Purification process of G-type natriuretic peptide

Abstract

The invention provides a purification process of G-type natriuretic peptide. The purification process comprises the following steps: S5, treating a GNP cracking intermediate crude product; s6, first-step purification: taking 0.2% TFA.H2O as a mobile phase A and acetonitrile as a mobile phase B, preliminarily separating the oxidized crude product solution obtained in the step S5, collecting to obtain a component 1, a component 2 and a component 3, and continuously and circularly purifying the component 1 and the component 3 until the component 2 is obtained; s7, second-step purification: a mobile phase A is 50mmol / L Na2SO4, the pH is 2.3, a mobile phase B is acetonitrile, the mobile phase A is used for purifying and separating the component 2 collected in the step S6 into components 4-6, and the component 4 and the component 6 continue to be circularly purified into a component 5; s8, third-step purification: a mobile phase A is 50mmol / L Na2SO4, the pH is 2.3, a mobile phase B is acetonitrile, the mobile phase A is used for purifying and separating the component 5 collected in the step S7 into components 7-9, the component 7 and the component 9 are continuously and circularly purified into a component 10, and the component 10 is recycled to the component 8; s9, purification in the fourth step: a mobile phase A is 0.2% HAC.H2O, and a mobile phase B is acetonitrile. The problems that GNP obtained through chemical synthesis is low in purity and poor in yield are solved.

Description

Technical Field

[0001] The present invention relates to the field of purification, and in particular to a purification process for 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 transformants are 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 purity of chemically synthesized GNPs is low, failing to meet quality requirements. Summary of the Invention

[0004] In view of this, the present invention aims to propose a purification process for G-type natriuretic peptide to solve the problems of low purity and poor yield of GNPs obtained by chemical synthesis in the prior art.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] A purification process for G-type natriuretic peptide comprises the following steps:

[0007] S5. Treatment of crude GNP cleavage intermediates;

[0008] S6. The first step of purification: mobile phase A is 0.2% TFA.H2O, mobile phase B is acetonitrile, the crude oxidation solution obtained in step S5 is initially separated, and fraction 1, fraction 2, and fraction 3 are collected. Fraction 1 and fraction 3 are further recycled to fraction 2;

[0009] 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;

[0010] 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;

[0011] S9. Fourth step purification: Mobile phase A is 0.2% HA C .H2O, mobile phase B is acetonitrile.

[0012] Furthermore, in step S5, the crude GNP cleavage intermediate is prepared with a 20% acetic acid aqueous solution to a concentration of 5 mmol / L and a volume of V1; it is concentrated under reduced pressure at 32±3°C for 8 to 12 min to 10% to 20% of the original solution, filtered with a mixed membrane 0.45 μm, and the filtrate is diluted to 10V1 with 0.5 mmol / L (oxidation concentration) 20% acetic acid aqueous solution. The solution is stirred evenly, sampled before oxidation, and tested by HPLC. Saturated I2 / acetic acid solution is added dropwise while stirring in a water bath until the solution turns reddish brown, and stirring is continued for 35 to 45 min. VC aqueous solution is added until the reddish brown color disappears to obtain the oxidized crude product solution.

[0013] Furthermore, the I2 / acetic acid solution is prepared by mixing iodine, ethanol, and acetic acid in a ratio of 40 g: 250 ml: 250 ml.

[0014] Furthermore, in step S6, the following purification procedure is used for the first elution purification: the chromatographic column filler is Kromasil 100-10-C18, the column inner diameter is 77 mm, the detection wavelength is 210 nm, the mobile phase A is 0.2% TFA.H2O, the mobile phase B is CH3CN, and the gradient elution conditions are: 0-10 min: 5% mobile phase B; 10-35 min: 7% mobile phase B; 35-50 min: 15% mobile phase B; 50-80 min: 18% mobile phase B; 80 min-80 min 30 s: 25% mobile phase B; 80 min 30 s-84 min: 80% mobile phase B; 84-96 min: 5% mobile phase B; the flow rate is 90 ml / min.

[0015] Furthermore, in step S7, the following purification procedure was used for a second elution purification: the chromatographic column filler was Kromasil 100-10-C18, the column inner diameter was 77 mm, the detection wavelength was 230 nm, the mobile phase A was 50 mmol / L Na2SO4, pH = 2.3 ± 0.2, the mobile phase B was CH3CN, and the gradient elution conditions were: 0-15 min: 5% mobile phase B; 15-25 min: 14% mobile phase B; 25-65 min: 17% mobile phase B; 65 min-70 min 30 s: 25% mobile phase B; 70 min 30 s-74 min: 70% mobile phase B; 74 min-86 min: 5% mobile phase B; the flow rate was 90 ml / min.

[0016] Furthermore, in step S8, the following purification procedure was used for the third elution purification: the chromatographic column filler was Kromasil 100-10-C18, the column inner diameter was 77 mm, the detection wavelength was 230 nm, the mobile phase A was 50 mmol / L Na2SO4, pH = 2.3 ± 0.2, the mobile phase B was CH3CN, and the gradient elution conditions were: 0-15 min: 5% mobile phase B; 15-35 min: 14% mobile phase B; 35-70 min: 17% mobile phase B; 70 min-70 min 30 s: 25% mobile phase B; 70 min 30 s-74 min: 80% mobile phase B; 74-86 min: 5% mobile phase B; the flow rate was 90 ml / min.

[0017] In step S9, a 0.2% acetic acid / water solution is used to replace the salt.

[0018] Furthermore, when step S6 is used for the first step of purification, the injection volume is 4.5 to 5.5 g / needle, and the chromatographic column diameter is 77 mm. When step S7 is used for the second step of purification, the injection volume is 3.0 to 4.0 g, and the chromatographic column diameter is 77 mm. When step S8 is used for the third step of purification, the injection volume is 3.0 to 4.0 g, and the chromatographic column diameter is 77 mm.

[0019] Furthermore, each fraction needs to be concentrated before being subjected to the next purification step.

[0020] Furthermore, the purified intermediate filtrate obtained after step S9 is freeze-dried to obtain a loose powdered GNP product.

[0021] Compared with the existing technology, the purification process of G-type natriuretic peptide described in the present invention has the following advantages: it can obtain the final active ingredient to the greatest extent, and the yield of each purification step is high, the total yield of the purification process is also high, and the purity meets the requirements, reducing the influence of impurities, and providing a stable and reliable process for the subsequent purification of chemically synthesized GNPs. DETAILED DESCRIPTION

[0022] 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.

[0023] 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.

[0024] Experimental Example 1 Treatment of crude GNP cracking intermediates

[0025] Considering the stability of the crude GNP cleavage intermediate during oxidation and storage, different treatment methods and storage time comparisons were adopted, as shown in Table 1.

[0026] Table 1

[0027]

[0028] Table 1 shows that the crude GNP cleavage intermediate, dissolved in 20% HAc / H2O and oxidized with 20% iodine / acetic acid, showed superior quantitative results compared to dissolved in 20% CH3OH / H2O, and its purity remained unchanged even after prolonged storage. Therefore, prior to purification, the following dissolution method was established: dissolution in 20% HAc / H2O, oxidation with 20% iodine / acetic acid, and reduction with VC.

[0029] Experimental Example 2: First step of purification mobile phase and injection volume screening

[0030] While keeping all other parameters unchanged, only the mobile phase for the first purification step was changed. When the mobile phase was an acetate system (i.e., phase A: 50 mmol / L NaAc, pH 6.0, phase B: acetonitrile) and the injection volume was 1.5 g / injection, the main peak purity was 83.81%. When the mobile phase was a TFA system (i.e., phase A: 0.2% TFA.H2O, phase B: acetonitrile) and the injection volume was 1.5 g / injection, the main peak purity was 93.23%. Therefore, 0.2% TFA.H2O was selected as the mobile phase for the first purification step to ensure product yield and minimize waste.

[0031] Based on the first-step purification method (including Kromasil-100-10-C18 filler and mobile phase), the first-step purification injection volume was examined. Based on sample purity and yield, the appropriate injection volume was selected to optimize the purification preparation cycle and reduce costs. The specific results are shown in Table 2.

[0032] Table 2

[0033] Injection volume Precursor quantity / purity Main peak quantification / purity Post-contamination quantification / purity Main peak yield 2.83g / injection 0.06g / 28.15％ 1.98g / 84.73％ 0.63g / 87.73％ 69.96％ 5.0g / injection 0.58g / 66.59％ 3.58g / 87.72％ 0.33g / 55.58％ 71.60％ 6.0g / injection 0.59g / 63.99％ 3.85g / 87.42％ 1.25g / 67.76％ 64.16％

[0034] As shown in Table 2, under the same chromatographic column and mobile phase, the yield, loss, and purification cost are compared. An injection volume of 5.0 g can ensure higher yield, purity, and lower loss. Therefore, the injection volume in the first step of purification is 4.5-5.5 g / needle (chromatographic column diameter 77 mm).

[0035] Experimental Example 3 Second step purification mobile phase and injection volume screening

[0036] While keeping all other parameters unchanged, only the mobile phase in the second purification step was changed. The screening results are shown in Table 3. Based on the second-step purification method (including Kromasil-100-10-C18 filler, 41.4 mm, and mobile phase), the second-step purification injection volume was examined. The specific results are shown in Table 4.

[0037] Table 3

[0038]

[0039] Table 4

[0040]

[0041] The above results show that:

[0042] (1) The purified and collected components of each mobile phase system did not meet the design requirements (total purity ≥98.5%, single impurity ≤0.3%). It was tentatively decided that a third step of purification would be required after the second step of purification. Compared with other mobile phases, the sulfate system did not show degradation or peak tailing when used as the mobile phase. Therefore, the sulfate system was selected as the mobile phase for the second and third steps of purification.

[0043] (2) Under the same chromatographic column and mobile phase conditions, the injection volume was 1.1 g, and the main peak yield was 64.1%, which is a high yield with low loss. The injection volume was 1.5 g, and the main peak yield was 23.3%, which is a low yield. By comparing the injection volume of 1.1 g, under the premise of ensuring a high yield and low loss, the injection volume of 3.0-4.0 g (chromatographic column diameter: 77 mm) should be selected.

[0044] Experimental Example 4: Screening of the third step purification injection volume

[0045] While ensuring that other parameters remain the same (the filler is Kromasil-100-10-C18, 41.4 mm), only the injection volume of the third step purification is changed. The screening results are shown in Table 5.

[0046] Table 5

[0047]

[0048] Under the same column and mobile phase conditions, with a column straight line of 41.4 mm and an injection volume of 1.0 g, the main peak yield was 79.0%, which is a high yield with minimal loss. However, with an injection volume of 1.4 g, the main peak yield was low, and no qualified main peak was obtained. A 1.0 g injection volume ensures a high yield and minimal loss. Therefore, the injection volume for the third step should be 3.0-4.0 g (based on a 77 mm column diameter).

[0049] Experimental Example 5

[0050] In the second step of purification, the results of the mobile phase and sample pH comparison experiment (50 mmol / L Na2SO4 pH = 2.3, 50 mmol / L Na2SO4 pH = 6.0) are shown in Table 6. The gradient conditions of the mobile phase are shown in Table 15.

[0051] Table 6

[0052]

[0053] Under the condition of 50mmol / L Na2SO4pH=6.0, the product was not completely eluted, the peak was tailing, and the main peak was present in a high proportion; under the condition of 50mmol / LNa2SO4pH=2.3, there was no product residue and the yield was relatively high, so the second step purification method was adjusted to: 50mmol / LNa2SO4pH=2.3.

[0054] Comparing the samples without pH adjustment and those adjusted to pH 3.0, there was no significant difference in yield and main peak quantification, indicating that pH had no effect on the sample, so the pH did not need to be adjusted in the second step of sample purification.

[0055] Experimental Example 6 Sample pH Comparison and Stability Experiment

[0056] During the storage of the qualified fractions collected during the second step of purification, degradation was observed. Therefore, storage experiments were conducted at different pH values. The fractions were adjusted to pH 2.3, 3.0, 4.0, 5.0, and 6.0, refrigerated (2-8°C), and monitored at different times to observe their stability. The results are shown in Table 7.

[0057] At pH 2.3 and 3.0, the sample showed an increase in the RRT≈0.67 miscellaneous peak, while the miscellaneous peaks following the main peak remained unchanged. At pH 4.0, 5.0, and 6.0, the RRT≈0.67 miscellaneous peak remained unchanged, while the miscellaneous peaks following the main peak (RRT≈1.01 and RRT≈1.03) increased. While the RRT≈0.67 miscellaneous peak can be removed during salt exchange, the miscellaneous peaks following the main peak are difficult to remove. Therefore, samples should be refrigerated and salt exchanged at pH 2.3–3.0.

[0058] Table 7

[0059]

[0060] Experimental Example 7 Salt Replacement Comparative Experiment

[0061] During the initial purification process, the sample showed a tendency to degrade, so the sample's relatively stable salt form was considered. A comparison was performed using 0.2% acetate and 5 mmol / L citrate. The mobile phase gradient conditions were as shown in Table 22. The results of the salt exchange comparison experiment are shown in Table 8.

[0062] Table 8

[0063]

[0064] Qualified salt products were obtained by using acetic acid and citric acid, but the content of citrate was higher, so 0.2% acetic acid / water solution was selected to replace the salt.

[0065] The synthesis process of GNPs of the present invention comprises the following steps:

[0066] S1. Preparation of HMP link-Nle-MBHAResin, comprising:

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] S2. Connect the first Fmoc protected amino acid

[0073] 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.

[0074] S3. Connecting the 2nd to 38th Fmoc-protected amino acids or amino acid fragments

[0075] S31. Protective amino acid pretreatment

[0076] 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;

[0077] S32. Deprotection

[0078] 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;

[0079] S33. Condensation reaction

[0080] 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.

[0081] 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 9.

[0082] Table 9

[0083]

[0084]

[0085]

[0086] Obtain natriuretic peptide GNP resin:

[0087] 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;

[0088] S34. Deprotection

[0089] 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;

[0090] 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%.

[0091] S4. Peptide resin cleavage reaction

[0092] A cleavage reagent was added to a reaction vessel at a ratio of 1 g:13 ml, wherein the cleavage reagent was TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5 (V / V / V / V / W / V). The peptide resin prepared in step S34 was slowly added under stirring and the reaction was stirred for 3 h. The reaction temperature was controlled at 25°C.

[0093] The reaction solution was washed with TFA at a ratio of 1 ml:3 ml, and the operation was repeated 2-3 times. The washings were combined and concentrated under reduced pressure to 1 / 3 of the original volume. Then, methyl tert-butyl ether at a temperature of -7°C was slowly added. After solid-liquid separation, the solution was washed with an appropriate amount of pre-cooled methyl tert-butyl ether and dried. The cracking yield was determined to be 39.2%.

[0094] Specifically, when the cleavage reagent is TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5, the final yield and purity content are both high. At this time, a large impurity in the crude product (enriched impurities, AA component analysis, MS) was found to be a chain break (Gly27-Pro28), and the chain break was significantly reduced.

[0095] GNP undergoes the above-mentioned synthesis process to obtain a crude GNP cracking intermediate, which is then subjected to multiple steps of purification, separation, concentration, and salt exchange to obtain a GNP purified intermediate. The raw material drug is obtained after freeze-drying, and the raw material drug is packaged, labeled, and stored.

[0096] The present invention mainly relates to a purification process for G-type natriuretic peptide (GNP), that is, a crude cleavage intermediate is purified to obtain a GNP purified intermediate, and the GNP purification process comprises the following steps:

[0097] S5. Treatment of crude GNP cleavage intermediates;

[0098] Specifically, in step S5, the crude GNP cleavage intermediate is prepared with a 20% acetic acid aqueous solution to a concentration of 5 mmol / L and a volume of V1; concentrated under reduced pressure at 32±3°C for 8 to 12 min to 10% to 20% of the original solution, filtered with a mixed membrane 0.45 μm, and the filtrate is diluted to 10V1 with 0.5 mmol / L (oxidation concentration) 20% acetic acid aqueous solution. The solution is stirred evenly, sampled before oxidation, and tested by HPLC. Saturated I2 / acetic acid solution is added dropwise while stirring in a water bath until the solution turns reddish brown, and stirring is continued for 35 to 45 min. VC aqueous solution is added until the reddish brown color disappears, thereby obtaining the oxidized crude product solution.

[0099] The I2 / acetic acid solution was prepared as follows: 40 g iodine, 250 mL ethanol, and 250 mL acetic acid.

[0100] The crude oxidation product solution obtained after treatment is sampled and the purity and peptide content of the cleavage intermediate are tested according to the current quality standards and detection methods for GNP cleavage intermediates. The mass-to-volume ratio concentration of GNP and related substances (purity) in the cleavage intermediate solution is calculated. Then, based on the mass-to-volume ratio concentration and the volume of the cleavage intermediate solution, the pure peptide content of the GNP is calculated for purification. The calculation formula is as follows:

[0101]

[0102] Where, Ct: GNP mass volume concentration in the test solution (mg / mL, minus water and salt content);

[0103] At: GNP peak area in the test solution for GNP content determination;

[0104] As: average peak area of ​​GNPs in GNP working reference solution;

[0105] Ws: weight of GNP working reference sample (mg);

[0106] D: dilution multiple of the test solution for GNP content determination;

[0107] P: GNP working reference content (%, excluding water and salt content);

[0108] Vt: The total volume of the GNP test solution.

[0109] 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.

[0110] 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;

[0111] 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;

[0112] S9. Fourth step purification: Mobile phase A is 0.2% HA C .H2O, mobile phase B is acetonitrile.

[0113] As a specific example of the present invention, the purification in step S6 is carried out according to the following conditions:

[0114] S61. Column equilibration;

[0115] Specifically, before running the complete purification gradient elution program for the first time, equilibrate the column using Table 10 and then wait for injection.

[0116] Table 10

[0117]

[0118] S62. Injection;

[0119] 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.

[0120] 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.

[0121] S63.HPLC purification;

[0122] Specifically, after the sample is added, the first step of elution purification is performed according to the purification procedure in Table 11.

[0123] Table 11

[0124]

[0125] 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.

[0126] S64. Component collection;

[0127] Specifically, the sample collection location was determined based on the absolute retention time (Tr) of the absorption peak formed by the UV detector signal, and the desired 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 12 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).

[0128] Table 12

[0129]

[0130] 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.

[0131] 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.

[0132] The flushing method is shown in Table 13

[0133] Table 13

[0134]

[0135] Components 1 (precursor impurities) and 3 (postcursor impurities) were purified separately to the main peak (see Table 11). If the amount was small, they could be mixed and purified together. Generally, the precursor impurities were collected after the main peak, resulting in a small amount of precursor impurities and some postcursor impurities. During the initial purification screening, precursor impurities were generally found to elute at high concentrations. 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.

[0136] S65. Component concentration;

[0137] 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.

[0138] 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.

[0139] Component 1 (premix): Concentration degree is 20% to 40% of the original volume, and the peptide content is 5 to 10 mg / mL;

[0140] Fraction 2 (main peak): The concentration is 25% to 40% of the original volume and contains 15 to 25 mg / mL of peptide.

[0141] 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.

[0142] 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.

[0143] As a specific example of the present invention, the purification in step S7 is carried out according to the following conditions:

[0144] S71. Column equilibration;

[0145] Specifically, before running the complete purification gradient elution program for the first time, equilibrate the column using Table 14 and then wait for injection.

[0146] Table 14

[0147]

[0148] S72. Injection;

[0149] 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.

[0150] 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.

[0151] S73.HPLC purification;

[0152] Specifically, after the sample is added, the first elution procedure is performed according to the purification procedure in Table 15.

[0153] Table 15

[0154]

[0155] 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.

[0156] S74. Component collection;

[0157] 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 16 below (collection vials were switched according to the collection location during component collection, and all subsequent collection operations were performed in the same manner).

[0158] Table 16

[0159]

[0160] 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 17.

[0161] Table 17

[0162]

[0163] Components 4 (pre-impurities) and 6 (post-impurities) are each purified separately to component 5 (main peak) as shown in Table 15. 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.

[0164] S75. Component concentration;

[0165] 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.

[0166] 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.

[0167] Component 4 (premix): Concentration level is 55% to 65% of the original volume, and the peptide content is 10 to 15 mg / mL;

[0168] Fraction 5 (main peak): The concentration is 15% to 30% of the original volume and contains 15 to 25 mg / mL of peptide;

[0169] 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.

[0170] As a specific example of the present invention, the purification in step S8 is carried out according to the following conditions:

[0171] S81. Column equilibration;

[0172] Specifically, before running the complete purification gradient elution program for the first time, equilibrate the column using Table 14 and then wait for injection.

[0173] S82. Injection;

[0174] Specifically, the injection operation is the same as step S72.

[0175] S83.HPLC purification;

[0176] Specifically, after the sample is added, the first elution procedure is performed according to the purification procedure in Table 18.

[0177] Table 18

[0178]

[0179] 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.

[0180] S84. Component collection;

[0181] 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 19 below.

[0182] Table 19

[0183]

[0184] 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 finally re-equilibrate the column using the method shown in Table 20.

[0185] Table 20

[0186]

[0187] Component 7 (pre-impurity) and component 9 (post-impurity) are both purified separately to the main peak as shown in Table 18. 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 component 7 (post-impurity) is the pre-impurity and a portion of component 9 (post-impurity).

[0188] 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.

[0189] S85. Component concentration;

[0190] 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.

[0191] 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.

[0192] Fraction 7 (premix): Concentration level is 20% to 40% of the original volume, and the peptide content is 5 to 15 mg / mL;

[0193] Fraction 8 (main peak): Concentration degree is 15% to 35% of the original volume, and the peptide content is 15 to 25 mg / mL;

[0194] Fraction 9 (post-mix): Concentration level is 10% to 20% of the original volume, and the peptide content is 5 to 10 mg / mL;

[0195] 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.

[0196] 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.

[0197] 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.

[0198] The step S9 is purified according to the following conditions:

[0199] S91. Column equilibration;

[0200] Specifically, before running the complete purification gradient elution program for the first time, equilibrate the column using Table 21 and then wait for injection.

[0201] S92. Injection;

[0202] 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.

[0203] Table 21

[0204]

[0205] S93.HPLC purification;

[0206] Specifically, after the sample is added, the fourth elution procedure is performed according to the purification procedure in Table 22.

[0207] Table 22

[0208]

[0209] 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.

[0210] S94. Component collection;

[0211] Specifically, clean component collection bottles were prepared, and the absolute retention time of the absorption peak formed by the UV detector signal was used to determine the sample collection location. During collection, each identical sample was collected on the same preparative HPLC chromatograph using the first injection purification sample collection method as a basic reference. The collected sample components were measured for purity on an Agilent 1260 high-performance liquid chromatograph, and the GNP purity was calculated by area normalization. The components were classified and stored according to the component classification method based on the purity of the tested samples. The subsequent purification sample collection method for the components referred to the purity of the first injection. A reference table for the collection of the first injection purification components is shown in Table 23.

[0212] Table 23

[0213]

[0214] All sample collection fluids that do not meet the classification criteria for each component are discarded.

[0215] S95. Component concentration;

[0216] 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.

[0217] 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.

[0218] 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.

[0219] In addition, for steps S6 to S9,

[0220] Injection volume (mL) = single injection volume (mg) / concentration (mg / mL),

[0221] Calculate the injection time based on the calculated injection volume.

[0222] Injection time (min) = injection volume (mL) / flow rate (mL / min),

[0223] Note: The injection flow rate is the normal elution flow rate of the preparative column.

[0224] 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.

[0225] The calculation formula for the first step purification yield is:

[0226] Yield = ∑ (concentration of component 2 * volume of the component) / (concentration of cleavage intermediate * volume of cleavage intermediate) * 100%.

[0227] The main peak yield of the first step is 80% to 95%.

[0228] The calculation formula for the second step purification yield is:

[0229] Yield = ∑ (concentration of component 5 * volume of the component) / (concentration of component 2 * volume of component 2) * 100%.

[0230] The main peak yield in the second step is 75% to 90%.

[0231] The calculation formula for the purification yield in the third step is:

[0232] Yield = ∑ (concentration of component 8 * volume of the component) / (concentration of component 5 * volume of the component) * 100%.

[0233] The main peak yield in the third step is 75% to 95%.

[0234] The calculation formula for the fourth step purification yield is:

[0235] Yield = Σ(amount of component 11) / (amount of component 8)*100%.

[0236] The purification yield in the fourth step is ≥90%.

[0237] Purification yield = (peptide content of purified intermediate / peptide content of cleavage intermediate) * 100%.

[0238] The total yield of the purification process is 50% to 80%.

[0239] Open the freeze drying box in advance and pre-freeze at -40°C. The purified intermediate filtrate obtained after step S9 is freeze-dried using the method shown in Table 24 below to obtain a loose powdered GNP product, which is then packaged after being unpacked.

[0240] Table 24

[0241]

[0242] Example 1

[0243] The crude GNP cleavage intermediate obtained through the synthesis process was prepared with 20% aqueous acetic acid to a concentration of 5 mmol / L in a volume of 2.9 L. After concentrating under reduced pressure at 32°C for 10 minutes, the mixture 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 evenly and, while stirring in a water bath, saturated I2 / acetic acid solution was added until the solution turned reddish-brown. Stirring was continued for 40 minutes, and an appropriate amount of aqueous VC was added until the reddish-brown color disappeared. This yielded the crude oxidized product solution.

[0244] 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.

[0245] The first purification step was performed using the conditions in Tables 10 to 13, ultimately separating component 1 (precursor), component 2 (main peak), and component 3 (postcursor). The concentration of each component is shown in Table 25, and the purity is shown in Table 26.

[0246] Table 25

[0247]

[0248] Table 26

[0249]

[0250] The second purification step employed the conditions shown in Tables 14 to 17, ultimately separating component 4, component 5, and component 6. The concentration of each component is shown in Table 27, and the purity is shown in Table 28.

[0251] Table 27

[0252]

[0253] Table 28

[0254]

[0255] Table 29

[0256]

[0257] Table 30

[0258]

[0259] Table 31

[0260]

[0261] Table 32

[0262]

[0263] In the third step of purification, the conditions in Tables 14 and 18-20 were used to finally separate and obtain component 7, component 8, component 9, and component 10. The concentration of each component is shown in Table 29, and the purity is shown in Table 30.

[0264] The fourth step of purification was performed using the conditions shown in Tables 21 to 23. The concentration of the separated fraction 11 is shown in Table 31, and the purity is shown in Table 32. The fraction was then freeze-dried using the freeze-drying method shown in Table 4 to obtain a loose powdered GNP product.

[0265] 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 purification process for G-type natriuretic peptide, characterized in that: The steps include: S5. Treatment of crude GNP cleavage intermediates; S6. The first step of purification: mobile phase A is 0.2% TFA.H2O, mobile phase B is acetonitrile, the crude oxidation solution obtained in step S5 is initially separated, and fraction 1, fraction 2, and fraction 3 are collected. Fraction 1 and fraction 3 are further recycled to fraction 2; 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; 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; S9. Fourth step purification: Mobile phase A is 0.2% HA C .H2O, mobile phase B is acetonitrile.

2. The purification process of G-type natriuretic peptide according to claim 1, characterized in that In step S5, the crude GNP cleavage intermediate is prepared with a 20% acetic acid aqueous solution to a concentration of 5 mmol / L and a volume of V1; it is concentrated under reduced pressure at 32±3°C for 8-12 min to 10%-20% of the original solution, filtered with a mixed membrane 0.45 μm, and the filtrate is diluted to 10V1 with 0.5 mmol / L (oxidation concentration) 20% acetic acid aqueous solution. The solution is stirred evenly, sampled before oxidation, and tested by HPLC. Saturated I2 / acetic acid solution is added dropwise while stirring in a water bath until the solution turns reddish brown, and stirring is continued for 35-45 min. VC aqueous solution is added until the reddish brown color disappears to obtain the oxidized crude product solution.

3. The purification process of G-type natriuretic peptide according to claim 2, characterized in that The I2 / acetic acid solution was prepared by mixing iodine, ethanol, and acetic acid in a ratio of 40 g: 250 ml: 250 ml.

4. The purification process of G-type natriuretic peptide according to claim 1, characterized in that In step S6, the following purification procedure is used for the first elution purification: The chromatographic column filler is Kromasil 100-10-C18, the column inner diameter is 77 mm, the detection wavelength is 210 nm, the mobile phase A: 0.2% TFA.H2O, the mobile phase B: CH3CN, the gradient elution conditions are: 0-10 min: 5% mobile phase B; 10-35 min: 7% mobile phase B; 35-50 min: 15% mobile phase B; 50-80 min: 18% mobile phase B; 80 min-80 min30 s: 25% mobile phase B; 80 min30 s-84 min: 80% mobile phase B; 84-96 min: 5% mobile phase B; the flow rate is 90 ml / min.

5. The purification process of G-type natriuretic peptide according to claim 1, characterized in that In step S7, the following purification procedure was used for the second elution purification: the chromatographic column filler was Kromasil 100-10-C18, the column inner diameter was 77 mm, the detection wavelength was 230 nm, the mobile phase A was 50 mmol / L Na2SO4, pH = 2.3 ± 0.2, the mobile phase B was CH3CN, and the gradient elution conditions were: 0-15 min: 5% mobile phase B; 15-25 min: 14% mobile phase B; 25-65 min: 17% mobile phase B; 65 min-70 min 30 s: 25% mobile phase B; 70 min 30 s-74 min: 70% mobile phase B; 74 min-86 min: 5% mobile phase B; the flow rate was 90 ml / min.

6. The purification process of G-type natriuretic peptide according to claim 1, characterized in that In step S8, the following purification procedure is used for the third elution purification: The chromatographic column packing material is Kromasil 100-10-C18, the column inner diameter is 77 mm, the detection wavelength is 230 nm, the mobile phase A is 50 mmol / L Na2SO4, pH = 2.3 ± 0.2, the mobile phase B is CH3CN, and the gradient elution conditions are: 0-15 min: 5% mobile phase B; 15-35 min: 14% mobile phase B; 35-70 min: 17% mobile phase B; 70 min-70 min30 s: 25% mobile phase B; 70 min30 s-74 min: 80% mobile phase B; 74-86 min: 5% mobile phase B; the flow rate is 90 ml / min.

7. The purification process for G-type natriuretic peptide according to claim 1, characterized in that: In step S9, a 0.2% acetic acid / water solution is used to replace the salt.

8. The purification process for G-type natriuretic peptide according to claim 1, characterized in that: When step S6 is used for the first step of purification, the injection volume is 4.5-5.5 g / needle, and the chromatographic column diameter is 77 mm. When step S7 is used for the second step of purification, the injection volume is 3.0-4.0 g, and the chromatographic column diameter is 77 mm. When step S8 is used for the third step of purification, the injection volume is 3.0-4.0 g, and the chromatographic column diameter is 77 mm.

9. The purification process of G-type natriuretic peptide according to claim 1, characterized in that: Each fraction needs to be concentrated before the next purification step.

10. The purification process of G-type natriuretic peptide according to claim 1, characterized in that: The purified intermediate filtrate obtained after step S9 is freeze-dried to obtain a loose powdered GNP product.

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