A method for preparing anti-coronavirus polypeptide

Abstract

The present invention relates to a method for preparing an anti-coronavirus polypeptide. The method comprises the following steps: 1) synthesis: preparing a polypeptide; 2) purification: purifying a solution of the polypeptide by column chromatography, collecting fractions; the fractions or their samples or mixtures thereof enter the next purification process or circulate the current purification process; the purification process comprises one-step purification and two-step purification. The method can prepare a product with high purity, few impurities, excellent separation effect, and high purification efficiency through the purification process; and can significantly improve its solubility, which is suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide drug synthesis, and in particular relates to a method for preparing a polypeptide. Background Art

[0002] The virus is mainly transmitted through respiratory droplets, close contact droplets, object-to-mouth, etc. After entering the human body, the virus rapidly replicates and multiplies in the respiratory tract, eventually causing symptoms of respiratory infection in the patient.

[0003] The World Health Organization (WHO) has proposed five "variants of concern" (VOC), namely Alpha, Beta, Gamma, Delta and Omicron. Currently, cases of infection with the Omicron strain (especially the BA.5 subclade) have replaced the Delta strain as the main global epidemic strain. Globally, people of all ages and regions are generally susceptible.

[0004] Clinically, traditional Chinese medicine, immunotherapy and supportive care are the main treatments. Due to the limitations of therapeutic drugs, they are far from meeting current clinical needs.

[0005] ① Variants continue to emerge and neutralizing antibodies are inactivated: Neutralizing antibodies target the RBD region (which is more prone to mutation). Since they are ineffective against the Omicron variant, the US FDA has revised the EUA statements of Roche / Regeneron and Eli Lilly, and revoked the Sotrovimab EUA authorization of GSK / VIR.

[0006] ②Currently, only three new coronavirus-specific drugs have been approved for marketing: Pfizer's Paxlovid, Real Biopharma's Azvudine Tablets, and Fosun Pharma's Ambavirizumab / Romivir Monoclonal Antibody Injection.

[0007] ③ Many adverse drug reactions: (1) Paxlovid: During medication, symptoms such as diarrhea, taste perversion, indigestion, gastroesophageal reflux disease, vomiting, myalgia, and dizziness may occur; it has liver toxicity (increased liver transaminase, clinical hepatitis and jaundice), and there is a risk of inducing HIV-1 resistance. (2) Azvudine: During medication, neurological diseases (dizziness, headache, drowsiness, somnolence, memory impairment, etc.), liver and kidney dysfunction, gastrointestinal system diseases (nausea, vomiting, diarrhea, abdominal distension, abdominal discomfort, abdominal pain, increased bowel movement frequency, indigestion, etc.), abnormal blood parameters, increased blood lactate, etc. may occur. (3) Molnupiravir: During medication, adverse reactions such as allergies (urticaria, toxic rash, erythema), diarrhea, nausea, vomiting, floating dizziness, and headache may occur.

[0008] ④ Potential drug resistance: It is only a matter of time before small molecule drugs develop drug resistance. Recently, there have been reports of multiple cases of nucleic acid re-test positive in patients infected with the new coronavirus after taking Paxlovid.

[0009] Based on the limitations of the above-mentioned therapeutic drugs and unmet clinical needs, it is urgent to develop broad-spectrum, highly effective, and highly accessible anti-COVID-19 drugs for the currently prevalent and new mutant strains that will emerge in the future.

[0010] Membrane fusion is an extremely important biological phenomenon. Many type I enveloped viruses that seriously endanger human health infect host cells through membrane fusion, such as HIV, influenza virus, Ebola virus, SARS-CoV, MERS-CoV, and the currently prevalent SARS-CoV-2.

[0011] The envelope glycoprotein (S protein) of human coronavirus plays a key role in the process of infecting target cells. The S protein can be divided into S1 subunit and S2 subunit. The S1 subunit contains two functional domains, the N-terminal domain (NTD) and the receptor binding domain (RBD), both of which are responsible for the binding of the virus to the host cell receptor (human angiotensin-converting enzyme 2, ACE2). They also contain several conformational neutralizing epitopes and are targets for the development of neutralizing antibodies and vaccines. The S2 subunit contains three functional domains, the fusion peptide (FP) and two heptad repeat regions (HR) - HR1 and HR2. After the RBD in S1 binds to the receptor, the S2 subunit changes conformation by inserting the FP into the host cell membrane, followed by HR1 forming a trimeric helix, while HR2 folds inversely in the groove formed by the HR1 trimer, resulting in a typical six-helix bundle structure (6-HB), thereby bringing the viral membrane and the cell membrane closer for a fusion reaction, allowing the viral genetic material to enter the target cell through the fusion pore to replicate and produce new viral particles.

[0012] Studies have found that peptides derived from the HR1 and HR2 regions of many viruses can act as viral membrane fusion inhibitors, and their mechanism of action is to competitively bind to the fusion protein in the pre-fusion state, thereby blocking the formation of the 6-HB structure. At present, the fusion inhibitors that act on the transmembrane subunit of the viral membrane fusion protein and inhibit the formation of homologous 6-HB are Roche's Enfuvirtide (T20, approved by the US FDA in 2003) and Frontier Bio's Abovirtide (approved by China's NMPA in 2018), which are used to treat HIV infection; in addition, Fusu Bio's Sifuvirtide and Kangbao Bio's Lipvirtide are in clinical phase II and phase I, respectively. Membrane fusion inhibitor antiviral drugs have clear targets and their drugability has been verified. In order to improve the half-life of polypeptides and enhance antiviral activity, lipopeptides modified with lipid compounds (such as fatty acids and cholesterol) have been the focus of drug research and development of viral membrane fusion inhibitors in recent years.

[0013] YKYY017 is a coronavirus membrane fusion inhibitor that acts on the HR1 region of the S2 subunit of the SARS-CoV-2 virus S protein. It can competitively interact with the viral HR1 region to form a heterologous six-helix bundle structure (6-HB), thereby inhibiting the formation of homologous 6-HB between the HR1 and HR2 domains of the virus itself, blocking the fusion process between the virus and the host cell, exerting antiviral activity, and has a broad spectrum.

[0014] The peptide sequence of YKYY017 is shown in SEQ ID NO.1:

[0015] Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile-Asp-Arg-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu-Asn-Glu-Ser-Leu-Ile-Asp-Leu-Gln-Glu-Leu-Gly-Lys-Tyr-Glu-Gln-Ty r-Ile-Lys-Glu-Ala-Ala-Ala-Lys-Lys(Chol)-NH2

[0016] Among them, acetyl (Ac-) is the amino terminal protecting group; the terminal amino group (-NH2) is the carboxyl terminal protecting group; Chol is the cholesterol succinate monoester group modified on the C-terminal amino acid lysine (Lys).

[0017] The existing patent CN 114736272 A discloses the preparation method of YKYY017 peptide, which is only in the laboratory research stage; however, as the laboratory research stage is expanded to the production stage, the purity and yield of the product will show a significant downward trend as the production scale expands. According to the experience of preparation and production in the field of peptides, when laboratory research is expanded to the production stage, the purity and other product properties can be reduced by about 10% to 20%.

[0018] Therefore, how to improve the purification yield and product purity of YKYY017, and produce high-purity lipopeptides on a large scale to meet the needs of small-batch laboratory research to large-scale production is a key technical problem that urgently needs to be solved. Summary of the invention

[0019] In view of the above problems, the present invention provides a method for preparing a polypeptide, which significantly improves the yield of the purification process (up to 9.3%), the purity of the purified product (98.63%) and the dissolution rate of the raw material (<1 min) through a specific purification process and parameters.

[0020] At the same time, ① through two-step purification, the impurity content can be significantly reduced, and the product purity and yield can be improved; compared with the product obtained by only one-step purification, the purity is increased by more than 25%, and the maximum single impurity content is reduced by 1.75%; the purity of the preparation product is significantly improved; ② through the three-step purification process, the prepared product has the best purity (98.63%), the least impurities (as low as 0.32%), and the yield (8.9%) is significant; ③ in the two-step purification process, a specific phenylhexyl is selected as the chromatographic column filler, and ④ in the three-step purification process, a specific C 18 As a chromatographic column filler, the prepared product has high purity (up to 98.63%), few impurities (as low as 0.32%), excellent separation effect (tailing factor as low as 1.220), and high purification efficiency (theoretical plate number as high as 17442); ⑤ Glacial acetic acid is used as a diluent for dilution before the flow fraction is loaded to obtain a preparation raw material with high solubility (<1min); ⑥ The method is suitable for large-scale production stage to obtain a high-purity, high-yield preparation product. The details are as follows:

[0021] A method for preparing a polypeptide, wherein:

[0022] 1) Synthesis: Preparation of peptides by solid phase synthesis;

[0023] 2) Purification: Purify the polypeptide solution by column chromatography and collect the fractions;

[0024] The fraction or its sample or mixture thereof enters the next purification process or circulates the current purification process;

[0025] The purification process includes one-step purification and two-step purification.

[0026] In some embodiments, the type of preparative column chromatography filler for the one-step purification and the two-step purification is phenylhexyl filler.

[0027] In some preferred embodiments, the fractions of the purification process cycle, the samples thereof, or the mixture of the samples are diluted with glacial acetic acid before loading.

[0028] In some more preferred embodiments, the amount of glacial acetic acid added is 5-15% of the volume of the original fraction.

[0029] In some preferred embodiments, in the one-step purification, the fractions meeting the criteria in 1.1 are collected;

[0030] Table 1

[0031]

[0032] In some preferred embodiments, the remaining fractions or a portion thereof are subjected to a cyclic one-step purification process.

[0033] In some preferred embodiments, the remaining fractions are selected from the fractions meeting the criteria of 1.2 and / or the fractions meeting the criteria of 1.3;

[0034] Table 2

[0035]

[0036] In some preferred embodiments, the fractions meeting the 1.1 standard are combined into the 1.1 standard sample. In some embodiments, the fractions meeting the 1.1 standard are the 1st fraction; the fractions meeting the 1.1 standard are combined into the 1st sample.

[0037] In some preferred embodiments, the fractions meeting the 1.2 standard are combined into the 1.2 standard sample. In some embodiments, the fractions meeting the 1.2 standard are the 2nd fraction; the fractions meeting the 1.2 standard are combined into the 2nd sample.

[0038] In some preferred embodiments, the fractions meeting the 1.3 standard are combined into the 1.3 standard sample. In some embodiments, the fractions meeting the 1.3 standard are the 3rd fraction; the fractions meeting the 1.3 standard are combined into the 3rd sample.

[0039] In some preferred embodiments, the 1.2 standard sample and the 1.3 standard sample are combined into the 1st standard residual fraction sample mixture. In some embodiments, the 2nd sample and the 3rd sample can be mixed into a mixture of the 2nd sample and the 3rd sample.

[0040] In some preferred embodiments, the one-step purification comprises the following steps:

[0041] i) Column washing: washing the column with a one-step purification elution mobile phase to prepare the chromatographic column;

[0042] ii) Pre-loading equilibrium: Use one-step purification starting mobile phase to prepare pre-loading equilibrium;

[0043] iii) Sample loading: Load the peptide solution

[0044] iv) Top loading: After loading, use 5-30% acetonitrile solution to carry the sample into the preparative chromatographic column; the top loading time is about 2-10 minutes;

[0045] v) Elution: elution is performed using a one-step purification mobile phase gradient;

[0046] vi) Detection and fraction collection: the eluted sample is detected by liquid chromatography, and the first fraction, the second fraction and / or the third fraction are collected, the first fraction is used for subsequent two-step purification; the second fraction and the third fraction are used for subsequent one-step purification;

[0047] vii) except for the first fraction, cyclically purifying the remaining fractions or their samples or their mixtures: returning to the purification process, repeating steps i) to vi) and collecting the first fraction; wherein the peptide solution in step iii) is replaced by the remaining fractions or their samples or their mixtures for loading, and before loading, 10% of the volume of the remaining fractions or their samples or their mixtures is added with glacial acetic acid for dilution; the remaining fractions or their samples or their mixtures are selected from the second fraction or their samples, the third fraction or their samples and their mixtures;

[0048] Preferably, the number of times of cyclic purification of the remaining fraction or its sample or sample mixture in step vii) is 1 to 3 times, preferably 1 time;

[0049] viii) combining the first fraction collected in step vi) and the first fraction collected in step vii) to obtain a first combined sample.

[0050] In some preferred embodiments, in the two-step purification, the fractions meeting the 1.1 standard or the 1.1 standard and the sample are purified by column chromatography, the fractions meeting the 2.1 standard are collected, and the remaining fractions are recycled in the two-step purification process;

[0051] Table 3

[0052]

[0053] In some preferred embodiments, the remaining fractions or a sample thereof are subjected to a cyclic two-step purification process;

[0054] In some preferred embodiments, the remaining fractions are selected from the fractions meeting the criteria of 2.2 and / or the fractions meeting the criteria of 2.3;

[0055] Table 4

[0056] standard Requirements for fractions before and after the main peak Section 2.2 Standard The fraction before the main peak meets the main peak purity ≥ 85.0% Standard 2.3 The fraction after the main peak meets the main peak purity ≥ 75.0%

[0057] In some preferred embodiments, the fractions meeting the 2.1 standard are combined into the 2.1 standard sample. In some embodiments, the fractions meeting the 2.1 standard or the 2.1 standard sample are used as finished products. In some embodiments, the fractions meeting the 2.1 standard are the 4th fraction; the fractions meeting the 2.1 standard are combined into the 4th sample.

[0058] In some preferred embodiments, the fractions meeting the 2.2 standard are combined into the 2.2 standard sample. In some embodiments, the fractions meeting the 2.2 standard are the 5th fraction; the fractions meeting the 2.2 standard are combined into the 5th sample.

[0059] In some preferred embodiments, the fractions meeting the 2.3 standard are combined into the 2.3 standard sample. In some embodiments, the fractions meeting the 2.3 standard are the 6th fraction; the fractions meeting the 2.3 standard are combined into the 6th sample.

[0060] In some preferred embodiments, the standard sample No. 2.2 and the standard sample No. 2.3 are combined into the remaining standard sample No. 2. In some embodiments, the sample No. 5 and the sample No. 6 can be mixed into a mixture of the sample No. 5 and the sample No. 6.

[0061] In some preferred embodiments, the two-step purification comprises the following steps:

[0062] i) Column washing: washing the column with a two-step purification elution mobile phase to prepare the chromatographic column;

[0063] ii) Pre-loading equilibrium: using the two-step purification starting mobile phase to prepare the pre-loading equilibrium;

[0064] iii) loading: loading the first fraction or its aliquot;

[0065] iv) Top loading: After loading, use 5-30% acetonitrile solution to carry the sample into the preparative chromatographic column; the top loading time is about 2-10 minutes;

[0066] v) Elution: elution is performed using a two-step purification mobile phase gradient;

[0067] vi) Detection and fraction collection: the eluted sample is detected by liquid chromatography, and the fourth fraction, the fifth fraction and / or the sixth fraction are collected;

[0068] vii) except for the 4th fraction, purifying the remaining fraction or its sample or its mixture in a cycle: returning to the second-step purification process, repeating steps i) to vi) and collecting the 4th fraction; wherein the 4th fraction or its sample in step iii) is replaced by the remaining fraction or its sample or its mixture for loading, and before loading, 10% of the volume of the remaining fraction or its sample or its mixture is diluted with glacial acetic acid; the remaining fraction or its sample or its mixture is selected from the 5th fraction or its sample, the 6th fraction or its sample and the mixture of the 5th and 6th fractions;

[0069] Preferably, the number of times of cyclic purification of the remaining fraction or its sample or sample mixture in step vii) is 1 to 3 times, preferably 1 time;

[0070] viii) combining the fourth fraction collected in step vi) and the fourth fraction collected in step vii) to obtain a fourth sample.

[0071] In some preferred embodiments, the purification process further comprises three steps of purification.

[0072] In some more preferred embodiments, the type of preparative column chromatography filler for the three-step purification is C 18 filler.

[0073] In some preferred embodiments, in the three-step purification, the fractions meeting the criterion 2.1 or the criterion 2.1 and the sample are purified by column chromatography, and the fractions meeting the criterion 3.1 are collected;

[0074] Table 5

[0075]

[0076] In some preferred embodiments, the remaining fractions or their pools are subjected to a cyclic three-step purification process;

[0077] In some preferred embodiments, the remaining fractions are selected from the fractions meeting the criteria of 3.2 and / or the fractions meeting the criteria of 3.3;

[0078] Table 6

[0079]

[0080]

[0081] In some preferred embodiments, the fractions meeting the 3.1 standard are combined into the 3.1 standard sample. In some embodiments, the fractions meeting the 3.1 standard or the 3.1 standard sample are used as finished products. In some embodiments, the fraction meeting the 3.1 standard is the 7th fraction; the fractions meeting the 3.1 standard are combined into the 7th sample.

[0082] In some preferred embodiments, the fractions meeting the 3.2 standard are combined into the 3.2 standard sample. In some embodiments, the fraction meeting the 3.2 standard is the 8th fraction; the fractions meeting the 3.2 standard are combined into the 8th sample.

[0083] In some preferred embodiments, the fractions meeting the 3.3 standard are combined into the 3.3 standard sample. In some embodiments, the fraction meeting the 3.3 standard is the 9th fraction; the fractions meeting the 3.3 standard are combined into the 9th sample.

[0084] In some preferred embodiments, the 3.2 standard sample and the 3.3 standard sample are combined into the 3rd standard residual fraction sample mixture. In some embodiments, the 8th sample and the 9th sample can be mixed into a mixture of the 8th sample and the 9th sample.

[0085] In some preferred embodiments, the three-step purification comprises the following steps:

[0086] i) Column washing: washing the column with a three-step purification elution mobile phase to prepare the chromatographic column;

[0087] ii) Pre-loading equilibrium: A three-step purification of the starting mobile phase was used to prepare the pre-loading equilibrium;

[0088] iii) loading: loading the fourth fraction or its combined sample;

[0089] iv) Top loading: After loading, use 5-30% acetonitrile solution to carry the sample into the preparative chromatographic column; the top loading time is about 2-10 minutes;

[0090] v) Elution: elution is performed using a three-step purification mobile phase gradient;

[0091] vi) Detection and fraction collection: the eluted sample is detected by liquid chromatography, and the 7th fraction, the 8th fraction and / or the 9th fraction are collected;

[0092] vii) except for the 7th fraction, purifying the remaining fraction or its sample or its mixture in a cycle: returning to the three-step purification process, repeating steps i) to vi) and collecting the 7th fraction; wherein the 4th fraction or its sample in step iii) is replaced by the remaining fraction or its sample or its mixture for loading, and before loading, 0.5-2 times the volume of the remaining fraction is added with water for dilution, preferably 1 times the volume of the remaining fraction is added with purified water; the remaining fraction or its sample or its mixture is selected from the 8th fraction or its sample, the 9th fraction or its sample and the mixture of the 8th and 9th fractions;

[0093] Preferably, the number of times of cyclic purification of the remaining fraction or its sample or sample mixture in step vii) is 1 to 3 times, preferably 1 time;

[0094] viii) combining the seventh fraction collected in step vi) and the seventh fraction collected in step vii) to obtain a seventh sample.

[0095] In some preferred embodiments, the one-step purification elution mobile phase is: mobile phase A1: 0.05% to 0.3% TFA aqueous solution, preferably 0.1% TFA aqueous solution; mobile phase B: acetonitrile.

[0096] In some preferred embodiments, the two-step purification elution mobile phase is: mobile phase A2: 2mM-20mM ammonium bicarbonate solution, preferably 10mM ammonium bicarbonate solution; mobile phase B: acetonitrile.

[0097] In some preferred embodiments, the three-step purification elution mobile phases are: mobile phase A3: 0.5 wt% to 5 wt% acetic acid solution, preferably 2 wt% acetic acid solution; mobile phase B: acetonitrile.

[0098] In some preferred embodiments, the method further comprises freeze-drying the purified sample to obtain a freeze-dried powder.

[0099] In some embodiments, the polypeptide comprises 20 to 60 amino acids.

[0100] In some preferred embodiments, the polypeptide is used to treat the new coronavirus.

[0101] In some more preferred embodiments, the polypeptide comprises YKYY017, and the polypeptide sequence of YKYY017 is:

[0102] Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile-Asp-Arg-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu-Asn-Glu-Ser-Leu-Ile-Asp-Leu-Gln-Glu-Leu-Gly-Lys-Tyr-Glu-Gln-Ty r-Ile-Lys-Glu-Ala-Ala-Ala-Lys-Lys(Chol)-NH2.

[0103] In some preferred embodiments, the polypeptide is synthesized as follows:

[0104] a) solid phase synthesis of fully protected peptides by sequentially coupling all amino acids from the carbon end to the nitrogen end; preferably, Rink Amide-MBHA Resin is used;

[0105] b) selectively removing the side chain protecting group of Lys; preferably using hydrazine as a catalyst to selectively remove the side chain protecting group of Lys;

[0106] c) coupling the side chain cholesterol succinate monoester in the presence of a coupling agent;

[0107] d) In the presence of a cleavage agent, the peptide resin is cleaved and the side chain is deprotected to obtain the polypeptide.

[0108] If the loss is not considered, the remaining fractions or their samples or mixtures thereof may not be subjected to cyclic purification. For example, after the crude peptide is purified in one step, the first fraction is obtained; the first fraction is purified in two steps to obtain the fourth fraction; the fourth fraction is purified in three steps to obtain the seventh fraction.

[0109] The remaining fractions can be subjected to one-step, two-step or three-step purification separately, or can be combined and then subjected to one-step, two-step or three-step purification.

[0110] In addition, if the experiment is non-continuous and the time interval exceeds 24 hours, and the intermediate products in the experiment need to be stored, the collected fractions or samples should be promptly supplemented with 1 volume of purified water and stored in a cold storage at 2-8°C for ≤3 days (room temperature storage for ≤1 hour). If the experiment is completed within 24 hours, the intermediate products do not need to be stored.

[0111] At the same time, when storing the fractions or samples of the product, one volume of purified water should be added in time and stored in a cold storage at 2-8°C for ≤3 days (room temperature storage for ≤1h).

[0112] The beneficial effects of the present invention are as follows:

[0113] 1. Two-step purification process

[0114] During the purification process, a two-step purification process (for example, one-step purification + two-step purification) can be used to prepare polypeptide products with high yield and purity, good separation effect, and high purification efficiency (for example, anti-new crown polypeptide YKYY017), which are specifically manifested as follows:

[0115] The yield of the purified product is as high as 9.3%, and the purity is 97.55%. The content of each impurity in the adjacent front impurity, adjacent rear impurity, and maximum single impurity is controlled within 0.56%. Compared with the product obtained by only one-step purification, the purity is increased by more than 25%, and the maximum single impurity content is reduced by 1.75%. The purity of the preparation product is significantly improved;

[0116] The tailing factor was controlled at 1.274, significantly lower than the limit standard ≤2.0, and the separation effect was significant;

[0117] The number of theoretical plates in the purification process is as high as 16840, significantly higher than the limit standard ≥5000, and the purification efficiency is high;

[0118] The solubility is significantly improved. The polypeptide (for example, the anti-COVID-19 polypeptide YKYY017) is dissolved in methanol with a dissolution time of <3 minutes and excellent solubility.

[0119] 2. Three-step purification process

[0120] Using a three-step purification process (e.g., one-step purification + two-step purification + three-step purification), the prepared polypeptide (e.g., anti-COVID-19 polypeptide YKYY017) has the best product yield and purity, the best separation effect, and the highest purification efficiency, as shown in the following:

[0121] The yield of the purified product was as high as 8.9%, the purity was 98.63%, and the content of adjacent front impurities was as low as 0.32%, which was significantly better than the limit standard ≤1.0; compared with the one-step purification + two-step purification method, the purity of the product was increased by 1.08%, the content of adjacent front impurities was reduced by 0.15%, and the content of adjacent rear impurities was reduced by 0.12%; the purity of the preparation product was significantly improved;

[0122] The tailing factor is only 1.220, significantly lower than the limit standard ≤2.0, and the separation effect is optimal;

[0123] The number of theoretical plates in the purification process was as high as 17442, significantly higher than the limit standard ≥5000, and the purification efficiency was the highest;

[0124] The solubility is optimal, and the dissolution time of the polypeptide (for example, the anti-new coronavirus polypeptide YKYY017) in methanol is controlled at <1min. Compared with the two-step purification, the solubility is reduced to 1 / 3, and the reduction effect is significant.

[0125] 3. Column chromatography fillers for one-step purification and two-step purification

[0126] a) During the one-step and two-step purification, the 18 ), using phenylhexyl as the filler for preparative chromatography, the prepared polypeptide (for example, the anti-new crown polypeptide YKYY017) has high purity, few impurities, excellent separation effect, and high purification efficiency, which is specifically manifested as follows:

[0127] After purification, the product purity is increased to more than 97%, significantly higher than the purity limit standard;

[0128] The content of each impurity in the adjacent front impurity, adjacent back impurity and the largest single impurity is controlled within 0.47-0.56%, which is significantly lower than the impurity content limit standard of ≤1.0%;

[0129] The tailing factor was controlled at 1.274, significantly lower than the limit standard ≤2.0, and the separation effect was significant;

[0130] The number of theoretical plates in the purification process is as high as 16840, and the purification efficiency is high.

[0131] b) On the contrary, when other preparative column chromatography filler types (e.g., C8 or C 18 ), the purified product has low purity, high impurity content, poor separation effect and low purification efficiency; therefore, it cannot be used as a qualified filler in the process of purifying polypeptides (for example, the new crown polypeptide YKYY017), which is specifically manifested as follows:

[0132] After purification, the main peak purity of the product is only 60-90%, which cannot meet the purity standard of ≥97.0%, and it is impossible to purify a qualified preparation product;

[0133] The content of each impurity increased significantly, with the adjacent front impurity, adjacent back impurity and the largest single impurity reaching about 1-11%, significantly higher than the impurity content standard of ≤1.0%, and the impurity content of the purified product was unqualified;

[0134] The tailing factor increases to 1.435-3.412, and the separation effect is poor;

[0135] During the purification process, the number of theoretical plates decreased significantly, the column efficiency decreased, and the purification efficiency was significantly reduced.

[0136] 4. Column chromatography packing for three-step purification

[0137] a) In the three-step purification process, compared with other types of prepared fillers (e.g., C8 or phenylhexyl), C 18 As a filler for preparative chromatography, the prepared peptides (for example, the anti-COVID-19 peptide YKYY017) have the best purity, the least impurities, and the highest purification efficiency, as shown in the following:

[0138] The purity of the purified product was 98.63%, and the adjacent impurity content was as low as 0.32%, which was significantly better than the limit standard ≤1.0;

[0139] The tailing factor is only 1.220, significantly lower than the limit standard ≤2.0, and the separation effect is optimal;

[0140] The number of theoretical plates in the purification process was as high as 17442, significantly higher than the limit standard ≥5000, and the purification efficiency was the highest;

[0141] b) On the contrary, when other types of fillers are used (for example, C8 or phenylhexyl), the purified product has poor purity and cannot be used as a qualified preparation product; and there is a phenomenon of low purification efficiency, or even dead adsorption that makes purification impossible, which is specifically manifested as follows:

[0142] The purity of the main peak was reduced to 90.82%, which failed to meet the purity limit standard (≥97.0%). Therefore, it was impossible to purify a qualified preparation product.

[0143] The impurity content of adjacent impurities and the largest single impurity increased to 1.53%, significantly higher than the impurity content limit standard (≤1.0%), and it was impossible to purify a qualified preparation product;

[0144] The number of theoretical plates during the purification process was 9175, and the purification efficiency was significantly reduced;

[0145] If other preparation fillers are used, the sample is easily deadly adsorbed and cannot be washed out, and the purified product cannot be obtained.

[0146] 5. Types of diluent

[0147] a) Before the fraction was subjected to cyclic purification and loaded, it was diluted with glacial acetic acid. The product obtained by the two-step purification had good solubility, as shown in the following:

[0148] The dissolution time of the peptide (e.g., anti-COVID-19 peptide YKYY017) in methanol is controlled at <3 min, with excellent solubility;

[0149] b) Before the fractions were subjected to cyclic purification and loading, they were diluted with glacial acetic acid and water (glacial acetic acid was used for dilution in the first and second steps, and water was used for dilution in the third step). The solubility of the preparation product obtained by the three-step purification was further improved compared with that of the two-step purification, as shown in the following:

[0150] The dissolution time of the peptide (e.g., anti-COVID-19 peptide YKYY017) in methanol is controlled at <1 min, and the solubility is the best;

[0151] c) On the contrary, when other types of substances are used as diluents (e.g., methanol, acetonitrile), the solubility of the final product is significantly reduced, as shown in the following:

[0152] Under the same conditions, using other types of solvents as diluents significantly increases the dissolution time to more than 6 to 9 minutes, greatly prolongs the dissolution time, and results in poor solubility of the product.

[0153] 6. Diluent dosage

[0154] Before the fraction is subjected to the cyclic purification process, 5-15% of the original volume (i.e. the original fraction or the original sample volume) of glacial acetic acid is used for dilution. During the two-step purification process, the dissolution time of the purified product in the methanol solvent is controlled to be less than 3 minutes, and the solubility is excellent.

[0155] During the three-step purification process, the dissolution time of the purified product in methanol solvent was controlled to be less than 1 min, and the solubility was the best.

[0156] 7. Application

[0157] The preparation method of the present invention is suitable for large-scale production, and the prepared product has high purity and yield and low impurity content. BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Figure 1 It is a schematic diagram of the process flow of the present invention. DETAILED DESCRIPTION

[0159] Example

[0160] Example 1: Preparation of crude YKYY017

[0161] Chemical reagents required in the preparation process:

[0162] All chemical reagents, such as various Fmoc amino acids, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), N,N-dimethylformamide (DMF), piperidine (PIPE), ninhydrin, acetic anhydride (Ac2O), N,N-diisopropylethylamine (DIEA), hydrazine hydrate, cholesterol monosuccinate, trifluoroacetic acid (TFA), ethanedithiol (EDT), thioanisole (TA), and phenol, were purchased from major chemical reagent suppliers without further purification before use.

[0163] The protected amino acid raw materials used in the peptide synthesis process include Fmoc-Lys(Dde)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, and Fmoc-Arg(Pbf)-OH. The abbreviations have well-known definitions: Fmoc is 9-fluorenylmethoxycarbonyl, Dde is 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, Boc is tert-butyloxycarbonyl, tBu is tert-butyl, OtBu is tert-butoxy, Trt is trityl, and Pbf is (2,3-dihydro-2,2,4,6,7-pentamethylbenzofuran-5-yl)sulfonyl.

[0164] The solid phase synthesis method of YKYY017 is as follows:

[0165] 1) Select Rink Amide-MBHA Resin (amino resin) as the carrier, Fmoc-Lys (Dde) -OH as the starting amino acid, HOBt / DIC as the coupling condensation agent, and perform a coupling reaction to form Fmoc-Lys (Dde) -Rink Amide MBHA Resin amino acid resin. According to the YKYY017 peptide sequence, use Fmoc protected amino acids as the starting material, HOBt / DIC as the coupling agent, and sequentially couple the Fmoc protected amino acids to complete the main chain:

[0166] Ac-Ser(tBu)-Val-Val-Asn(Trt)-Ile-Gln(Trt)-Lys(Boc)-Glu(OtBu)-Ile-Asp(Ot Bu)-Arg(Pbf)-Leu-Asn(Trt)-Glu(OtBu)-Val-Ala-Lys(Boc)-Asn(Trt)-Leu-Asn(Trt)-Glu(OtBu)-Ser(tBu)-Leu-Ile-Asp(OtBu)-Leu-Gln(Trt )-Glu(OtBu)-Leu-Gly-Lys(Boc)-Tyr(tBu)-Glu(OtBu)-Gln(Trt)-Tyr(tBu)-Ile-Lys(Boc)-Glu(OtBu)-Ala-Ala-Ala-Lys(Boc)-Lys(Dde)-Rink Amide MBHA Resin.

[0167] The specific steps are:

[0168] Weigh 2000.00g of Rink Amide-MBHA Resin resin (Sub = 0.31mmol / g) and add it to a solid phase reactor, add 27L of dichloromethane (DCM) to swell the resin for 0.5h. Drain the solvent, add 15L v / v 20% piperidine / DMF solution for deprotection, and react for 30min. Drain and add 15L of DMF to wash 6 times. The indene test result is positive. Weigh 800g of Fmoc-Lys(Dde)-OH, 335g of HOBT, and 391g of DIC, add 10L of DMF to dissolve, activate for 10 minutes, and the activation temperature does not exceed 10°C. Add the activated solution to the reactor, react until the indene test result is negative, and then drain. Add DMF to wash 3 times, 15L each time;

[0169] Repeat the above steps, according to the amino acid sequence Fmoc-Ala-OH; Fmoc-Ala-OH; Fmoc-Ala-OH; Fmoc-Glu(OtBu)-OH; Fmoc-Lys(Boc )-OH; Fmoc-Ile-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Glu(OtBu)-OH; Fmoc-Tyr(tB u)-OH; Fmoc-Lys(Boc)-OH; Fmoc-Gly-OH; Fmoc-Leu-OH; Fmoc-Glu(OtBu)-OH; Fmoc-Gln(Trt)-O H; Fmoc-Leu-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Ser(tBu)-OH; Fmoc-Gl u(OtBu)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Leu-OH; Fmoc-Asn(Trt)-OH; Fmoc-Lys(Boc)-OH; Fmoc-A la-OH; Fmoc-Val-OH; Fmoc-Glu(OtBu)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Leu-OH; Fmoc-Arg(Pbf)-O H; Fmoc-Asp(OtBu)-OH; Fmoc-Ile-OH; Fmoc-Glu(OtBu)-OH; Fmoc-Lys(Boc)-OH; Fmoc-Gln(Trt) -OH; Fmoc-Ile-OH; Fmoc-Asn(Trt)-OH; Fmoc-Val-OH; Fmoc-Val-OH; Fmoc-Ser(tBu)-OH for coupling reaction;

[0170] Add 15L v / v 20% piperidine / DMF solution for deprotection again and react for 30min. Drain and add 15L DMF to wash 6 times. The result of indene test is positive. Add 214mL acetic anhydride and 271mL DIEA to acetylate for 30min. After the result of indene test is negative, drain. Add DMF to wash 3 times, 15L each time.

[0171] 2) Using hydrazine as a catalyst, selectively remove the side chain protecting group of Lys to obtain:

[0172] Ac-Ser(tBu)-Val-Val-Asn(Trt)-Ile-Gln(Trt)-Lys(Boc)-Glu(OtBu)-Ile-Asp(Ot Bu)-Arg(Pbf)-Leu-Asn(Trt)-Glu(OtBu)-Val-Ala-Lys(Boc)-Asn(Trt)-Leu-Asn(Trt)-Glu(OtBu)-Ser(tBu)-Leu-Ile-Asp(OtBu)-Leu-Gln( Trt)-Glu(OtBu)-Leu-Gly-Lys(Boc)-Tyr(tBu)-Glu(OtBu)-Gln(Trt)-Tyr(tBu)-Ile-Lys(Boc)-Glu(OtBu)-Ala-Ala-Ala-Lys(Boc)-Lys-Rink Amide MBHA Resin.

[0173] The specific steps are:

[0174] Add 15L v / v 2% hydrazine / DMF solution for deprotection and react for 30min. Drain and add 15L DMF for washing 6 times. The result of indene test is positive.

[0175] 3) Coupling the side chain cholesterol succinate monoester in the presence of a coupling agent to obtain:

[0176] Ac-Ser(tBu)-Val-Val-Asn(Trt)-Ile-Gln(Trt)-Lys(Boc)-Glu(OtBu)-Ile-Asp(Ot Bu)-Arg(Pbf)-Leu-Asn(Trt)-Glu(OtBu)-Val-Ala-Lys(Boc)-Asn(Trt)-Leu-Asn(Trt)-Glu(OtBu)-Ser(tBu)-Leu-Ile-Asp(OtBu)-Leu-Gln(Trt )-Glu(OtBu)-Leu-Gly-Lys(Boc)-Tyr(tBu)-Glu(OtBu)-Gln(Trt)-Tyr(tBu)-Ile-Lys(Boc)-Glu(OtBu)-Ala-Ala-Ala-Lys(Boc)-Lys(Chol)-Rink Amide MBHA Resin.

[0177] The specific steps are:

[0178] Weigh 681.4g of cholesterol succinate, 559.0g of HATU, and 631.9mL of DIEA, add 10L of DMF to dissolve, activate for 10 minutes, the activation temperature does not exceed 10°C, add the activated solution to the reactor, react until the indene test result is negative, and then drain. Add DMF to wash 3 times, 15L each time;

[0179] After the coupling was completed, the mixture was washed alternately with DCM and methanol for 3 times, 20 L each time, and dried in vacuo to obtain YKYY017 resin.

[0180] 4) In the presence of a cleavage agent, the peptide resin is cleaved and the side chain is deprotected to obtain a crude product of YKYY017, to obtain:

[0181] Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile-Asp-Arg-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu-Asn-Glu-Ser-Leu-Ile-Asp-Leu-Gln-Glu-Leu-Gly-Lys-Tyr-Glu-Gln-Ty r-Ile-Lys-Glu-Ala-Ala-Ala-Lys-Lys(Chol)-NH2;

[0182] The specific steps are:

[0183] Prepare the cleavage reagent trifluoroacetic acid TFA: thioanisole TA: ethanedithiol EDT: anisole = 90:5:3:2, slowly add the cleavage agent to the YKYY017 peptide resin at ≤10°C, return to room temperature after 0.5h and continue to react for 2h. After the reaction is completed, add anhydrous ether for precipitation. Centrifuge the precipitate 4 times and dry to obtain the crude YKYY017.

[0184] Example 2: Purification method of crude YKYY017 (one-step purification + two-step purification)

[0185] After the crude YKYY017 is obtained in Example 1, subsequent purification, lyophilization and packaging processes are required. Purification can be performed by one-step purification, two-step purification or three-step purification, wherein the one-step purification and two-step purification are specifically as follows (see the process flow Figure 1 ):

[0186] 5) Purification

[0187] The crude YKYY017 obtained in Example 1 was weighed and crushed, and slowly added to a 20% acetonitrile aqueous solution under stirring and ultrasonic dissolving until no obvious solids were present, and then filtered using a 0.45 μm filter membrane, and the filtrate was collected to obtain a crude YKYY017 solution; the crude YKYY017 solution was purified by preparative column chromatography; the specific steps are as follows:

[0188] 5-1) One-step purification

[0189] Chromatographic conditions:

[0190] Type of preparative column packing: SILICAONE-10PH6-3;

[0191] Model of preparative chromatography: DAC-300;

[0192] Diameter of preparative chromatography column: 300 mm;

[0193] Column efficiency ≥4000 / column, column length 20~30cm;

[0194] Maximum sample flow rate: 1.4L / min (84L / H);

[0195] Maximum elution flow rate: 1.6L / min (96L / H);

[0196] Detection wavelength: 230nm;

[0197] Mobile Phase:

[0198] ① One-step purification elution mobile phase: mobile phase A1: 0.1% TFA aqueous solution; mobile phase B: acetonitrile;

[0199] ② One-step purification starting mobile phase: mobile phase A: purified water; mobile phase B: acetonitrile;

[0200] Density setting of mobile phase B: 0.79 g / mL (790 kg / m 3 );

[0201] The specific steps are as follows:

[0202] i) Column washing: washing the preparative chromatographic column with a one-step purification elution mobile phase (mobile phase A1 and mobile phase B), the washing time being ≥ 10 min;

[0203] Table 7: Column washing gradient

[0204] Time min <![CDATA[A1%]]> B％ 0 15 85 ≥10 15 85

[0205] ii) Equilibration before loading: Use one-step purification starting mobile phase (mobile phase A and mobile phase B) to prepare the equilibrium before loading for ≥10 min;

[0206] Table 8: Equilibrated gradient before loading

[0207]

[0208]

[0209] iii) Sample loading: Load the crude YKYY017 solution; sample loading amount: 20-40 g / needle; maximum sample loading flow rate: 1.4 L / min (84 L / H);

[0210] iv) Top sampling: After the sample loading is completed, use 20% acetonitrile solution to bring the sample in the loading pipeline into the preparative chromatographic column at the sample flow rate; the top sampling time is about 3 minutes;

[0211] v) Elution: elution is performed using a one-step purification mobile phase (mobile phase A1 and mobile phase B) gradient;

[0212] Table 9: Elution gradient

[0213] Time min <![CDATA[A1%]]> B％ 0 75 25 10 60 40 70 40 60

[0214] vi) Detection and fraction collection: The eluted sample is detected by liquid chromatography, and the first fraction that meets the 1.1 standard, the second fraction that meets the 1.2 standard and / or the third fraction that meets the 1.3 standard are collected according to the detection results and the fraction standards in the following table, and the rest are discarded; the first fraction is used for subsequent two-step purification; the second fraction is the fraction collected before the peak, and the third fraction is the fraction collected after the peak. The second and third fractions are used for subsequent one-step purification in a cycle;

[0215] Table 10: One-step purification fraction collection criteria

[0216]

[0217] Note: Relative retention time is the ratio of the impurity peak retention time to the main peak retention time; based on crude peptides or samples with low purity, there are many and complex characteristic peaks before and after the main peak in the liquid chromatogram, so multiple peaks within the relative retention time interval of specific impurities before the main peak (i.e., RRT = 0.93-0.97) and the relative retention time interval of specific impurities after the main peak (i.e., RRT = 1.03-1.13) are selected for measurement.

[0218] The 1.1 standard requires that the following conditions be met simultaneously: the purity of the main peak is ≥ 75.0%; the maximum single impurity content within the relative retention time interval of RRT = 0.93-0.97 is ≤ 3.0%; the maximum single impurity content within the relative retention time interval of RRT = 1.03-1.13 is ≤ 3.0%. Based on the 1.1 standard, the main peak has high purity and the maximum single impurity content is low, so the fraction that meets the 1.1 standard is the fraction that flows out within the retention time of the main peak;

[0219] The 1.2 standard requires that the following conditions be met simultaneously: the purity of the main peak is ≥ 60.0%; the maximum single impurity content within the relative retention time interval of RRT = 0.93-0.97 is ≤ 10.0%; the maximum single impurity content within the relative retention time interval of RRT = 1.03-1.13 is ≤ 3.0%. The fraction that meets the 1.2 standard is the fraction that flows out before the retention time of the main peak;

[0220] The 1.3 standard requires that the following conditions be met simultaneously: the purity of the main peak is ≥ 60.0%; the maximum single impurity content within the relative retention time interval of RRT = 0.93 to 0.97 is ≤ 3.0%; the maximum single impurity content within the relative retention time interval of RRT = 1.03 to 1.13 is ≤ 15.0%. The fraction that meets the 1.3 standard is the fraction that flows out after the retention time of the main peak.

[0221] vii) except the first fraction, the remaining fractions (the second and third fractions) are combined and circulated for purification: return to the one-step purification process of 5-1), repeat steps i) to vi), and collect the first fraction; wherein, the YKYY017 crude solution in step iii) is replaced with the combined sample of the remaining fractions for loading, and 10% of the volume of the remaining fraction is added with glacial acetic acid for dilution before loading;

[0222] viii) combining the first fraction collected in step vi) and the first fraction collected in step vii) to obtain a first combined sample.

[0223] 5-2) Two-step purification:

[0224] Chromatographic conditions:

[0225] Type of preparative column packing: SILICAONE-10PH6-3;

[0226] Model of preparative chromatography: DAC-300;

[0227] Diameter of preparative chromatography column: 300 mm;

[0228] Column efficiency ≥4000 / column, column length 20~30cm;

[0229] Maximum sample flow rate: 1.4L / min (84L / H);

[0230] Maximum elution flow rate: 1.6L / min (96L / H);

[0231] Detection wavelength: 230nm;

[0232] Mobile Phase:

[0233] ① Two-step purification elution mobile phase: mobile phase A2: 10mM ammonium bicarbonate solution; mobile phase B: acetonitrile;

[0234] ② Two-step purification starting mobile phase: mobile phase A: purified water; mobile phase B: acetonitrile;

[0235] Density setting of mobile phase B: 0.79 g / mL (790 kg / m 3 );

[0236] i) Column washing: Use two-step purification elution mobile phase (mobile phase A2 and mobile phase B) to wash the preparative chromatographic column, and the washing time is ≥ 10 min;

[0237] Table 11: Column wash gradient

[0238]

[0239]

[0240] ii) Equilibration before loading: Use the two-step purification starting mobile phase (mobile phase A and mobile phase B) to prepare the pre-loading equilibrium for ≥10 min;

[0241] Table 12: Equilibration gradient before loading

[0242] Time min A％ B％ 0 85 15 ≥10 85 15

[0243] iii) Sample loading: before loading the first sample, add 10% of the original volume of glacial acetic acid (i.e., add 10% of the volume of glacial acetic acid of the first sample), and then load the sample; sample loading amount: 20-50 g / needle; maximum sample loading flow rate: 1.4 L / min (84 L / H);

[0244] iv) Top sampling: After the sample loading is completed, use 20% acetonitrile solution to bring the sample in the loading pipeline into the preparative chromatographic column at the sample flow rate; the top sampling time is about 3 minutes;

[0245] v) Elution: elution is performed using a two-step purification elution mobile phase (mobile phase A2 and mobile phase B);

[0246] Table 13: Elution gradient

[0247] Time min <![CDATA[A2%]]> B％ 0 85 15 10 65 35 70 55 45

[0248] vi) Detection and fraction collection: The eluted sample is detected by liquid chromatography, and according to the detection results and the fraction standards in the following table, the 4th fraction that meets the 2.1 standard, the 5th fraction that meets the 2.2 standard and / or the 6th fraction that meets the 2.3 standard are collected, and the rest are discarded; the 4th fraction is used as the finished product for subsequent storage; or the 4th fraction is used for subsequent three-step purification; the 5th fraction is the pre-peak collection fraction, the 6th fraction is the post-peak collection fraction, and the 5th and 6th fractions are used for subsequent cyclic two-step purification;

[0249] Table 14: Second-step purification fraction collection criteria

[0250]

[0251] Table 15: Second-step purification fraction collection criteria

[0252]

[0253]

[0254] Note: The 2.1 standard requires the following conditions to be met at the same time: the purity of the main peak is ≥ 97.0%; the maximum single impurity content within the relative retention time interval of RRT = 0.93-0.97 is ≤ 2.0%; the maximum single impurity content within the relative retention time interval of RRT = 1.03-1.13 is ≤ 2.0%. Based on the 1.1 standard, the main peak has high purity and the maximum single impurity content is low, so the fraction that meets the 2.1 standard is the fraction that flows out within the retention time of the main peak;

[0255] Standard 2.2 requires that the following conditions be met simultaneously: the fractions eluting before the retention time of the main peak that satisfy the main peak purity ≥ 85.0%;

[0256] Standard 2.3 requires that the following conditions be met simultaneously: among the fractions eluting after the retention time of the main peak, the fractions satisfying the main peak purity ≥ 75.0% shall be included.

[0257] vii) except the 4th fraction, the remaining fractions (5th and 6th fractions) are combined and purified in a cycle: return to the two-step purification process of 5-2), repeat steps i) to vi) and collect the 4th fraction; wherein the first combined sample in step iii) is replaced with the combined sample of the remaining fractions for loading, and 10% of the volume of the remaining fractions is added with glacial acetic acid for dilution before loading;

[0258] viii) combining the fourth fraction collected in step vi) and the fourth fraction collected in step vii) to obtain a fourth sample.

[0259] Example 3: Purification method of crude YKYY017 (one-step purification + two-step purification + three-step purification)

[0260] On the basis of Example 2, a three-step purification was performed, that is, the fourth sample was used as the loading component and a three-step purification was performed according to the following steps.

[0261] 5-3) Three-step purification

[0262] Chromatographic conditions:

[0263] Preparative chromatographic column packing type: Daisogel C 18 ;

[0264] Model of preparative chromatography: DAC-300;

[0265] Diameter of preparative chromatography column: 300 mm;

[0266] Column efficiency ≥4000 / column, column length 20~30cm;

[0267] Maximum sample flow rate: 1.4L / min (84L / H);

[0268] Maximum elution flow rate: 1.6L / min (96L / H);

[0269] Detection wavelength: 230nm;

[0270] Mobile Phase:

[0271] ① Three-step purification elution mobile phase: mobile phase A3: 2wt% acetic acid solution; mobile phase B: acetonitrile;

[0272] ② Three-step purification starting mobile phase: mobile phase A: purified water; mobile phase B: acetonitrile;

[0273] Density setting of mobile phase B: 0.79 g / mL (790 kg / m 3 );

[0274] i) Column washing: The preparative chromatographic column is washed with a three-step purification elution mobile phase (mobile phase A3 and mobile phase B), and the washing time is ≥ 10 min;

[0275] Table 16: Column wash gradient

[0276] Time min <![CDATA[A3%]]> B％ 0 25 75 ≥10 25 75

[0277] ii) Equilibration before loading: The three-step purification starting mobile phase (mobile phase A and mobile phase B) was used to prepare the pre-loading equilibrium for ≥10 min;

[0278] Table 17: Equilibration gradient before loading

[0279] Time min A％ B％ 0 75 25 ≥10 75 25

[0280] iii) Sample loading: before loading the fourth sample, add 10% of the original volume of glacial acetic acid (i.e., add 10% of the volume of the fourth sample) and then load the sample; sample loading amount: 20-80 g / needle; maximum sample loading flow rate: 1.4 L / min (84 L / H);

[0281] iv) Top sampling: After the sample loading is completed, use 20% acetonitrile solution to bring the sample in the loading pipeline into the preparative chromatographic column at the sample flow rate; the top sampling time is about 3 minutes;

[0282] v) Elution: elution is performed using a three-step purification elution mobile phase (mobile phase A3 and mobile phase B);

[0283] Table 18: Elution gradient

[0284] Time min A3％ B％ 0 75 25 10 65 35 70 25 75

[0285] vi) Detection and fraction collection: The eluted sample is detected by liquid chromatography, and the 7th fraction that meets the 3.1 standard, the 8th fraction that meets the 3.2 standard and / or the 9th fraction that meets the 3.3 standard are collected according to the detection results and the fraction standards in the following table, and the rest are discarded; the 7th fraction is used as the finished product for subsequent storage; the 8th fraction is the pre-peak collection fraction; the 9th fraction is the post-peak collection fraction; the 8th fraction and the 9th fraction are used for the subsequent three-step purification cycle;

[0286] Table 19: Three-step purification fraction collection criteria

[0287]

[0288] Table 20: Three-step purification fraction collection criteria

[0289] standard Requirements for fractions before and after the main peak Standard 3.2 The fraction before the main peak meets the main peak purity ≥ 85.0% Standard 3.3 The fraction after the main peak meets the main peak purity ≥ 85.0%

[0290] Note: The 3.1 standard requires the following conditions to be met at the same time: the purity of the main peak is ≥ 98.0%; the maximum single impurity content within the relative retention time interval of RRT = 0.93-0.97 is ≤ 1.0%; the maximum single impurity content within the relative retention time interval of RRT = 1.03-1.13 is ≤ 1.0%. Based on the 1.1 standard, the main peak has high purity and the maximum single impurity content is low, so the fraction that meets the 3.1 standard is the fraction that flows out within the retention time of the main peak;

[0291] Standard 3.2 requires that the following conditions be met simultaneously: the fractions eluting before the retention time of the main peak must have a main peak purity of ≥85.0%;

[0292] Standard 3.3 requires that the following conditions be met simultaneously: among the fractions eluting after the retention time of the main peak, the fractions satisfying the main peak purity ≥ 85.0% shall be included.

[0293] vii) except the 7th fraction, the remaining fractions (the 8th and 9th fractions) are combined and circulated for purification: returning to the three-step purification process of 5-3), repeating steps i) to vi) and collecting the 7th fraction; wherein the 4th combined sample in step iii) is replaced with the combined sample of the remaining fractions for loading, and purified water of 1 times the volume of the remaining fractions is added for dilution before loading;

[0294] viii) The seventh fraction collected in step vi) and the seventh fraction collected in step vii) are combined to obtain the seventh sample. The seventh sample is used as the finished product

[0295] Note: In step viii) of the three-step purification (5-3), the main peak purity of the related substances detected in the combined fractions (the 7th sample) should be ≥98.0%, and the maximum single impurity should be ≤1.0%.

[0296] In addition, if the experiment is non-continuous and the time interval exceeds 24 hours, the intermediate products in Examples 2 and 3 above need to be stored, then the collected fractions or samples are promptly supplemented with 1 volume of purified water and stored in a cold storage at 2-8°C for ≤3 days (room temperature storage for ≤1 hour). If the experiment is completed within 24 hours, the intermediate products do not need to be stored.

[0297] At the same time, when storing the 4th and 7th samples of the product, 1 volume of purified water should be added in time and stored in a cold storage at 2-8°C for ≤3 days (room temperature storage for ≤1h).

[0298] The production process of YKYY017 includes: peptide resin synthesis, crude product preparation, purification (one-step purification, two-step purification and three-step purification), lyophilization and packaging. The results showed that purification (one-step purification, two-step purification and three-step purification) had a significant impact on the key quality attributes and yield of the product. Therefore, on this basis, the following further studies were conducted.

[0299] Based on the three-step purification process, the one-step purification and the two-step purification are mainly used for purification, which significantly improves the purity and yield of the raw materials; and during the one-step purification and the two-step purification process, glacial acetic acid is added to prevent the physical polymerization of the raw materials while also improving the solubility to a certain extent.

[0300] The three-step purification process is the salt conversion stage. At this stage, the content of relevant substances (impurities) is basically qualified. The three-step purification mainly plays the role of column concentration, improving solubility, and further improving purity.

[0301] Therefore, in the following exploration process, the purification conditions of one-step purification, two-step purification and three-step purification can be explored separately, or the purification conditions of one-step purification and two-step purification can be explored together.

[0302] Example 4: Effect of filler type (one-step purification and two-step purification)

[0303] The present invention explores the influence of filler types during the purification process. Based on Example 2, the fillers in 5-1) one-step purification and 5-2) two-step purification are replaced by the phenylhexyl (SILICAONE-10PH6-3) in Example 2:

[0304] Example 4 Method 1: C18 (Daiso);

[0305] Example 4 Method 2: C 18 (Kromasil);

[0306] Example 4 Method 3: C8 (Kromasil).

[0307] Example 5: Effect of filler type (three-step purification)

[0308] The present invention explores the influence of filler types in the purification process. Based on Example 3, the filler in the three-step purification is changed from Daisogel C 18 Replace with:

[0309] Example 5 Method 1: C8 (Kromasil);

[0310] Example 5 Method 2: SILICA ONE-10PH6-3.

[0311] Example 6: Effect of diluent type

[0312] The fractions of the cyclic purification process need to be diluted with glacial acetic acid as a diluent before loading, otherwise physical polymerization is likely to occur, resulting in a prolonged dissolution time of the final product. In order to explore the effects of different diluents, based on Example 3 "adding 10% of the original volume of glacial acetic acid before loading", the diluent glacial acetic acid was replaced with:

[0313] Example 6 Method 1: Methanol.

[0314] Example 6 Method 2: Acetonitrile.

[0315] Example 7: Effect of diluent dosage (three-step purification)

[0316] The fractions of the cyclic purification process need to be diluted with glacial acetic acid as a diluent before loading, otherwise physical polymerization is likely to occur, resulting in a prolonged dissolution time of the final product. The above Example 6 explores the effects of different types of diluents. The following section further explores the effects of different amounts of diluents. Therefore, based on Example 3 "adding 10% of the original volume of glacial acetic acid before loading", the amount of diluent glacial acetic acid is replaced by:

[0317] Embodiment 7 Method 1:

[0318] Before loading, add 5% of the original volume of glacial acetic acid;

[0319] Embodiment 7 Method 2:

[0320] Before loading, add 15% volume of glacial acetic acid.

[0321] Example 8: Effect of diluent dosage (two-step purification)

[0322] Based on Example 7, and based on Example 2 "adding 10% of the original volume of glacial acetic acid before loading the sample", the amount of the diluent glacial acetic acid is replaced by:

[0323] Embodiment 8 Method 1:

[0324] Before loading, add 5% of the original volume of glacial acetic acid;

[0325] Embodiment 8 Method 2:

[0326] Before loading, add 15% of the original volume of glacial acetic acid.

[0327] Comparative Example 1:

[0328] Only the one-step purification method in Example 2 was used to purify the crude YKYY017 obtained in Example 1.

[0329] Comparative Example 2:

[0330] The crude YKYY017 product was purified by referring to the purification method of the pure product disclosed in the fourth part of Example 1 in patent CN 114736272 A. The specific steps are:

[0331] Take the crude lipopeptide, add water / acetonitrile, stir and dissolve, remove insoluble matter by centrifugation and set aside. Purify by reverse phase high performance liquid chromatography.

[0332] Chromatographic column model: Agela C 18 (10μm, 50×250mm);

[0333] Mobile phase: Mobile phase A: 0.05% TFA and 2% acetonitrile in water; Mobile phase B: 90% acetonitrile / water solution;

[0334] Mobile phase flow rate: 25 mL / min;

[0335] UV detection wavelength: 220nm.

[0336] The crude product solution was loaded onto a chromatographic column for gradient elution, and the corresponding purified components were collected and directly freeze-dried to remove the solvent, thereby obtaining a pure trifluoroacetate polypeptide in a fluffy state.

[0337] The trifluoroacetate peptide pure product was redissolved with water and acetonitrile, and a large amount of anion exchange resin (acetate form) was added and stirred for 3 hours. After filtering and washing the ion exchange resin with a water / acetonitrile mixed solvent, the filtrate was combined and freeze-dried to obtain a fluffy peptide acetate pure product.

[0338] Purification product performance determination method:

[0339] 1. Determination of impurity content

[0340] The freeze-dried powder products prepared in the above examples were respectively measured according to high performance liquid chromatography (General Rules 0512 of Part IV of the Chinese Pharmacopoeia).

[0341] Calculated by the area normalization method, the content of a single impurity (single impurity) is ≤1.0%, and the total amount of impurities (total impurities) is ≤3.0%.

[0342] (1) Preparation of solution:

[0343] Blank solution (diluent): phosphoric acid-water (0.15:100)

[0344] Reference solution: Take 10 mg of YKYY017 reference solution (self-made, analyzed by ultraviolet (UV), infrared (IR), amino acid ratio, mass spectrometry (MS), MS2 peptide sequence analysis, nuclear magnetic resonance (NMR) 1 H-NMR), X-ray powder diffraction, circular dichroism (CD), fluorescence chromatography and other means to confirm the structure, and determine the purity to be 98.6%). Weigh accurately, place in a 20mL volumetric flask, add diluent to dissolve and dilute to the scale to make a solution containing about 0.5mg per 1mL as the reference solution.

[0345] Test solution: Take 10 mg of the product, weigh accurately, place in a 20 mL volumetric flask, add diluent to dissolve and dilute to scale, shake well to make a solution containing about 0.5 mg per 1 mL as the test solution. Prepare two in parallel.

[0346] (2) Chromatographic conditions:

[0347] Chromatographic column: Agilent ZORBAX 300SB-C 18 Column (4.6 mm × 150 mm, 3.5 μm) or chromatographic column with equivalent performance;

[0348] Mobile Phase:

[0349] Mobile phase A: 75 mmol / L potassium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid);

[0350] Mobile phase B: acetonitrile-water (9:1);

[0351] Flow rate: 1.0 mL / min;

[0352] Column temperature: 40°C;

[0353] Detection wavelength: 220nm.

[0354] Perform gradient elution according to the table below;

[0355] Table 21: Chromatographic conditions for impurity determination - elution gradient

[0356] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 10 45 55 45 20 80 46 75 25 55 75 25

[0357] (3) Determination method: Accurately measure 10 μL of blank solution, reference solution, and test solution respectively, inject them into the chromatograph, and record the chromatogram.

[0358] According to the chromatogram results, the content of each impurity such as adjacent front impurity, adjacent back impurity, and maximum single impurity is calculated by area normalization method; and the tailing factor and theoretical plate number are calculated.

[0359] Among them, the adjacent front impurity is the impurity peak adjacent to the main peak (YKYY017 peak) and located before the retention time of the main peak; the adjacent back impurity is the impurity peak adjacent to the main peak and located after the retention time of the main peak; the separation between the main peak and the adjacent front impurity and adjacent back impurity peaks should be greater than 1.0.

[0360] Counted by YKYY017 peak, the theoretical plate number is ≥5000; the tailing factor is ≤2.0.

[0361] Main peak purity: the percentage of the main peak area in the liquid chromatogram; Main peak purity standard: main peak purity (product purity) ≥ 97.0%.

[0362] Impurity content limit standard:

[0363] Adjacent front impurity ≤1.0%;

[0364] Adjacent impurities ≤ 1.0%;

[0365] Maximum single impurity ≤1.0%.

[0366] 2. Yield calculation

[0367] Yield (B) = YKYY017 batch yield / purified crude product nominal feed amount × 100%

[0368] (YKYY017 purification yield (B) limit standard ≥ 6.0%)

[0369] 3. Determination of product dissolution time

[0370] Take 0.05g of the YKYY017 product to be tested and dissolve it in 100mL of methanol. Shake it moderately and record the time taken for complete dissolution, which is the product dissolution time. Dissolution time limit standard: ≤3min.

[0371] Result Analysis 1:

[0372] By comparing and analyzing the above-mentioned Example 2 and Example 3, it was found that the two-step purification process and the three-step purification process had an important influence on the purification effect of YKYY017.

[0373] The details are as follows:

[0374] Table 22

[0375] Example Purification process Example 2 One-step purification + two-step purification Example 3 One-step purification + two-step purification + three-step purification

[0376] Table 23

[0377] parameter Limit Standard Example 2 Example 3 Yield (%) ≥6.0 9.3 8.9 Main peak purity (%) ≥97.0 97.55 98.63 Adjacent front impurity (%) ≤1.0 0.47 0.32 Adjacent impurities (%) ≤1.0 0.56 0.44 Maximum single impurity (%) ≤1.0 0.56 0.46 Tailing Factor ≤2.0 1.274 1.220 Theoretical plates ≥5000 16840 17442 Adsorption process changes / Column pressure is stable and elution is normal Column pressure is stable and elution is normal

[0378] From the above experimental data results, it can be seen that Example 2 (one-step purification + two-step purification) adopts a two-step purification process and Example 3 (one-step purification + two-step purification + three-step purification) adopts a three-step purification process, both of which can achieve good purification effects on the YKYY017 crude product.

[0379] The purification process of Example 2 and Example 3 has a yield as high as 8.9-9.3%, which exceeds the yield limit standard (≥6%) by more than 50%.

[0380] The main peak purity is 97.55-98.63%, significantly higher than the purity standard of 97.0%. Among them, the content of each impurity is significantly reduced, the adjacent front impurity is only 0.32-0.47%, the adjacent rear impurity is as low as 0.44-0.56%, and the maximum single impurity is controlled at 0.46-0.56%. The content of the above impurities is significantly lower than the impurity content standard ≤1.0%.

[0381] After purification, YKYY017 has a tailing factor of 1.220-1.274, which is significantly lower than the limit of ≤2.0, indicating that the separation between the peaks is good and significant.

[0382] During the purification process, the number of theoretical plates is as high as 16840-17442, which is significantly higher than the limit standard ≥5000, and the purification efficiency is high.

[0383] summary:

[0384] During the purification process, a two-step purification process (e.g., one-step purification + two-step purification in Example 2) and a three-step purification process (e.g., one-step purification + two-step purification + three-step purification in Example 3) were used to prepare YKYY017 products with high yield and purity, good separation effect and high purification efficiency.

[0385] a) In the purification process, a two-step purification process (e.g., one-step purification + two-step purification in Example 2) is adopted to prepare a YKYY017 product with high yield and purity, good separation effect and high purification efficiency, which is specifically manifested as follows:

[0386] The yield of the purified product was as high as 9.3%, and the purity was 97.55%. The contents of adjacent front impurities, adjacent rear impurities, and the largest single impurity were all controlled within 0.56%. Compared with the product obtained by only one-step purification (the effect of one-step purification is shown in Result Analysis 6), the purity was increased by more than 25%, and the maximum single impurity content was reduced by 1.75%. The purity of the preparation product was significantly improved.

[0387] The tailing factor was controlled at 1.274, significantly lower than the limit standard ≤2.0, and the separation effect was significant;

[0388] The number of theoretical plates in the purification process is as high as 16840, significantly higher than the limit standard ≥5000, and the purification efficiency is high;

[0389] The solubility was significantly improved. The dissolution time of YKYY017 in methanol was less than 3 minutes, and the solubility was excellent (for the solubility effect, see Result Analysis 4).

[0390] b) A three-step purification process (e.g., one-step purification + two-step purification + three-step purification in Example 3) was used to prepare the YKYY017 product with the best yield and purity, the best separation effect, and the highest purification efficiency, as shown in the following:

[0391] The yield of the purified product was as high as 8.9%, the purity was 98.63%, and the content of adjacent front impurities was as low as 0.32%, which was significantly better than the limit standard ≤1.0; compared with the one-step purification + two-step purification method, the purity of the product was increased by 1.08%, the content of adjacent front impurities was reduced by 0.15%, and the content of adjacent rear impurities was reduced by 0.12%; the purity of the preparation product was significantly improved;

[0392] The tailing factor is only 1.220, significantly lower than the limit standard ≤2.0, and the separation effect is optimal;

[0393] The number of theoretical plates in the purification process was as high as 17442, significantly higher than the limit standard ≥5000, and the purification efficiency was the highest;

[0394] The solubility is optimal, and the dissolution time of YKYY017 in methanol is controlled at <1 min. Compared with the two-step purification, the solubility is reduced to 1 / 3, and the reduction effect is significant (see Result Analysis 4 for the solubility effect).

[0395] Result Analysis 2:

[0396] In combination with the above-mentioned Example 2 and Example 4, the influence of the type of filler prepared in the one-step purification and two-step purification processes on the purification effect of YKYY017 was first studied.

[0397] The details are as follows:

[0398] Table 24

[0399] Example filler Example 2 Phenylhexyl (SILICAONE-10PH6-3) Example 4 Method 1 <![CDATA[C 18 (Daiso)]]> Example 4 Method 2 <![CDATA[C 18 (Cromasil)]]> Example 4 Method 3 <![CDATA[C8(Kromasil)]]>

[0400] Table 25

[0401]

[0402] Through the screening of prepared fillers in the above experiments, the purification effects of different types of fillers on the prepared products were investigated. The results showed that:

[0403] 1) Using phenylhexyl as the filler in the purification process (eg, Example 2), the main peak purity of the purified product was 97.55%, which was higher than the purity standard of 97.0%.

[0404] Among them, the content of each impurity is significantly reduced, and the adjacent front impurity, adjacent rear impurity and the maximum single impurity are controlled within the range of 0.47-0.56%, and the content of each impurity is significantly lower than the impurity content standard ≤1.0%.

[0405] After purification, YKYY017 has a tailing factor of 1.274, which is significantly lower than the limit of ≤2.0, indicating that the separation between the peaks is good and significant.

[0406] During the purification process, the number of theoretical plates reached 16840, significantly higher than the limit standard ≥5000, and the purification efficiency was high.

[0407] 2) On the contrary, other materials are used as fillers in the purification process (for example, C 18 and C8), the main peak purity of the purified product was only about 60-90%, significantly lower than the purity standard of 97.0%; the main peak purity was as low as 60.82%, which was about 37% lower than the effect of purification using phenylhexyl as the filler in the purification process, and it was impossible to prepare a YKYY017 product with qualified purity.

[0408] Among them, the content of each impurity increased significantly, with the adjacent front impurities, adjacent rear impurities and the maximum single impurity reaching about 1-11%, and the content of each single impurity was significantly higher than the impurity content standard of ≤1.0%; the content of adjacent rear impurities and the maximum single impurity reached up to 11.42%, which was more than 20 times higher than the impurity content of the product purified using phenylhexyl as the filler in the purification process.

[0409] From the chromatogram test results, it can be seen that the tailing factor of YKYY017 after purification shows an increasing trend compared with phenylhexyl as filler, increasing to about 1.4-3.4, and up to 3.412, which significantly exceeds the tailing factor limit standard (≤2.0) and has a poor separation effect.

[0410] The number of theoretical plates in the purification process is 9800-14300. Although the composite limit standard is ≥5000, the theoretical plate number is reduced by as much as 7040 compared with phenylhexyl as the filler in the purification process, and the purification efficiency is significantly reduced.

[0411] summary:

[0412] a) During the one-step purification and two-step purification, compared with other preparation filler types (e.g., Example 4C8 or C 18 ), using phenylhexyl (for example, Example 2) as the filler for preparative chromatography, the prepared YKYY017 product has high purity, few impurities, excellent separation effect, and high purification efficiency, which is specifically manifested as follows:

[0413] After purification, the product purity was increased to 97.55%, which was further higher than the purity limit standard;

[0414] The content of each impurity in the adjacent front impurity, adjacent back impurity and the largest single impurity is controlled within 0.47-0.56%, which is significantly lower than the impurity content limit standard of ≤1.0%;

[0415] The tailing factor was controlled at 1.274, significantly lower than the limit standard ≤2.0, and the separation effect was significant;

[0416] The number of theoretical plates in the purification process is as high as 16840, and the purification efficiency is high.

[0417] b) On the contrary, when other filler types (e.g., Example 4C8 or C 18 ), the purified product has low purity, high impurity content, poor separation effect and low purification efficiency; therefore, it cannot be used as a qualified filler in the process of purifying YKYY017, which is specifically manifested as follows:

[0418] After purification, the main peak purity of the product is only 60-90%, which cannot meet the purity standard of ≥97.0%, and it is impossible to purify a qualified preparation product;

[0419] The content of each impurity increased significantly, with the adjacent front impurity, adjacent back impurity and the largest single impurity reaching about 1-11%, which was significantly higher than the impurity content standard of ≤1.0%, and the impurity content of the purified product was unqualified;

[0420] The tailing factor increases to 1.435-3.412, and the separation effect is poor;

[0421] During the purification process, the number of theoretical plates decreased significantly, the column efficiency decreased, and the purification efficiency was significantly reduced.

[0422] Result Analysis 3:

[0423] In combination with the above-mentioned Example 3 and Example 5, the influence of the filler type of the chromatographic column prepared in the three-step purification process on the purification effect of YKYY017 was further studied.

[0424] Table 26

[0425] Example filler Example 3 <![CDATA[C 18 (Daisogel)]]> Example 5 Method 1 <![CDATA[C8(Kromasil)]]> Example 5 Method 2 Phenylhexyl (SILICAONE-10PH6-3)

[0426] Table 27

[0427]

[0428] Through the screening of fillers prepared in the three-step purification process in the above experiment, the purification effect of different types of fillers on the prepared product was investigated. The results showed that:

[0429] 1) In the three-step purification process, C 18 (For example, Example 3) When used as a filler type, the elution is normal, and the column pressure is stable without obvious increase, and its three-step purification effect is significantly better than other filler types.

[0430] Among them, the purity of the main peak is as high as 98.63%, which is significantly higher than the purity limit standard (≥97.0%);

[0431] The content of each impurity in the adjacent front impurity, adjacent rear impurity and the largest single impurity is controlled within the range of 0.32-0.46%, which is significantly lower than the impurity content limit standard (≤1.0%);

[0432] As can be seen from the chromatogram, the tailing factor is only 1.220, indicating an excellent separation effect;

[0433] During the purification process, the number of theoretical plates reached 17442, significantly higher than the limit standard ≥5000, and the purification efficiency was the highest.

[0434] 2) On the contrary, as the number of purification times increases, when a three-step purification process is performed and the filler type used is C8 (for example, method 1 in Example 5), the column pressure gradually increases, resulting in a significant decrease in the purity of the purified YKYY017 product, a significant increase in the impurity content, a deterioration in the separation degree, and a significant decrease in the purification efficiency.

[0435] Among them, the purity of the main peak was reduced to 90.82%, which was higher than that of C 18 When used as a filler, the purity was reduced by 7.81% and could not reach the purity limit standard (≥97.0%).

[0436] Moreover, the impurity contents of adjacent subsequent impurities and the largest single impurity both increased to 1.53%, significantly exceeding the impurity content limit standard (≤1.0%); therefore, using C8 as a filler, it is impossible to purify a qualified preparation product.

[0437] The number of theoretical plates in the purification process was 9175, which was higher than the limit of ≥5000, but compared with C 18 When used as a filler, the number of theoretical plates decreased by 47%, and the purification efficiency was significantly reduced.

[0438] 3) When the filler used is phenylhexyl (for example, method 2 in Example 5), the sample to be purified will be deadly adsorbed, which is not conducive to the purification of the sample and the purified product cannot be obtained.

[0439] summary:

[0440] a) In the three-step purification process, compared with other types of prepared fillers (e.g., C8 or phenylhexyl in Example 5), C18 (e.g., Example 3) was used as the filler for preparing the chromatographic column, and the prepared YKYY017 had the best purity, the least impurities, and the highest purification efficiency, as shown in the following:

[0441] The purity of the purified product was 98.63%, and the adjacent impurity content was as low as 0.32%, which was significantly better than the limit standard ≤1.0;

[0442] The tailing factor is only 1.220, significantly lower than the limit standard ≤2.0, and the separation effect is optimal;

[0443] The number of theoretical plates in the purification process was as high as 17442, significantly higher than the limit standard ≥5000, and the purification efficiency was the highest;

[0444] b) On the contrary, in the three-step purification process, other types of fillers (for example, Example 5C8 or phenylhexyl) are used to prepare the purified product, and the purity is poor and cannot be used as a qualified preparation product; and there is a phenomenon of low purification efficiency, and even dead adsorption cannot complete the purification, which is specifically manifested as follows:

[0445] The purity of the main peak was reduced to 90.82%, which failed to meet the purity limit standard (≥97.0%). Therefore, it was impossible to purify a qualified preparation product.

[0446] The impurity content of adjacent impurities and the largest single impurity increased to 1.53%, significantly higher than the impurity content limit standard (≤1.0%), and it was impossible to purify a qualified preparation product;

[0447] The number of theoretical plates during the purification process was 9175, and the purification efficiency was significantly reduced;

[0448] When other fillers are used, the sample is prone to dead adsorption and cannot be washed out, and no purified product can be obtained. Result Analysis 4:

[0449] In combination with the above-mentioned Example 2, Example 3 and Example 6, the types of diluents before loading the fractions in the cyclic purification process were investigated to study the effects of different diluents on the dissolution time of the product.

[0450] Table 28

[0451] Example Thinner Dissolution time Example 2 10% original volume glacial acetic acid <3min Example 3 10% original volume glacial acetic acid <1min Example 6 Method 1 10% original volume of methanol 9min Example 6 Method 2 10% original volume of acetonitrile 6min

[0452] Note: The above dissolution times are the time it takes for 0.05g YKYY017 to dissolve in 100mL methanol (with moderate shaking).

[0453] Through the above experiment, different types of diluents were used to investigate the fractions of the cyclic purification process before loading. The results showed that:

[0454] Before the fractions were subjected to the cyclic purification process, glacial acetic acid (e.g., Example 2 and Example 3) was used for dilution, and the dissolution time of the product obtained by the three-step purification in the methanol solvent was controlled to be <1 min, and the dissolution rate was optimal. The dissolution time of the product obtained by the two-step purification in the methanol solvent was within 3 min; since the product of the two-step purification was also partially present as the TFA salt of the polypeptide, and ammonium bicarbonate (in the mobile phase) was present in the system, the solubility of the two-step purification was slightly lower than that of the three-step purification, but still met the standard of ≤3 min.

[0455] On the contrary, when other types of diluents are used for dilution (for example, methanol in method 1 of Example 6, acetonitrile in method 2 of Example 6), under the same conditions, the dissolution time is significantly increased to more than 6 to 9 minutes; compared with the case where glacial acetic acid is used as the diluent, the dissolution time is increased by more than 2 to 9 times, and the dissolution rate is significantly reduced.

[0456] summary:

[0457] a) Before the fraction is subjected to cyclic purification and loaded, it is diluted with glacial acetic acid (for example, Example 2). The preparation product obtained by the two-step purification has good solubility, which is specifically manifested as follows:

[0458] The dissolution time of YKYY017 in methanol is less than 3 minutes, and its solubility is excellent;

[0459] b) Before the fraction is subjected to cyclic purification and loaded, it is diluted with glacial acetic acid (for example, Example 3). The solubility of the preparation product obtained by the three-step purification is further improved compared with the two-step purification, as shown in the following specific performance:

[0460] The dissolution time of YKYY017 in methanol is controlled at <1min, and the solubility is optimal;

[0461] c) On the contrary, when other types of substances are used as diluents (e.g., methanol in method 1 of Example 6, acetonitrile in method 2 of Example 6), the solubility of the preparation product is significantly reduced, as shown in the following specific manifestations:

[0462] Under the same conditions, using other types of solvents as diluents significantly increases the dissolution time to more than 6 to 9 minutes, greatly prolongs the dissolution time, and results in poor solubility of the product.

[0463] Result Analysis 5:

[0464] In combination with the above-mentioned Examples 3 and 7 as well as Examples 2 and 8, the amount of diluent used in the fractions of the cyclic purification process before loading was investigated to study the effect of different amounts of diluent on the dissolution time of the product.

[0465] Table 29

[0466] Example Diluent dosage Dissolution time Example 3 10% original volume glacial acetic acid <1min Example 7 Method 1 5% original volume glacial acetic acid <1min Example 7 Method 2 15% original volume glacial acetic acid <1min Example 2 10% original volume glacial acetic acid <3min Example 8 Method 1 5% original volume glacial acetic acid <3min Example 8 Method 2 15% original volume glacial acetic acid <3min

[0467] Note: The above dissolution times are the time it takes for 0.05g YKYY017 to dissolve in 100mL methanol (with moderate shaking).

[0468] Through the above experiment, different amounts of diluent were used to investigate the fractions of the cyclic purification process before loading. The results showed that:

[0469] In the two-step purification process, before the fraction is subjected to the cyclic purification process, 5-15% of the original volume of glacial acetic acid (for example, Example 2 and Example 8) is used for dilution, and the dissolution time of the purified product in methanol solvent is controlled to be ≤3min, and the solubility is excellent.

[0470] In the three-step purification process, before the fractions are subjected to the cyclic purification process, 5 to 15% of the original volume of glacial acetic acid (for example, Example 3 and Example 7) is used for dilution, and the dissolution time of the purified product in methanol solvent is controlled to be ≤ 1 min, and the solubility is the best.

[0471] Result Analysis 6:

[0472] In combination with the above-mentioned Example 2, Example 3 and Comparative Example 1 and Comparative Example 2, the purification effects of different purification methods were explored.

[0473] Table 30

[0474]

[0475] The effects of the preparations obtained by the above-mentioned different purification methods were investigated. Different purification methods affected the product purity and yield of YKYY017. The results showed that:

[0476] The purification process was carried out using the methods of Example 2 (one-step purification + two-step purification) and Example 3 (one-step purification + two-step purification + three-step purification) of the present invention, and the yield was about 9%, and the highest could reach 9.3%; the product purity was above 97.5%, and the highest was 98.63%; the maximum single impurity was controlled below 0.56%, and the minimum was only 0.45%.

[0477] Using the purification method of Comparative Example 1 which only undergoes a one-step purification process, the product purity is only about 70%, which is far below the limit standard of ≥97.0%; and the maximum impurity content is as high as 2.31%, which exceeds the limit standard (≤1.0%) by nearly 1.5 times, and a preparation product with qualified purity cannot be obtained.

[0478] The yield of Comparative Example 2, which is different from the purification method of the present invention, is only 6.5%, which is about 30% lower than that of the present invention, and the yield is significantly reduced. The purity of the product is only 95.32%, which cannot meet the purity limit standard of ≥97.0% of the present application; compared with the purification method of the present application, the purity also shows a trend of significant decrease; at the same time, the maximum single impurity shows a significant increase, and the maximum single impurity purity of the purified product of Comparative Example 1 is more than doubled compared with the purification method of the present application, and the impurity content increases significantly.

[0479] Moreover, Example 2, Example 3 and Comparative Example 1 are all purification processes in the process-scaled production stage, and Comparative Example 2 is only in the laboratory research stage; the expansion from the laboratory research stage to the production stage is better than the expansion of the production scale, which will cause the purity and yield of the product to show a significant downward trend.

[0480] On this basis, the product purity, yield and impurity content of Examples 2 and 3 are better than those of Comparative Example 2. Therefore, in the same production stage, the product purity, yield and other performance indicators of the present invention are more significantly improved than those of Comparative Example 2.

[0481] summary:

[0482] For different purification methods, the raw material product is prepared by the method of the present invention, and its product yield and purity are significantly improved compared with other methods; and the impurity content is significantly reduced.

Claims

1. A method for preparing a polypeptide, wherein: The method comprises the following steps: 1) Synthesis: preparation of peptides; 2) Purification: Purify the polypeptide solution by column chromatography and collect fractions; The fraction or its sample or mixture thereof enters the next purification process or circulates the current purification process; The purification process includes one-step purification and two-step purification; The polypeptide is YKYY017, and the polypeptide sequence of YKYY017 is: Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile-Asp-Arg-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu-Asn-Glu-Ser-L eu-Ile-Asp-Leu-Gln-Glu-Leu-Gly-Lys-Tyr-Glu-Gln-Tyr-Ile-Lys-Glu-Ala-Ala-Ala-Lys-Lys(Chol)-NH2; The filler type of the preparative chromatographic column for the one-step purification and the two-step purification is phenylhexyl filler; The fractions of the purification process, the samples thereof or the mixture of the samples are diluted with glacial acetic acid before loading; The amount of glacial acetic acid added is 5-15% of the original fraction volume; In the one-step purification, the fractions meeting the criteria in 1.1 are collected; Combine the fractions that meet the criteria of Section 1.1 into the sample of Section 1.1; In the two-step purification, the fractions meeting the criteria of step 1.1 or the combination of step 1.1 and step 1.1 are purified by column chromatography, and the fractions meeting the criteria of step 2.1 are collected; Combine the fractions that meet the criteria of Section 2.1 into the sample of Section 2.1; The one-step purification elution mobile phase is: mobile phase A1: 0.05% to 0.3% TFA aqueous solution; mobile phase B: acetonitrile; The two-step purification elution mobile phase is: mobile phase A2: 2mM-20mM ammonium bicarbonate solution; mobile phase B: acetonitrile.

2. The preparation method according to claim 1, wherein In step 1), the polypeptide is prepared by solid phase synthesis method.

3. The preparation method according to claim 1 or 2, wherein In the one-step purification, after the fractions meeting the criteria in 1.1 are collected, the remaining fractions or their pools are subjected to a cyclic one-step purification process; The remaining fractions are selected from the fractions meeting the criteria of 1.2 and / or the fractions meeting the criteria of 1.3; Combine the fractions that meet the criteria of Section 1.2 into the sample of Section 1.2; Combine the fractions that meet the criteria of Section 1.3 into the sample of Section 1.3; Combine the standard sample No. 1.2 and the standard sample No. 1.3 to form the remaining fraction and sample mixture of the first standard.

4. The preparation method according to claim 1 or 2, wherein: In the two-step purification, after the fractions meeting the criteria in 2.1 are collected, the remaining fractions or their combined samples are subjected to a cyclic two-step purification process; The remaining fractions are selected from the fractions meeting the criteria of 2.2 and / or the fractions meeting the criteria of 2.3; Combine the fractions that meet the criteria of Section 2.2 into the sample of Section 2.2; Combine the fractions that meet the criteria of Section 2.3 into the sample of Section 2.3; Combine the standard sample No. 2.2 and the standard sample No. 2.3 to form the remaining fraction and sample mixture of the second standard.

5. The preparation method according to claim 3, wherein: In the two-step purification, after the fractions meeting the criteria in 2.1 are collected, the remaining fractions or their combined samples are subjected to a cyclic two-step purification process; The remaining fractions are selected from the fractions meeting the criteria of 2.2 and / or the fractions meeting the criteria of 2.3; Combine the fractions that meet the criteria of Section 2.2 into the sample of Section 2.2; Combine the fractions that meet the criteria of Section 2.3 into the sample of Section 2.3; Combine the standard sample No. 2.2 and the standard sample No. 2.3 to form the remaining fraction and sample mixture of the second standard.

6. The preparation method according to claim 1 or 2, wherein: The purification process also includes three steps of purification; The type of preparative chromatographic filler for the three-step purification is C 18 filler; In the three-step purification, the fractions meeting the criteria of step 2.1 or the combination of step 2.1 and step 2.1 are purified by column chromatography, and the fractions meeting the criteria of step 3.1 are collected; Combine the fractions that meet the criteria of Section 3.1 into the sample of Section 3.1; The three-step purification elution mobile phases are: mobile phase A3: 0.5wt% to 5wt% acetic acid solution; mobile phase B: acetonitrile.

7. The preparation method according to claim 6, wherein: In the three-step purification, after the fractions meeting the criteria in Section 3.1 are collected, the remaining fractions or their pools are subjected to a three-step purification process in a cycle; The remaining fractions are selected from the fractions meeting the criteria of 3.2 and / or the fractions meeting the criteria of 3.3; Combine the fractions that meet the criteria of Section 3.2 into the sample of Section 3.2; Combine the fractions that meet the criteria of Section 3.3 into the sample of Section 3.3; Combine the standard sample No. 3.2 and the standard sample No. 3.3 to form the remaining fraction and sample mixture of the third standard.

8. The preparation method according to claim 1 or 2, wherein: In the one-step purification elution mobile phase, mobile phase A1 is 0.1% TFA aqueous solution.

9. The preparation method according to claim 3, wherein: In the one-step purification elution mobile phase, mobile phase A1 is 0.1% TFA aqueous solution.

10. The preparation method according to claim 4, wherein: In the one-step purification elution mobile phase, mobile phase A1 is 0.1% TFA aqueous solution.

11. The preparation method according to claim 5, wherein: In the one-step purification elution mobile phase, mobile phase A1 is 0.1% TFA aqueous solution.

12. The preparation method according to claim 6, wherein: In the one-step purification elution mobile phase, mobile phase A1 is 0.1% TFA aqueous solution.

13. The preparation method according to claim 7, wherein: In the one-step purification elution mobile phase, mobile phase A1 is 0.1% TFA aqueous solution.

14. The preparation method according to claim 1 or 2, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

15. The preparation method according to claim 3, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

16. The preparation method according to claim 4, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

17. The preparation method according to claim 5, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

18. The preparation method according to claim 6, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

19. The preparation method according to claim 7, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

20. The preparation method according to claim 8, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

21. The preparation method according to claim 9, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

22. The preparation method according to claim 10, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

23. The preparation method according to claim 11, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

24. The preparation method according to claim 12, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

25. The preparation method according to claim 13, wherein: In the two-step purification elution mobile phase, mobile phase A2: 10 mM ammonium bicarbonate solution.

26. The preparation method according to claim 6, wherein: In the three-step purification elution mobile phase, mobile phase A3: 2wt% acetic acid solution.

27. The preparation method according to claim 7, wherein: In the three-step purification elution mobile phase, mobile phase A3: 2wt% acetic acid solution.

28. The preparation method according to claim 12, wherein: In the three-step purification elution mobile phase, mobile phase A3: 2wt% acetic acid solution.

29. The preparation method according to claim 13, wherein: In the three-step purification elution mobile phase, mobile phase A3: 2wt% acetic acid solution.

30. The preparation method according to claim 18, wherein: In the three-step purification elution mobile phase, mobile phase A3: 2wt% acetic acid solution.

31. The preparation method according to claim 19, wherein In the three-step purification elution mobile phase, mobile phase A3: 2wt% acetic acid solution.

32. The preparation method according to claim 24, wherein: In the three-step purification elution mobile phase, mobile phase A3: 2wt% acetic acid solution.

33. The preparation method according to claim 25, wherein: In the three-step purification elution mobile phase, mobile phase A3: 2wt% acetic acid solution.

34. The preparation method according to claim 1 or 2, wherein: The method also includes freeze-drying the purified sample to obtain freeze-dried powder.

35. The preparation method according to claim 1 or 2, wherein: The polypeptide is used to treat novel coronavirus infection.

36. The preparation method according to claim 1 or 2, wherein: The synthesis method of the polypeptide is as follows: a) using a resin to sequentially couple all amino acids from the carbon end to the nitrogen end to synthesize a fully protected peptide in solid phase; b) selectively removing the side chain protecting group of Lys; c) coupling the side chain cholesterol succinate monoester in the presence of a coupling agent; d) In the presence of a cleavage agent, the peptide resin is cleaved and the side chain is deprotected to obtain the polypeptide.

37. The preparation method according to claim 36, wherein In step a), the resin is Rink Amide-MBHAResin.

38. The preparation method according to claim 36, wherein In step b), hydrazine is used as a catalyst to selectively remove the side chain protecting groups.

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