrying out suction filtration on the cracked reaction liquid, washing with TFA (Trifluoroacetic Acid), combining washing liquids, carrying out reduced pressure concentration on the filtrate at 32 + / -3 DEG C until the volume is 30-40% of the initial volume, and then precipitating with methyl tert-butyl ether. The cracking process of the G-type natriuretic peptide has the advantages that the cracking yield is high and can reach 20.0-30.0%, a stable and reliable process is provided for chemical synthesis of GNP cracking, and a high-quality cracking product is provided for subsequent preparation of GNP raw material medicines.

Description

Technical Field

[0001] The present invention relates to the field of resin cracking, and in particular to a cracking process of G-type natriuretic peptide. Background Art

[0002] Natriuretic peptides are a class of polypeptide hormones with biological activities such as natriuresis, diuresis, vasodilation and anti-proliferation. They activate the cyclic guanosine monophosphate signaling pathway by binding to specific receptors, antagonizing the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system, thereby regulating blood pressure, fluid balance and vascular tension. They include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), etc.

[0003] G-type natriuretic peptide (GNP), a new member of the natriuretic peptide family, is derived from the East African green mamba snake and is a novel polypeptide hormone. Chinese Patent Application No. 201310127277.6 discloses a G-type natriuretic peptide derived from the East African green mamba snake. This new member of the natriuretic peptide family is obtained by constructing a recombinant expression vector using the gene encoding GNP, which is then transformed into host cells to form a transformant. The transformant is then cultured, extracted, and purified. GNP can be used to treat acute or chronic heart failure and myocardial infarction combined with heart failure. However, the cell-based recombinant method has a long production cycle and requires significant equipment investment. Furthermore, impurities such as host proteins, endotoxins, and residual nucleic acids must be removed through complex chromatography processes. The separation efficiency of hydrophobic peptides (such as Pro / Gly containing GNPs) is low, resulting in a significant decrease in yield. Therefore, GNPs can be prepared through chemical synthesis. However, the GNPs obtained through chemical synthesis still face the challenge of cleavage.

[0004] Since there are few reports on chemically synthesized GNPs in the prior art, there is also a lack of a GNP cracking process that is stable, reliable, and has a high cracking yield. Summary of the Invention

[0005] In view of this, the present invention aims to propose a cleavage process for G-type natriuretic peptide to solve the problem in the prior art of lacking a method for cleaving chemically synthesized GNPs with a high cleavage yield.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] A G-type natriuretic peptide cleavage process is used to cleave a synthesized G-type natriuretic peptide resin to obtain a crude linear peptide product, comprising the following steps:

[0008] S41. Preparation of lysis reagent;

[0009] S42. Concentration;

[0010] S43. precipitation;

[0011] S44. Filter;

[0012] S45. Drying.

[0013] Furthermore, in step S41, the lysis reagent includes the following components in the following ratio: TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5 (V / V / V / V / W / V).

[0014] Furthermore, in step S41, the usage ratio of the cleavage reagent and the G-type natriuretic peptide resin is 12-15 ml / g.

[0015] Furthermore, in step S41, after the cleavage reagent is prepared, it is pre-cooled to 10±3°C, and the dry peptide resin is added under nitrogen protection and stirred evenly. After the temperature stabilizes, the mixture is stirred at 25±3°C and cleavage is performed.

[0016] Furthermore, the reaction was stirred at 25±3°C for 3 h for cleavage.

[0017] Furthermore, in step S42, the reaction solution after cleavage in step S41 is filtered and washed with TFA, the washings are combined, and the filtrate is concentrated under reduced pressure at 32±3°C to 30% to 40% of the initial volume.

[0018] Furthermore, in step S43, methyl tert-butyl ether is frozen at -7±3°C, and the concentrated solution obtained in step S42 is added while stirring for precipitation.

[0019] Furthermore, in step S44, the precipitate obtained in step S43 is vacuum filtered until no liquid flows out, and then pre-cooled methyl tert-butyl ether is added to the precipitate, and the solid is stirred and washed, and filtered until no liquid flows out. The washing is repeated 7 to 9 times to obtain a white solid, which is the crude GNP cleavage product.

[0020] Furthermore, the synthesis process of the G-type natriuretic peptide resin comprises the following steps:

[0021] S1. Fmoc-Nle, HMP linker-OH and a solid phase support are coupled to obtain HMPlink-Nle-MBHA Resin, wherein the solid phase support is MBHA Resin resin with a degree of substitution of 0.40 mmol / g to 0.62 mmol / g;

[0022] S2. Connect Fmoc-Arg(Pbf)-OH to obtain Fmoc-Arg(Pbf)-HMPAResin;

[0023] S3. According to the sequence of natriuretic peptide GNP, remove the Fmoc protecting group of each amino acid or fragment, and couple the amino acids or fragments one by one, wherein Gly 33 -Asp 34 Using the Fmoc-Asp(OtBu)-Gly-OH fragment, the natriuretic peptide GNP peptide resin was obtained;

[0024] Compared with the existing technology, the G-type natriuretic peptide cleavage process of the present invention has the following advantages: the cleavage yield is high, which can reach 20.0-30.0%, providing a stable and reliable process for the cleavage of chemically synthesized GNPs, and providing high-quality cleavage products for the subsequent preparation of GNP raw materials. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with specific embodiments. It should be noted that the data in the following experimental examples are obtained by the inventor through a large number of experiments. Due to space limitations, only a portion thereof is shown in the specification, and those skilled in the art can understand and implement the present invention under these data. These embodiments are merely intended to illustrate the present invention and are not intended to limit the scope of the present invention. It should also be understood that, after having read the contents of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these changes or modifications also fall within the scope protected by this application.

[0026] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0027] The synthesis process of GNPs of the present invention comprises the following steps:

[0028] S1. Preparation of HMP link-Nle-MBHAResin, comprising:

[0029] S11. 57.7 g of MBHA resin (with a degree of substitution of 0.6 mml / g and a cross-linking degree of 1%) was added to 500 mL of DCM and stirred for 30 minutes. The solution was drained and the resin was washed once with DCM at a ratio of 1 ml:1 ml. Then, 5% DIPEA / DCM was added to the resin at a ratio of 1 ml:1 ml, stirred for 3 minutes, and the solution was drained. This process was repeated twice. The resin was then washed three times with DCM at a ratio of 1 ml:1 ml and set aside.

[0030] S12. Take 400 ml of DMF / DCM (2:1) and cool it to below -5°C. Add 26.5 g of the protected amino acid Fmoc-Nle-OH and 11.6 g of HOBt, then cool and stir for 30 min. Take another 24 ml of DIC / DCM (volume ratio 1:1), cool it to below -5°C, and slowly add it to the Fmoc-Nle-OH / HOBt / DMF / DCM solution. Stir at -5°C for 60 min. Then, stir and add it to the resin treated with S11 for condensation reaction for 120 min. Control the stirring speed to 120 r / min and the reaction temperature to 24°C. Take a sample and test it with ninhydrin solution. A negative test result (colorless or almost colorless or light yellow, the solution is colorless) indicates that the connection is complete, and the reaction solution is removed.

[0031] S13. Add the above resin to 500 mL of DMF and stir for 2 minutes. Drain the solution and repeat the resin washing once. Record the volume of peptide resin V2. Add PIP / DMF (VPIP:VDMF = 1:4) solution to the resin at a volume ratio of 1:1. Stir and react for 30 minutes. Drain the solution and repeat the resin washing once. Wash the resin twice with DMF and then four times with DCM.

[0032] S14. Cool 400 ml of DMF / DCM (2:1) to below -5°C, add 10.9 g of the protected amino acid HMP linker-OH and 9.3 g of HOBt, and stir under reduced pressure for 40 min. Take another 20 ml of DIC / DCM (1:1), cool to below -5°C, and slowly add it to the HMP linker-OH / HOBt / DMF / DCM solution. Stir at -5°C for 60 min. Then, add the resin treated in S13 and carry out a condensation reaction for 120 min. Control the stirring speed at 120 rpm and the reaction temperature at 24°C. Take a sample and test it with ninhydrin solution. A negative test (the resin is light blue or almost blue) indicates that the connection is complete. Remove the reaction solution.

[0033] S15. Add DMF to the resin at a ratio of 1 ml:1 ml, stir for 2 minutes, and drain the solution. Wash the resin once with DMF and drain. Then, add DCM at a ratio of 1 ml:1 ml, stir for 2 minutes, and drain the solution. Repeat the washing and draining once.

[0034] S2. Connect the first Fmoc protected amino acid

[0035] Take 58.3g Fmoc-Arg(Pbf)-OH and dissolve it in 0.2L DCM under stirring. Add it to the HMP link-Nle-MBHAResin resin prepared in S1 and stir and swell for 30min. Control the temperature at 22℃. Then slowly add DCB dropwise and react for 5h. Remove the reaction solution. Add DCM at a ratio of 1g:2ml and stir for 2min to drain. Repeat the washing of the resin twice. Then add DCM / MeOH / DIPEA (V) at a ratio of 1g:1ml. DCM :V MeOH :V DIPEA =17:2:1) the capped solution was stirred for 5 min, the solution was drained, and the process was repeated twice; DCM was added at a ratio of 1 ml:1 ml, stirred for 2 min, and the solution was drained, and the washing and draining were repeated twice to obtain Fmoc-Arg(Pbf)-HMPAResin.

[0036] S3. Connecting the 2nd to 38th Fmoc-protected amino acids or amino acid fragments

[0037] S31. Protective amino acid pretreatment

[0038] Let the volume be V R2 Cool the DMF to below -5°C, add 35.6 g of protected amino acid Fmoc-Gly-OH and 18.6 g of HOBt, and then cool and stir for 40 minutes; cool 38 ml of DIC / DCM (volume ratio 1:1) to below -5°C, then slowly add it to the Fmoc-Gly-OH / HOBt / DMF solution, continue stirring at -5-0°C for 1-2 hours, and set aside;

[0039] S32. Deprotection

[0040] Add a volume of V to the Fmoc-Arg(Pbf)-HMPAResin prepared in S2. R2 DMF, stir for 2 minutes and then drain the solution, repeat once; then add a volume of V R2 PIP / DMF (volume ratio of 1:1), stirred for 2 minutes and then drained the solution; added a volume of V R2 PIP / DMF(V PIP :V DMF =1:1) solution, stirred with resin for 8 minutes, drained; then washed the resin twice with DMF, then washed the resin 4 times with DCM and set aside;

[0041] S33. Condensation reaction

[0042] The protected amino acid Fmoc-Gly-OH / HOBt / DMF / DIC / DCM solution treated with S31 was added to the resin deprotected by S32 for condensation reaction for 120 min, with a stirring speed of 120 r / min and a reaction temperature of 10°C; a negative result of ninhydrin test (the resin solution was all light yellow) indicated that the coupling was completed, and the solution was drained and then heated with a volume of V R2 The resin was washed three times with DMF to obtain Fmoc-Gly-Arg(Pbf)-HMPA Resin.

[0043] According to the above steps S31, pretreatment of protected amino acids, S32, deprotection, S33, and condensation reaction, the remaining amino acids or fragments are coupled in sequence according to the order of the main chain amino acids. Different coupling times and coupling temperatures are set as shown in Table 1:

[0044] Table 1

[0045]

[0046]

[0047] Obtain natriuretic peptide GNP resin:

[0048] Fmoc-Lys(Boc)-Ser(tBu)-Thr(tBu)-Pro-Asp(OtBu)-Gly-Cys(Trt)-Phe-Gly-His(Trt)-Lys(Boc)-Leu-Asp(OtBu)-Pro-Ile-Gly-Ser(tBu)-His( Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Pro-Gly-Ala-Gly-Pro-His(Trt)-Pro-Lys(Boc)-ProThr(tBu)-Pro-Gly-Ala-Gly-Arg(pbf)-HMPAResin;

[0049] S34. Deprotection

[0050] Add VR2 volume of DMF to the natriuretic peptide GNP resin prepared by S33, stir for 2 minutes and then drain the solution. Repeat once and record the volume V of the peptide resin. R3 ; Then add a volume of V R3 PIP / DMF (volume ratio of 1:4), stirred for 2 minutes and then drained the solution; added a volume of V R3 PIP / DMF(V PIP :V DMF=1:4) solution, stirred with resin at 10°C for 30 min, drained; then washed the resin twice with DMF, then washed the resin 4 times with DCM and set aside;

[0051] The peptide resin was transferred to a vacuum drying oven and dried at 30° C. and a vacuum degree ≥-0.092 MPa for 8 to 12 hours to constant weight. The purity of the crude product was 55.13% and the yield was 79.12%.

[0052] The GNP peptide resin used in the present invention is prepared by the above-mentioned GNP synthesis process. The synthesized G-type natriuretic peptide resin is cracked to obtain a crude linear peptide product.

[0053] It should be noted that the membrane used for lysis was a 0.45 μm mixed membrane.

[0054] Experimental Example 1 Screening of lysis reagent types and formulations

[0055] Based on the GNP synthesis process, the peptide resin cleavage process conditions were investigated and cleavage process screening was performed. The peptide resin side chains contained 5 equivalents of Trt, 2 equivalents of OtBu, 5 equivalents of tBu, 3 equivalents of Boc, and 1 equivalent of Pbf. Furthermore, considerations included carrier cleavage, side chain protection removal, and ion scavenging agents.

[0056] Specifically, the amount of cleavage reagent used was: 13 ml / g peptide resin (35 ml / experiment);

[0057] Cracking reaction temperature: 22±3℃;

[0058] Cleavage reaction time: 3h;

[0059] Peptide resin dosage: 2 mmol (13 g), 5 samples;

[0060] Precipitation solvent: methyl tert-butyl ether.

[0061] Add the cleavage reagent to the cleavage reaction flask. Prepare the cleavage reagents (order of addition: H2O, TFA, phenol, thioanisole, EDT, TIS. If no reagent is available, do not add. Nitrogen is introduced during addition). Precool to 10±3°C. Add 0.4mmol (2.6g) of dry peptide resin under nitrogen and stir evenly. After the temperature stabilizes, maintain the temperature at 22±3°C and stir for 3 hours. After the reaction is complete, precipitate the reaction solution directly with methyl tert-butyl ether and extract the sample with 200ml of 20% acetic acid aqueous solution.

[0062] The extract was oxidized by adding iodine / ethanol / acetic acid solution dropwise for 30-40 min (from colorless to brownish red), and then reduced with VC (from brownish red to colorless, and the volume of the solution was measured for testing).

[0063] The results of the screening of lysis reagent types and formulations are shown in Table 2.

[0064] Table 2

[0065]

[0066] The precipitate and washing were extracted once with methyl tert-butyl ether and a 20% aqueous acetic acid solution, and the extract was added to the oxidation solution for oxidation.

[0067] The analysis method is as follows: chromatographic column PHenomennex XB-C18 2.6um 250mm×4.6mm, column temperature: 50℃, wavelength: 214nm, flow rate: 0.5ml / min, mobile phase A: 100mmol / L Na2SO4 pH=2.3, mobile phase B: CH3CN.

[0068] The elution program was as follows: 0-10 min: 83% mobile phase A, 17% mobile phase B; 10-30 min: 81% mobile phase A, 19% mobile phase B; 30-40 min: 80% mobile phase A, 20% mobile phase B; 40-43 min: 78% mobile phase A, 22% mobile phase B; 43-43.5 min: 30% mobile phase A, 70% mobile phase B; 43.5-50 min: 83% mobile phase A, 17% mobile phase B.

[0069] The peak emission time of the cleavage reagent under the above analysis method is phenol: 20 minutes, thioanisole: 11-12 minutes.

[0070] The elution results showed that samples 1-5 had slightly lower purity, which may be affected by the phenol solvent, but their content and yield were higher than the other four samples. Therefore, the type and formula of the cleavage reagent were TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5 (V / V / V / V / W / V).

[0071] Experimental Example 2 Screening of the amount of lysis reagent

[0072] Peptide resin cleavage is a solid-phase reaction that requires a large number of reagents. Considering cost control, this application is for a medium-length peptide that contains a pair of disulfide bonds, so the amount of cleavage reagent used is very important.

[0073] Type and ratio of lysis reagents: TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5;

[0074] Cracking reaction temperature: 22±3℃;

[0075] Cleavage reaction time: 3 hours;

[0076] Peptide resin dosage: 1.2 mmol (7.8 g), 3 samples;

[0077] Precipitation solvent: methyl tert-butyl ether.

[0078] The specific operation is the same as that of Experimental Example 1, and the amount of lysis reagent used is shown in Table 3.

[0079] Table 3

[0080]

[0081]

[0082] The precipitate and washing were extracted once with methyl tert-butyl ether and a 20% aqueous acetic acid solution, and the extract was added to the oxidation solution for oxidation.

[0083] The analysis method was the same as that of Experiment 1. The results showed that the purity of the three samples was similar, and the peptide content of sample 2-3 was slightly higher. Considering that the synthesized GNPs were medium-length peptides and could be completely precipitated, the dosage of the lysis reagent was selected to be 12-15 ml / g.

[0084] Experimental Example 3 Screening of cracking reaction temperature and time

[0085] Type and ratio of lysis reagents: TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5;

[0086] Amount of lysis reagent: 13 ml / g peptide resin (35 ml / experiment);

[0087] Peptide resin dosage: 3.6 mmol (23.4 g), 9 samples;

[0088] Precipitation solvent: methyl tert-butyl ether.

[0089] Add the cleavage reagent to the cleavage reaction bottle. Prepare the cleavage reagent (order of addition: H2O, TFA, phenol, thioanisole, EDT, TIS, purging with nitrogen during addition). Precool to 10±3°C. Add 0.4mmol (2.6g) of dry peptide resin under nitrogen and stir evenly. After the temperature stabilizes, react for the corresponding time according to the temperature requirements in Tables 4-1, 4-2, and 4-3 below. After the reaction is completed, the reaction solution is directly precipitated with methyl tert-butyl ether, and the sample is extracted with 200ml of 20% acetic acid aqueous solution.

[0090] The extract was oxidized by adding iodine / ethanol / acetic acid solution dropwise for 30-40 min (from colorless to brownish red), and then reduced with VC (from brownish red to colorless, and the volume of the solution was measured for testing).

[0091] The cracking reaction temperatures and times are shown in Tables 4-1, 4-2, and 4-3. Table 4-1 shows the first set of experiments, with a reaction temperature of 10±3°C and reaction times of 2, 3, and 4 hours, respectively. Table 4-2 shows the second set of experiments, with a reaction temperature of 22±3°C and reaction times of 2, 3, and 4 hours, respectively. Table 4-3 shows the third set of experiments, with a reaction temperature of 28±3°C and reaction times of 2, 3, and 4 hours, respectively.

[0092] Table 4-1

[0093]

[0094]

[0095] Table 4-2

[0096]

[0097] Table 4-3

[0098]

[0099] The precipitate and washing were extracted once with methyl tert-butyl ether and a 20% aqueous acetic acid solution, and the extract was added to the oxidation solution for oxidation.

[0100] The chromatographic analysis method was the same as in Experimental Example 1. The first set of experiments showed that lower reaction temperatures and longer reaction times did not necessarily result in complete cleavage. The second set of experiments showed that increasing the reaction temperature to 22±3°C and longer reaction times led to more complete reactions and higher peptide content. The third set of experiments showed that increasing the reaction temperature to 28±3°C and shorter reaction times resulted in better purity and higher peptide content. However, as the reaction time increased, purity deteriorated and the peptide content decreased.

[0101] Taking all factors into consideration, different batches release different amounts of heat when the resin just reacts. At the beginning of the reaction, a smooth transition is required, so the temperature is lowered. After the temperature stabilizes, it needs to be raised to 25±3°C, which is conducive to increasing the reaction rate while preventing the temperature from being too high and risking the production of by-products. The reaction time is controlled at 3h.

[0102] Therefore, the cracking temperature and time are controlled as follows: first prepare the cleavage reagent (the order of addition is H2O, TFA, phenol, thioanisole, EDT, TIS, and nitrogen is introduced during addition) and pre-cool it to 10±3°C. Then, add the dry peptide resin under nitrogen protection and stir evenly. After the temperature stabilizes, stir and react at 25±3°C for 3 hours.

[0103] Experimental Example 4: Concentration and Precipitation Solution Selection

[0104] Type and ratio of lysis reagents: TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5;

[0105] Amount of lysis reagent: 13 ml / g peptide resin (35 ml / experiment);

[0106] Peptide resin dosage: 1.2 mmol (7.8 g), divided into 4 samples;

[0107] Precipitation solvents: methyl tert-butyl ether, ethyl acetate.

[0108] Add the cleavage reagent to the cleavage reaction bottle as required. After the cleavage reagent is prepared (addition order: H2O, TFA, phenol, thioanisole, EDT, TIS, and nitrogen is introduced), pre-cool it to 10±3°C, add 1.2 mmol (7.8 g) of dry peptide resin under nitrogen protection and stir evenly. After the temperature stabilizes, react at 25±3°C for 3h. After the reaction is completed, transfer the reaction filtrate to a round-bottom flask.

[0109] Stir the reaction mixture evenly and divide it into 4 equal parts:

[0110] 1. Add chilled methyl tert-butyl ether (MTBE) to one portion (12 ml MTBE / ml concentrate), shake well, let stand, and extract (200 ml 20% acetic acid / water);

[0111] 2. Add chilled ethyl acetate (EA) to one portion for precipitation (12 ml EA / ml concentrate), shake well, let stand, and extract (200 ml 20% acetic acid / water);

[0112] 3. Concentrate one portion under reduced pressure at 32°C ± 3°C (15-30 min), then precipitate with chilled methyl tert-butyl ether (MTBE) (12 ml MTBE / ml concentrate). Shake well, let stand, filter, wash the solid with the appropriate amount of MTBE, evaporate the MTBE, dissolve it with a 20% acetic acid / water solution, and dilute the solution to 200 ml for testing.

[0113] 4. Concentrate one portion under reduced pressure at 32°C ± 3°C, then precipitate with chilled ethyl acetate (EA) (12 ml EA / ml concentrate), shake well, let stand, filter, wash the solid with the corresponding EA, evaporate the EA, dissolve it with 20% acetic acid / water solution, and dilute the solution to 200 ml for testing.

[0114] The specific results are shown in Table 5.

[0115] Table 5

[0116] project 4-1 Concentrated EA 4-2 Extraction EA 4-3 Concentrated MTBE 4-4 Extraction of MTBE Crude product purity 34.12％ 35.50％ 37.55％ 35.12％ content 0.160g 0.194g 0.164g 0.207g Yield 14.45％ 17.52％ 14.81％ 18.69％

[0117] The precipitate and washing were extracted once with methyl tert-butyl ether and a 20% aqueous acetic acid solution, and the extract was added to the oxidation solution for oxidation.

[0118] The chromatographic analysis method was the same as in Experimental Example 1. According to Table 5, the purity of the crude products was comparable. The content and yield of Groups 4-2 and 4-4 were higher than those of Groups 4-1 and 4-3, indicating that the extraction method was superior to the concentration method. However, considering large-scale production, further comparisons between the concentration and extraction methods were conducted later. For details, see Experimental Example 6.

[0119] The content of group 4-4 was higher than that of group 4-2, indicating that the precipitation reagent MTBE was better than EA, so MTBE was selected.

[0120] Experimental Example 5 GNP oxidation method

[0121] The Cys residues at positions 7 and 23 in the GNP sequence need to be oxidized to form a disulfide bond. This experiment compared three methods: DMSO oxidation, iodine / acetic acid oxidation, and buffered salt oxidation. The results are shown in Table 6.

[0122] For DMSO oxidation, the oxidation concentration was 0.6 mmol / L, 0.3 mmol. Dissolve the crude resin in 500 ml of a 10% DMSO / water solution, adjust the pH to 9.0 ± 0.2 with 2 M Na2CO3, and monitor the complete oxidation with HPLC. Adjust the pH to 5.5 with HAc.

[0123] For iodine / acetic acid oxidation, the oxidation concentrations are 0.6 mmol / L and 0.3 mmol. Dissolve the crude resin in 500 ml of 30% acetic acid solution (concentration 0.6 mol / L), filter with a membrane filter, and stir the solution thoroughly. Add saturated I2 / acetic acid solution dropwise while stirring in a water bath until the solution turns reddish-brown. Continue stirring for 30 minutes, then add a small amount of VC aqueous solution until the reddish-brown color disappears. This yields the crude oxidized product solution.

[0124] For buffered salt oxidation, the oxidation concentration is 0.6mmol / L and 0.3mmol. First, add 400ml of water to the reaction flask and introduce nitrogen for 15-30 minutes. Then, weigh the materials and add NH4Ac, EDTA, and Cys·HCl respectively. Stir evenly. Continue to introduce nitrogen and cool the flask by 4-10℃. Then, slowly add the ground crude GNPs and make up the volume with water to 500ml (oxidation concentration is 0.6mmol). Stir and dissolve for 2-4 hours. Then adjust the pH to 8.0±0.2. After 4 hours, naturally raise the temperature to 20℃±5℃ and continue the reaction.

[0125] Table 6

[0126]

[0127] Table 6 shows that the three oxidation methods achieved comparable purity, with some peak differences, but no significant advantage. Comparing only operational controllability, DMSO and buffered salt oxidation times were long, while iodine oxidation took a short time and was easier to operate. Therefore, iodine oxidation was selected.

[0128] Experimental Example 6 Oxidation Concentration Screening 1

[0129] Type and ratio of lysis reagents: TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5;

[0130] Amount of lysis reagent: 13 ml / g peptide resin (35 ml / experiment);

[0131] Peptide resin dosage: 4 mmol (25.4 g), divided into 7 samples;

[0132] Precipitation solvent: methyl tert-butyl ether.

[0133] Add the cleavage reagent to the cleavage reaction bottle as required. After the cleavage reagent is prepared (addition order: H2O, TFA, phenol, thioanisole, EDT, TIS, and nitrogen), pre-cool it to 10±3°C. Under nitrogen protection, add 4.0 mmol (25.4 g) of dry peptide resin and stir evenly. After the temperature stabilizes, react at 25±3°C for 3 hours. After the reaction is completed, transfer the reaction filtrate to a round-bottom flask.

[0134] 1. Take out 1 mmol of sample (sample #1), concentrate under reduced pressure at 32°C ± 3 for 30 min, then add frozen methyl tert-butyl ether (MTBE) to precipitate, filter, wash the solid with frozen MTBE, evaporate the MTBE, and dissolve it in 500 ml of 20% acetic acid / water (oxidation concentration 2.0 mmol / L), concentrate, oxidize with iodine, and monitor.

[0135] 2. The remaining 3 mmol of cleavage reaction solution was stirred evenly and divided into 6 equal parts, namely 2#, 3#, 4#, 5#, 6#, and 7# samples.

[0136] Each portion was precipitated with MTBE at -7±3°C (12 ml / ml concentrate), shaken well, allowed to stand, and extracted (20% acetic acid / water);

[0137] Sample #2 (0.5mmol): Extract the sample with 1.0L of 20% acetic acid / water, concentrate at 36℃±3 for 5-10min, control the oxidation concentration at 0.5mmol / L, retain the sample (before oxidation), oxidize it with iodine / ethanol / acetic acid solution for 30min, and then reduce it with Vc to detect before and after oxidation.

[0138] Sample #3 (0.5mmol): Extract the sample with 500ml of 20% acetic acid / water, concentrate at 32℃±3 for 5-10min, control the oxidation concentration at 1mmol / L, retain the sample (before oxidation), oxidize it with iodine / ethanol / acetic acid solution for 30min, and then reduce it with Vc. The samples before and after oxidation are to be tested.

[0139] Sample #4 (0.5mmol): Extract the sample with 250ml of 20% acetic acid / water, concentrate at 32℃±3 for 5-10min, control the oxidation concentration at 2mmol / L, retain the sample (before oxidation), oxidize it with iodine / ethanol / acetic acid solution for 30min, and then reduce it with Vc. The samples before and after oxidation are to be tested.

[0140] Sample #5 (0.5mmol): Extract the sample with 170ml of 20% acetic acid / water, concentrate at 32℃±3 for 5-10min, control the oxidation concentration at 3mmol / L, retain the sample (before oxidation), oxidize it with iodine / ethanol / acetic acid solution for 30min, and then reduce it with Vc. The samples before and after oxidation are to be tested.

[0141] Sample #6 (0.5mmol): Extract the sample with 100ml of 20% acetic acid / water, concentrate at 32℃±3 for 5-10min, control the oxidation concentration at 5mmol / L, retain the sample (before oxidation), oxidize it with iodine / ethanol / acetic acid solution for 30min, and then reduce it with Vc. The samples before and after oxidation are to be tested.

[0142] Sample #7 (0.5mmol): Extract the sample with 500ml of acetonitrile / water, concentrate at 32℃±3 for 5-10min, control the oxidation concentration at 1mmol / L, retain the sample (before oxidation), oxidize it with iodine / ethanol / acetic acid solution for 30min, and then reduce it with Vc. The samples before and after oxidation are to be tested.

[0143] The comparison results of oxidation concentration are shown in Table 7-1:

[0144] Table 7-1

[0145] project 1# 2# 3# 4# 5# 6# 7# Oxidation concentration 2mmol / l 0.5mmol / l 1mmol / l 2mmol / l 3mmol / l 5mmol / l 1mmol / l Crude product purity 38.28％ 25.69％ 30.55％ 39.79％ 38.67％ 38.31％ 33.43％ Crude product amount 1mmol 0.5mmol 0.5mmol 0.5mmol 0.5mmol 0.5mmol 0.5mmol content 0.726g 0.358g 0.337g 0.328g 0.295g 0.269g 0.32g Yield 19.67％ 19.40％ 18.26％ 17.77％ 15.98％ 14.57％ 17.34％

[0146] The precipitate and washing were extracted once with methyl tert-butyl ether and a 20% aqueous acetic acid solution, and the extract was added to the oxidation solution for oxidation.

[0147] The chromatographic analysis method was the same as in Experimental Example 1. According to the results in Table 7-1, comparing samples 2# to 6#, it was found that the lower the oxidation concentration, the higher the yield and peptide content, indicating that the lower the oxidation concentration, the better the product. Comparing samples 7# and 3#, it was found that the yield and content of the acetonitrile / water extraction sample were lower than those using 20% ​​acetic acid / water at the same extraction volume. Therefore, 20% acetic acid / water was selected as the sample oxidation solution. Sample 1# had the highest yield and content, indicating that the cleavage reaction solution was better concentrated, but it had to be concentrated to 30-40%. Therefore, methyl tert-butyl ether was extracted with 20% acetic acid and water, and the crude product solution was combined and oxidized together.

[0148] The above results show that the lower the oxidation concentration, the higher the yield and content of the product. Therefore, considering the oxidation concentration becoming smaller: 0.2mmol / L, 0.3mmol / L, 0.4mmol / L, 0.5mmol / L 4 samples were further screened for oxidation concentration. For details, refer to Experimental Example 7.

[0149] Experimental Example 7 Oxidation Concentration Screening 2

[0150] Type and ratio of lysis reagents: TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5;

[0151] Amount of lysis reagent: 13 ml / g peptide resin (35 ml / experiment);

[0152] Peptide resin dosage: 2 mmol (12.7 g), divided into 4 samples;

[0153] Precipitation solvent: methyl tert-butyl ether.

[0154] Add the cleavage reagent to the cleavage reaction bottle as required. After the cleavage reagent is prepared (addition order: H2O, TFA, phenol, thioanisole, EDT, TIS, and nitrogen is introduced), pre-cool it to 10±3°C, add 2.0mmol (12.7g) of dry peptide resin under nitrogen protection and stir evenly. After the temperature stabilizes, react at 25±3°C for 3h. After the reaction is completed, the reaction filtrate and washing liquid are combined and divided into 4 parts, which are transferred to round-bottom flasks respectively. The mixture is concentrated under reduced pressure at 32°C±3 to 30-40% of the volume, and then frozen methyl tert-butyl ether (MTBE) is added for precipitation. The solid is filtered, washed with frozen MTBE, and the MTBE is evaporated.

[0155] 1# (0.5mmol): Use 20% acetic acid / water 2.5L solution, control the oxidation concentration at 0.2mmol / L, retain the sample (before oxidation), concentrate at 32℃±3 for 5-10min (to take away the unvolatile MTBE), use iodine / ethanol / acetic acid solution (add slowly), oxidize for 30min, and then reduce with Vc, and test the samples before and after oxidation.

[0156] 2# (0.5mmol): Use 1.7L of 20% acetic acid / water solution, control the oxidation concentration at 0.3mmol / L, retain the sample (before oxidation), concentrate at 32℃±3 for 5-10min (to take away the unvolatile MTBE), use iodine / ethanol / acetic acid solution (add slowly), oxidize for 30min, and then reduce with Vc, and test the samples before and after oxidation.

[0157] 3# (0.5mmol): Use 1.25L of 20% acetic acid / water solution, control the oxidation concentration at 0.4mmol / L, retain the sample (before oxidation), concentrate at 32℃±3 for 5-10min (to take away the unvolatile MTBE), use iodine / ethanol / acetic acid solution (add slowly), oxidize for 30min, and then reduce with Vc, and test the samples before and after oxidation.

[0158] 4# (0.5mmol): Use 1.0L of 20% acetic acid / water solution, control the oxidation concentration at 0.5mmol / L, retain the sample (before oxidation), concentrate at 32℃±3 for 5-10min (to take away the unvolatile MTBE), use iodine / ethanol / acetic acid solution (add slowly), oxidize for 30min, and then reduce with Vc, and test the samples before and after oxidation.

[0159] The comparison results of oxidation concentration are shown in Table 7-2:

[0160] Table 7-2

[0161] project 1# 2# 3# 4# Oxidation concentration 0.2mmol / L 0.3mmol / L 0.4mmol / L 0.5mmol / L Crude product purity 36.59％ 36.28％ 36.88％ 34.99％ content 0.400g 0.410g 0.323g 0.393g Yield 21.68％ 22.22％ 17.50％ 21.30％

[0162] The chromatographic analysis method was the same as in Experiment 1. According to the results in Table 7-2, the peak of the pyrolysis reagent phenol occurs at 19 min. The data sets above show comparable content and purity. Sample 3# exhibited some elution during concentration. However, due to the large volumes of samples 1# and 2#, and the need for scale-up production, the large volume would be difficult to handle. Therefore, the oxidation concentration of sample 4# was selected to be 0.5 mmol / L.

[0163] Experimental Example 8 Oxidation Temperature Screening

[0164] Type and ratio of lysis reagents: TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5;

[0165] Amount of lysis reagent: 13 ml / g peptide resin (35 ml / experiment);

[0166] Peptide resin dosage: 1.5 mmol (9.5 g), divided into 3 samples;

[0167] Precipitation solvent: methyl tert-butyl ether.

[0168] The cleavage method is the same as in Example 7.

[0169] 1# (0.5mmol): Use 1.0L of 20% acetic acid / water solution, control the oxidation concentration at 0.5mmol / L, retain the sample (before oxidation), concentrate at 32℃±3 for 5-10min (take away the unvolatile MTBE), place the sample in a water bath, control the temperature at 10℃±3℃, use iodine / ethanol / acetic acid solution (add slowly), oxidize for 40min, and then reduce with Vc, and test the samples before and after oxidation.

[0170] 2# (0.5mmol): Use 1.0L of 20% acetic acid / water solution, control the oxidation concentration at 0.5mmol / L, retain the sample (before oxidation), concentrate at 32℃±3 for 5-10min (take away the unvolatile MTBE), place the sample in a water bath, control the temperature at 20℃±3℃, use iodine / ethanol / acetic acid solution (add slowly), oxidize for 40min, and then reduce with Vc, and test the samples before and after oxidation.

[0171] 3# (0.5mmol): Use 1.0L of 20% acetic acid / water solution, control the oxidation concentration at 0.5mmol / L, retain the sample (before oxidation), concentrate at 32℃±3 for 5-10min (take away the unvolatile MTBE), place the sample in a water bath, control the temperature at 28℃±3℃, use iodine / ethanol / acetic acid solution (add slowly), oxidize for 40min, and then reduce with Vc, and test the samples before and after oxidation.

[0172] The results are shown in Table 8.

[0173] Table 8

[0174] project 1# 2# 3# Oxidation temperature 10℃±3℃ 20℃±3℃ 28℃±3℃ Crude product purity 33.27％ 33.01％ 31.34％ content 0.392g 0.405g 0.397g Yield 21.24％ 21.95％ 21.51％

[0175] The chromatographic analysis method was the same as that of Experimental Example 1. According to the results in Table 8, the purity and content were comparable at different oxidation temperatures. Considering the operability of scale-up in production and the ease of temperature control, the oxidation temperature was selected to be 20±3°C.

[0176] Experimental Example 9 Oxidation Time Screening

[0177] The cleavage was performed using the previously determined cleavage and oxidation conditions. Samples were taken at 10, 20, 30, 40, 50, and 60 minutes of oxidation time, and the progress of oxidation was monitored using Vc reduction. Since the bulk oxidation time was tentatively set at 40 minutes, the bulk sample needed to be reduced after 40 minutes of oxidation. Before Vc reduction, two samples were taken, and the oxidation reaction was stirred for 50 and 60 minutes, followed by Vc reduction.

[0178] The oxidation results at different oxidation times are shown in Table 9.

[0179] Table 9

[0180]

[0181] The chromatographic analysis method was the same as that of Experimental Example 1. According to the results in Table 9, the sample was completely oxidized after 30 minutes of HPLC detection, and there was no significant change after 60 minutes of later detection. Therefore, the oxidation time was selected to be 30 to 40 minutes.

[0182] The calculation formulas for the volumes of the lysis reagents that need to be explained are as follows:

[0183] Total volume of lysis reagent (ml) = theoretical weight of peptide resin (g) × 12-15 (ml)

[0184] Required volume of TFA (ml) = total volume of lysis reagent required × 0.815 (ml)

[0185] Required volume of H2O (ml) = total volume of lysis reagent required × 0.05 (ml)

[0186] Required volume of EDT (ml) = total volume of lysis reagent required × 0.025 (ml)

[0187] Required volume of TIS (ml) = total volume of lysis reagent required × 0.01 (ml)

[0188] Required volume of ArOH (g) = total volume of cleavage reagents required × 0.05 (g)

[0189] Required volume of ArSCH (ml) = total volume of lysis reagent required × 0.05 (ml)

[0190] The cleavage reaction of the present invention is as follows:

[0191]

[0192] The cracking process of the present invention comprises the following steps:

[0193] S41. Preparation of lysis reagent;

[0194] Specifically, H2O, TFA (trifluoroacetic acid), ArOH (phenol), ArSCH3 (thioanisole), EDT (1,2-ethanedithiol), and TIS (triisopropylsilane) were added in sequence, pre-cooled in a water bath under stirring conditions, the temperature was controlled at 10±3°C, and stirred evenly. After the temperature stabilized, the GNP peptide resin was slowly added under stirring. After the temperature stabilized, the reaction was continued to stir at 25±3°C for 3 hours (timed after the addition of the resin).

[0195] Since the process of adding GNP peptide resin is a temperature-increasing process, the speed of adding the resin can affect the temperature. Therefore, it is necessary to control the temperature of the entire system not exceeding 20°C by controlling the addition speed of the GNP peptide resin.

[0196] S42. Concentration;

[0197] Specifically, after the reaction is completed, the reaction solution is filtered and washed with TFA 2 to 3 times, each time stirring for 1 to 3 minutes, and the washing solution is combined and concentrated. The filtrate is concentrated under reduced pressure at 32±3° C. (vacuum degree greater than 0.090 MPa) to 30% to 40% of the initial volume.

[0198] It should be noted that due to the large volume of the precipitate, the reaction solution can be divided into multiple portions for concentration and precipitation.

[0199] Resin washing after cracking: Stir thoroughly and drain as much as possible to fully dissolve the product in TFA and wash it clean to reduce the risk of resin wrapping the product.

[0200] S43. precipitation;

[0201] Specifically, methyl tert-butyl ether is frozen at -7±3° C., then added to a precipitation reaction container, and stirred while adding the concentrated solution to perform precipitation.

[0202] During precipitation, a white solid precipitates out, and the precipitation reaction container is placed at -7±3°C and allowed to stand until the supernatant becomes clear and transparent.

[0203] The volume ratio of methyl tert-butyl ether and concentrate is 1 ml:8-10 ml.

[0204] S44. Filter;

[0205] Specifically, the precipitate obtained in step S43 is vacuum filtered until no liquid flows out, and then pre-cooled methyl tert-butyl ether is added to the precipitate, and the white solid is stirred and washed for 2 to 4 minutes, and filtered until no liquid flows out. The washing is repeated 7 to 9 times, and the liquid is filtered as much as possible to obtain a white solid, which is the crude GNP cleavage product.

[0206] The precipitate and filter were extracted once with methyl tert-butyl ether and 20% acetic acid aqueous solution, and the extract was added to the oxidation solution for oxidation.

[0207] S45. Drying;

[0208] Specifically, the crude GNP cracking product obtained in step S44 is dried under reduced pressure at room temperature, vacuumed online, and controlled to have a vacuum degree of ≥0.09 MPa, for 4 to 6 hours. A small amount of NaOH may be added during drying.

[0209] In order to facilitate the stabilization and subsequent purification of the crude GNP pyrolysis product, the GNP pyrolysis process further includes S5. oxidation.

[0210] Specifically, the solid obtained in step S44 was diluted with 20% aqueous acetic acid to a concentration of 5 mmol / L, with a volume of V1. The solution was then concentrated under reduced pressure at 32±3°C for 8-12 min to a volume of 10-20% of the original solution. The solution was then filtered through a membrane filter, and the filtrate was diluted to 10 V1 with 0.5 mmol / L (oxidation concentration) of 20% aqueous acetic acid. (The solution was stirred evenly, and a sample was taken before oxidation for HPLC analysis.)

[0211] Saturated I2 / ethanol / acetic acid solution was added dropwise to the oxidizing solution while stirring at 20±5°C until it turned red-brown. Stirring was continued for 35-45 min. A small amount of VC aqueous solution was added until the red-brown color disappeared to obtain a crude oxidizing solution.

[0212] The I2 / acetic acid solution was prepared as follows: 40 g iodine, 250 mL ethanol, and 250 mL acetic acid.

[0213] Pyrolysis yield = (peptide content of pyrolysis intermediate / theoretical content of GNP) × 100%

[0214] Theoretical content of GNP = weight of feed resin × resin substitution value × GNP molecular weight

[0215] The yield of cracking should meet 20.0-30.0%.

[0216] The cleaved sample, i.e., the crude cleavage intermediate, is purified to obtain a GNP purified intermediate. The GNP purification process comprises the following steps:

[0217] S6. First purification step: Mobile phase A is 0.2% TFA.H2O, and mobile phase B is acetonitrile. The crude oxidation product solution is initially separated and the components are collected as component 1, component 2, and component 3. Components 1 and 3 are then recycled to purify component 2.

[0218] S7. The second step of purification: mobile phase A was 50 mmol / LNa2SO4, pH = 2.3, and mobile phase B was acetonitrile, for purifying and separating the component 2 collected in step S6 into component 4, component 5 and component 6, and the components 4 and 6 were further purified in a cycle to component 5;

[0219] S8. The third step of purification: mobile phase A is 50 mmol / LNa2SO4, pH = 2.3, mobile phase B is acetonitrile, for the step S7 collected component 5 to purify and separate into component 7, component 8, component 9, component 7 and component 9 continue to circulate and purify to component 10, component 10 is recycled to component 8;

[0220] S9. Fourth step purification: Mobile phase A is 0.2% HA C .H2O, mobile phase B is acetonitrile.

[0221] As a specific example of the present invention, the purification in step S6 is carried out according to the following conditions:

[0222] S61. Column equilibration;

[0223] S62. Injection;

[0224] Specifically, the single injection volume is calculated as 4.5 to 5.5 g (based on the amount of powdered peptide, if there is peptide content, it is adjusted appropriately) based on the inner diameter of the 77 mm column. The injection volume is calculated according to the concentration information, and then the corresponding volume of the crude oxidation solution is measured, and the entire sample is injected into the preparative chromatographic column using the injection valve.

[0225] At the end of each injection, rinse the injection container and injection pipe with purified water to ensure that all the samples are injected into the chromatographic column.

[0226] S63.HPLC purification;

[0227] Specifically, after the sample is added, the first step of elution purification is performed according to the purification procedure in Table 10.

[0228] Table 10

[0229]

[0230] Note: When subsequent purifications are performed using the same HPLC conditions and method, there is no need to run the S61. column equilibration method separately.

[0231] S64. Component collection;

[0232] Specifically, the absolute retention time (Tr) of the absorption peak formed by the UV detector signal was used to determine the sample collection location, and the required sample components were collected in sections. During collection, the first-shot purification sample collection method was used as a basic reference for each identical sample on the same preparative HPLC chromatograph. The basic reference method for the first-shot collection method for the crude oxidation product is shown in Table 11 below (the collection bottles were switched according to the collection location during component collection, and all subsequent collection operations were performed in the same manner).

[0233] The collected sample components were tested for purity on an Agilent 1260 HPLC. GNP purity was calculated using the area normalization method. The components were then classified and stored based on the purity of the sample. Subsequent sample collection methods followed the same purity criteria as the first injection. Any sample collection fluid that did not meet the classification criteria for each component was discarded.

[0234] It should be noted that: because the crude product contains a lot of cleavage substances after dissolution and filtration, the liquid appears light yellow. The chromatographic column must be flushed after every 4 to 5 injections to ensure that the column is clean, otherwise a lot of yellow substances will remain in the preparation column.

[0235] Table 11

[0236]

[0237] Components 1 (precursor impurities) and 3 (postcursor impurities) were purified separately to the main peak (see Table 10). If the amount was small, they could be mixed and purified together. Generally, the precursor impurities were collected after the main peak, and a small amount remained. During the initial purification screening, the precursor impurities were generally found to be present at a high peak. Therefore, during purification, the precursor impurities required a 10% HAc.H2O flush for 12 minutes after injection. The postcursor impurities remaining after the main peak were collected and recirculated.

[0238] S65. Component concentration;

[0239] Specifically, the collected components were concentrated, with component 1 and component 3 concentrated first, followed by component 2, depending on the purification progress. The water bath temperature was 34 ± 2°C, the rotation speed was 50 ± 5 r / s, and the vacuum degree was ≤ -0.09 MPa. The concentrated volume was determined based on the sample estimate, injection concentration, and injection volume.

[0240] Component 1 and component 3 are purified to component 2 in step S6. If the yield is not high, a recycling method can be adopted to collect component 2 to ensure the yield.

[0241] Component 1 (premix): Concentration degree is 20% to 40% of the original volume, and the peptide content is 5 to 10 mg / mL;

[0242] Fraction 2 (main peak): Concentration degree is 25% to 40% of the original volume, and the peptide content is 15 to 25 mg / mL;

[0243] Component 3 (post-mixture): The concentration level is 5% to 15% of the original volume, and the peptide content is 5 to 15 mg / mL.

[0244] It should be noted that the collected components should not be stored for a long time at room temperature and need to be sealed and refrigerated.

[0245] As a specific example of the present invention, the purification in step S7 is carried out according to the following conditions:

[0246] S71. Column equilibration;

[0247] S72. Injection;

[0248] Specifically, the single injection volume is 3.0-4.0 g (based on the crude peptide amount) with a 77 mm column inner diameter. The injection volume is calculated based on the concentration information, and then the corresponding volume of the purified intermediate solution is measured and all of the sample is injected into the preparative chromatographic column using an injection valve.

[0249] At the end of each injection, rinse the injection container and injection pipe with purified water to ensure that all the samples are injected into the chromatographic column.

[0250] S73.HPLC purification;

[0251] Specifically, after the sample is added, the first elution procedure is performed according to the purification procedure in Table 12.

[0252] Table 12

[0253]

[0254] Note: When subsequent purifications are performed using the same HPLC conditions and method, there is no need to run the S71. Column Equilibration method separately.

[0255] S74. Component collection;

[0256] Specifically, the absolute retention time (Tr) of the absorption peak formed by the UV detector signal was used to determine the sample collection location, and the required sample components were collected in sections. During collection, the first-shot purification sample collection method was used as a basic reference for each identical sample on the same preparative HPLC chromatograph. The basic reference method for the first-shot collection method for purified intermediates is shown in Table 13 below (collection vials were switched according to the collection location during component collection, and all subsequent collection operations were performed in the same manner).

[0257] Table 13

[0258]

[0259] To ensure that the product enters the next step of purification, the preparative column is flushed after the second step of purification. The flushing method is shown in Table 14.

[0260] Components 4 (pre-impurities) and 6 (post-impurities) are each purified separately to component 5 (main peak) as shown in Table 12. If the amount is small, they can be mixed and purified together. Generally, the pre-impurities are collected after the main peak, with a small amount remaining. The post-impurities are collected after the main peak, with some pre-impurities and some post-impurities remaining. The post-impurities are collected after the main peak, with some pre-impurities and post-impurities remaining, and need to be recycled again. When collecting the main peak, it is important to note that when the column efficiency is high, the main peak collection volume is large. Therefore, constant attention should be paid to column efficiency changes. If the column efficiency decreases, the column should be flushed.

[0261] Table 14

[0262]

[0263] S75. Component concentration;

[0264] Specifically, depending on the purification progress, fractions 4 and 6 were concentrated first, followed by fraction 5. The water bath temperature was 34 ± 2°C, the rotation speed was 50 ± 5 r / s, and the vacuum degree was ≤ -0.09 MPa. The volume of the concentrate was determined based on the sample estimate, injection concentration, and injection volume.

[0265] Component 4 and component 6 are purified to component 5 in step S7. If the yield is not high, a recycling method can be adopted to collect component 5 to ensure the yield.

[0266] Component 4 (premix): Concentration level is 55% to 65% of the original volume, and the peptide content is 10 to 15 mg / mL;

[0267] Fraction 5 (main peak): The concentration is 15% to 30% of the original volume and contains 15 to 25 mg / mL of peptide;

[0268] Component 6 (post-mixture): The concentration level is 15% to 30% of the original volume, and the peptide content is 5 to 10 mg / mL.

[0269] As a specific example of the present invention, the purification in step S8 is carried out according to the following conditions:

[0270] S81. Column equilibration;

[0271] S82. Injection;

[0272] Specifically, the injection operation is the same as step S72.

[0273] S83.HPLC purification;

[0274] Specifically, after the sample is added, the first elution procedure is performed according to the purification procedure in Table 15.

[0275] Table 15

[0276]

[0277] Note: When subsequent purifications are performed using the same HPLC conditions and method, there is no need to run the S81. Column Equilibration method separately.

[0278] S84. Component collection;

[0279] Specifically, the method for collecting components in step S84 is the same as that in step S74. The basic reference method for the first needle collection method of the purified intermediate is shown in Table 16 below.

[0280] Table 16

[0281]

[0282] To ensure that the product enters the next step of purification, the preparative column is flushed after the third step of purification. The flushing method is as follows: first use the mobile phase: mobile phase A: 95% 30% HAc.H2O, mobile phase B: 5% acetonitrile, flush for 12 minutes, then replace it with the following mobile phase: mobile phase A: 20% 30% HAc.H2O, mobile phase B: 80% acetonitrile, flush for 3 minutes, and then balance the column.

[0283] Component 7 (pre-impurity) and component 9 (post-impurity) are both purified separately to the main peak as shown in Table 15. If the amount is small, they can be mixed together for purification. Generally, after the main peak of component 7 (pre-impurity) is collected, a small amount of the remaining pre-impurity and a portion of component 9 (post-impurity) remain.

[0284] Since the yield of component 8 collected from the front impurity is low, it is first collected into component 10 (secondary main peak), and the remaining components 7 (front impurity) and 9 (later impurity) need to be recycled again. After the main peak of the rear impurity is collected, the remaining front impurities and rear impurities need to be recycled again.

[0285] S85. Component concentration;

[0286] Specifically, the collected components were concentrated, with components 7 and 9 concentrated first, followed by component 8, depending on the purification progress. The water bath temperature was 34 ± 2°C, the rotation speed was 50 ± 5 rpm, and the vacuum level was ≤ -0.09 MPa. The volume of the concentrated fraction was determined based on the sample estimate, injection concentration, and injection volume.

[0287] Components 7 and 9 are purified to component 8 by step S8. If the yield is not high, a recycling method can be adopted to first collect component 10, and then purify it to component 8 by step S8 to ensure the yield.

[0288] Fraction 7 (premix): Concentration level is 20% to 40% of the original volume, and the peptide content is 5 to 15 mg / mL;

[0289] Fraction 8 (main peak): The concentration level is 15% to 35% of the original volume, and the peptide content is 15 to 25 mg / mL;

[0290] Fraction 9 (post-mix): Concentration level is 10% to 20% of the original volume, and the peptide content is 5 to 10 mg / mL;

[0291] Component 10 (secondary main peak): The concentration degree is 35% to 45% of the original volume, and the peptide content is 5 to 15 mg / mL.

[0292] It should be noted that the collected components should not be stored for a long time at room temperature and need to be sealed and refrigerated.

[0293] As a specific example of the present invention, the concentrated fraction 8 obtained in step S8 is quantified and then purified in step S9, and is injected into a equilibrated preparative chromatography column in batches using an injection valve for purification.

[0294] The step S9 is purified according to the following conditions:

[0295] S91. Column equilibration;

[0296] S92. Injection;

[0297] Specifically, the single injection volume is calculated as 9.0-12.0 g (peptide content) based on the inner diameter of the 77 mm column. The injection volume is calculated according to the concentration information, and then the corresponding volume of the component sample solution is measured. The injection pump is used to inject all of this sample into the preparative chromatographic column. At the end of each injection, the injection container and injection pipeline are rinsed with purified water to ensure that the sample is completely injected into the chromatographic column.

[0298] S93.HPLC purification;

[0299] Specifically, after the sample is added, the fourth elution procedure is performed according to the purification procedure in Table 17.

[0300] Table 17

[0301]

[0302] Note: When subsequent purifications are performed using the same HPLC conditions and method, there is no need to run the S91 column equilibration method separately.

[0303] S94. Component collection;

[0304] Specifically, clean component collection bottles were prepared, and the sample collection location was determined based on the absolute retention time of the absorption peak formed by the UV detector signal. During collection, each identical sample was collected using the first injection purification sample collection method as a basic reference on the same preparative HPLC chromatograph. The collected sample components were measured for purity on an Agilent 1260 high-performance liquid chromatograph, and the GNP purity was calculated using the area normalization method. The components were then classified and stored according to the component classification method based on the purity of the tested samples. The component collection method for subsequent purification samples referred to the purity of the first injection collection components. A reference table for the collection of the first injection purification components is shown in Table 18.

[0305] Table 18

[0306]

[0307] All sample collection fluids that do not meet the classification criteria for each component are discarded.

[0308] S95. Component concentration;

[0309] Specifically, the collected fraction 11 is concentrated. During the concentration process, the water bath temperature is controlled at 34±2°C, the rotation speed is 50±5 r / s, and the vacuum degree is ≤-0.09 MPa. The concentrated volume is determined based on the sample weight, injection concentration, and injection volume. Fraction 11 does not precipitate during the concentration process.

[0310] Fraction 11 was concentrated (main desalted peak): the concentration degree was 50% to 60% of the original volume, and the peptide content was 35 to 45 mg / mL.

[0311] It should be noted that component 11 began to degrade after 5 hours of storage, so the storage time of component 11 needs to be calculated after collection.

[0312] In addition, for steps S6 to S9,

[0313] Injection volume (mL) = single injection volume (mg) / concentration (mg / mL),

[0314] Calculate the injection time based on the calculated injection volume.

[0315] Injection time (min) = injection volume (mL) / flow rate (mL / min),

[0316] Note: The injection flow rate is the normal elution flow rate of the preparative column.

[0317] Before each component is purified in the next step, it needs to be concentrated and quantified before purification to confirm the number of injection needles.

[0318] The calculation formula for the first step purification yield is:

[0319] Yield = ∑ (concentration of component 2 * volume of the component) / (concentration of the cleavage intermediate * volume of the cleavage intermediate) * 100%.

[0320] The main peak yield of the first step is 80% to 95%.

[0321] The calculation formula for the second step purification yield is:

[0322] Yield = ∑ (concentration of component 5 * volume of the component) / (concentration of component 2 * volume of component 2) * 100%.

[0323] The yield of the main peak in the second step is 75% to 90%.

[0324] The calculation formula for the purification yield in the third step is:

[0325] Yield = ∑ (concentration of component 8 * volume of the component) / (concentration of component 5 * volume of the component) * 100%.

[0326] The yield of the main peak in the third step is 75% to 95%.

[0327] The calculation formula for the fourth step purification yield is:

[0328] Yield = Σ(amount of component 11) / (amount of component 8)*100%.

[0329] The purification yield in the fourth step is ≥90%.

[0330] Purification yield = (peptide content of purified intermediate / peptide content of cleavage intermediate) * 100%.

[0331] The total yield of the purification process is 50% to 80%.

[0332] The freeze drying box is opened in advance and pre-frozen at -40°C. The purified intermediate filtrate obtained after step S9 is freeze-dried to obtain a loose powdered GNP product, which is packaged after being taken out of the box.

[0333] Examples 1 to 4 using the GNP cracking process of the present invention are shown in Table 19.

[0334] In each example, the ratio of the cleavage reagent to the G-type peptide resin was 1:13 ml / g.

[0335] Table 19

[0336]

[0337]

[0338] Total MBTE usage: washing product (two sub-batches combined and washed 7 times each) + precipitation usage

[0339] During dissolution and oxidation: dissolve the solid in 20% acetic acid water to a concentration of 5 mmol (2900 ml), concentrate under reduced pressure at 32°C ± 3°C for 10 min, and dilute to 0.5 mmol (29000 ml) before oxidation.

[0340] After testing and calculation, the purity of the sample after GNP pyrolysis in Example 1 was 48.82%, and the pyrolysis yield was 28.09%; the purity of the sample after GNP pyrolysis in Example 2 was 48.15%, and the pyrolysis yield was 28.93%; the purity of the sample after GNP pyrolysis in Example 3 was 51.88%, and the pyrolysis yield was 30.0%; the purity of the sample after GNP pyrolysis in Example 4 was 51.98%, and the pyrolysis yield was 27.63%.

[0341] Example 5

[0342] The crude GNP pyrolysis intermediate obtained in Example 1 after preparation of the pyrolysis reagent, concentration, precipitation, filtration, and drying was prepared with 20% aqueous acetic acid to a concentration of 5 mmol / L and a volume of 2.9 L. After concentration under reduced pressure at 32°C for 10 min, the product was filtered through a 0.45 μm mixed membrane. The filtrate was diluted to 29 L with 0.50.5 mmol / L (oxidation concentration) of 20% aqueous acetic acid. The solution was stirred uniformly and added to a saturated I2 / acetic acid solution while stirring in a water bath until the solution turned reddish-brown. Stirring was continued for 40 min, and an appropriate amount of VC aqueous solution was added until the reddish-brown color disappeared, thereby obtaining a crude oxidized product solution.

[0343] Due to the large volume, concentration was required, and the concentrated volume was controlled at 10% to 20% of the original volume. The temperature during concentration was 34°C, the rotation speed was 50 r / s, and the vacuum degree was ≤-0.090 MPa. After concentration was completed, the solution was diluted to half and filtered through filter paper, a 0.45 μm mixed membrane, and a 0.22 μm mixed membrane to obtain a clear filtrate. The crude product was measured to contain 62.21 g of peptide.

[0344] The first purification step was performed using the conditions shown in Table 10, resulting in the separation of component 1 (previous impurity), component 2 (main peak), and component 3 (latter impurity). The concentration of each component is shown in Table 20, and the purity is shown in Table 21.

[0345] The second purification step employed the conditions shown in Table 12, ultimately separating component 4, component 5, and component 6. The concentration of each component is shown in Table 22, and the purity is shown in Table 23.

[0346] Table 20

[0347]

[0348] Table 21

[0349]

[0350] Table 22

[0351]

[0352] Table 23

[0353]

[0354] Table 24

[0355]

[0356] The third purification step was performed using the conditions in Table 15, and finally fractions 7, 8, 9, and 10 were separated. The concentration of each fraction is shown in Table 24, and the purity is shown in Table 25.

[0357] The fourth step of purification was performed under the conditions shown in Table 17. The concentration of the isolated fraction 11 is shown in Table 26, and the purity is shown in Table 27. The product was then freeze-dried to obtain a loose powdered GNP product.

[0358] Table 25

[0359]

[0360] Table 26

[0361]

[0362] Table 27

[0363]

[0364] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A G-type natriuretic peptide cracking process, characterized in that: The method is used to cleave the synthetic G-type natriuretic peptide resin to obtain a crude linear peptide, comprising the following steps: S41. Preparation of lysis reagent; S42. Concentration; S43. precipitation; S44. Filter; S45. Drying.

2. The cleavage process of G-type natriuretic peptide according to claim 1, wherein In step S41, the lysis reagent includes the following components in the following ratio: TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5 (V / V / V / V / W / V).

3. The cleavage process of G-type natriuretic peptide according to claim 1, wherein In step S41, the usage ratio of the cleavage reagent and the G-type natriuretic peptide resin is 12-15 ml / g.

4. The cleavage process of G-type natriuretic peptide according to claim 1, wherein In step S41, after the cleavage reagent is prepared, it is pre-cooled to 10±3°C, and dry peptide resin is added under nitrogen protection and stirred evenly. After the temperature stabilizes, the mixture is stirred at 25±3°C and cleavage is performed.

5. The cleavage process of G-type natriuretic peptide according to claim 4, characterized in that: The reaction was stirred at 25±3°C for 3 h for cleavage.

6. The cleavage process of G-type natriuretic peptide according to claim 1, characterized in that: In step S42, the reaction solution after cleavage in step S41 is filtered and washed with TFA, the washings are combined, and the filtrate is concentrated under reduced pressure at 32±3°C to 30% to 40% of the initial volume.

7. The cleavage process of G-type natriuretic peptide according to claim 1, characterized in that: In step S43, methyl tert-butyl ether is frozen at -7±3°C, and the concentrated solution obtained in step S42 is added thereto while stirring for precipitation.

8. The cleavage process of G-type natriuretic peptide according to claim 1, characterized in that: In step S44, the precipitate obtained in step S43 is vacuum filtered until no liquid flows out, and then pre-cooled methyl tert-butyl ether is added to the precipitate, and the solid is stirred and washed, and filtered until no liquid flows out. The washing is repeated 7 to 9 times to obtain a white solid, which is the crude GNP cleavage product.

9. The cleavage process of G-type natriuretic peptide according to claim 1, characterized in that: The synthesis process of the G-type natriuretic peptide resin comprises the following steps: S1. Fmoc-Nle, HMP linker-OH and a solid phase support are coupled to obtain HMPlink-Nle-MBHA Resin, wherein the solid phase support is MBHA Resin resin with a degree of substitution of 0.40 mmol / g to 0.62 mmol / g; S2. Connect Fmoc-Arg(Pbf)-OH to obtain Fmoc-Arg(Pbf)-HMPAResin; S3. According to the sequence of natriuretic peptide GNP, remove the Fmoc protecting group of each amino acid or fragment, and couple the amino acids or fragments one by one, wherein Gly 33 -Asp 34 The natriuretic peptide GNP peptide resin was obtained using the Fmoc-Asp(OtBu)-Gly-OH fragment.

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