Patent Description:
Ularitide is a natriuretic peptide developed by Cardiorentis (AG) and composed of <NUM> amino acid residues. Ularitide was originally isolated from urine by Schulz-Knappe et al. in <NUM> as a renal natriuretic peptide belonging to the atrial natriuretic peptide (ANP) family, which is mainly used to treat acute heart failure.

The molecular formula of ularitide is as follows:
<CHM>.

Epidemiological data show that the number of heart failure patients worldwide has reached <NUM> million, and is still increasing at the rate of <NUM> million per year. And the <NUM>-year survival rate of heart failure patients is substantially equal to that of malignant tumor patients. <NUM>% of heart failure patients will be hospitalized again within <NUM> days after discharge, which adds medical and insurance expenses. The prevalence of heart failure in adults in China is <NUM>%, of which <NUM>% for men and <NUM>% for women. Currently, there are still about <NUM> million heart failure patients among adults aged <NUM>-<NUM> years old, and the number is increasing year by year.

Common causes include coronary heart disease, hypertension, cardiomyopathy and (or) valvular disease, diabetes, and others, among which coronary artery disease is an important factor in heart failure. According to statistics, the global annual expenditure on heart failure is <NUM> billion US dollars. According to a large-scale epidemiological survey in <NUM>, the prevalence rate of heart failure among adults in mainland China has reached <NUM>%, and there are about <NUM> million heart failure patients. Heart failure can be divided into acute heart failure and chronic heart failure. In the past <NUM> years, there have been <NUM> million cases of emergency treatment for acute heart failure in the United States, and about <NUM>-<NUM>% are first diagnosed with heart failure, while most are due to deterioration.

All diseases causing chronic heart failure can lead to acute heart failure. With the increasing number of chronic heart failure patients, chronic cardiac decompensation and acute heart failure episodes have also become the leading cause of hospitalization in patients with heart failure. The annual incidence rate of heart failure is <NUM>%-<NUM>%. The prognosis of acute heart failure is very poor. The in-hospital mortality rate is <NUM>%, the <NUM>-day mortality rate is <NUM>%, and the <NUM>-year and <NUM>-year mortality rates are as high as <NUM>% and <NUM>%, respectively. The mortality rate of acute heart failure caused by acute myocardial infarction is higher. The in-hospital mortality rate of patients with acute pulmonary edema is <NUM>%, and the one-year mortality rate is <NUM>%. Therefore, ularitide has a broad market prospect.

The existing peptide purification is mainly achieved by high performance liquid phase system, and the organic phase is acetonitrile, methanol, etc. The use amount of the organic phase is generally substantial, resulting in a large amount of waste liquid discharge. Waste liquid recovery is difficult and dangerous. With the extension of peptide sequence, the discharge amount of waste liquid will be larger, the purification cycle will be longer, and the enterprise cost will be higher. Environmental protection, safety and cost problems have restricted the development of pharmaceutical enterprises. A purification method to reduce the enterprise cost and discharge of waste liquid is urgently needed to minimize the risk of organic waste liquid storage.

<CIT> discloses a method for purifying sinapultide by a reversed-phase high-performance liquid chromatography in order to mainly solve the technical problems that more by-products are generated in synthesis of sinapopide and the purification is difficult. The method comprises the following steps: <NUM>) crude peptides obtained by solid phase synthesis are heated and dissolved with an aqueous solution of ethanol; <NUM>) two chromatographic columns in series are used, one stationary phase is a reverse-phase silica gel column of octalkyl-bonded silica gel, and the other stationary phase is a reverse-phase silica gel column of octadecyl-bonded silica gel; <NUM>) a mobile phase consisted of two phases, an ammonium sulfate-phosphate buffer is a phase A and chromatographically pure acetonitrile is a phase B, and a peptide solution with a peak of interest is collected by zigzag linear gradient elution and purification; <NUM>) the obtained peptide solution is concentrated by rotary evaporation under reduced pressure and a concentrate is prepared for use; <NUM>) the concentrate is converted into an acetate by an exchange of anion exchange resin; <NUM>) the ultimate high-purity peptide acetate solution is concentrated by rotary evaporation under reduced pressure again and freeze-dried to obtain a white powder finished product.

<CIT> discloses a method for preparing ularitide. The method includes the following steps that (<NUM>) full-protection linear peptide resin is synthesized; (<NUM>) splitting is performed to obtain linear peptide; (<NUM>) oxidation is conducted to obtain the ularitide; and (<NUM>) purification by reverse-phase high-performance liquid chromatography, salt rotation and freeze-drying are carried out to obtain the ularitide competitive product.

<CIT> discloses a purifying method of ularitide and the appropriate column separation conditions are further enlarged onto a dynamic axial compression industrial preparative chromatography system for separation for obtaining of high purity ularitide. The advantages of the method are that, good effect of separation and purification can be achieved by direct use of the dynamic axial compression industrial preparative chromatography for separation of an extracted crude product, the method has the advantages of simple operation, short period and high efficiency, and the purity can reach more than <NUM>%.

<CIT> discloses that high-purity cardiodilatin fragments, such as Ularitide can be prepared if the crude product is purified using a reversed-phase HPLC column, and the cardiodilatin fragment is eluted using a buffer system containing triethylammonium phosphate (TEAP) and acetonitrile in aqueous solution. Preferably, the pH value of the elution buffer is adjusted to a value of <NUM>-<NUM>, more specifically, of <NUM>-<NUM>. Elution of peptide is particularly advantageous if a continuous gradient of eluant is applied, <NPL>), discloses a method for purification of Ularitide by reverse-phase HPLC using C18 columns (3x25cm, <NUM> microm) with <NUM>% formic acid and water as mobile phase.

<CIT> refers to a method of purifying Liraglutide. The method comprises a two dimensional reversed phase high performance liquid chromatography protocol, wherein the first step is carried out at a pH value between <NUM> to <NUM> using a mobile phase comprising a phosphate buffer and acetonitrile, and the second step is carried out at a pH value below <NUM> using a mobile phase comprising trifluoroacetic acid and acetonitrile.

Ularitide is mainly used for acute heart failure, so its quality is particularly important. Ularitide has a long peptide sequence, and it has to undergo an oxidation step in the intermediate process, resulting in more impurities. In order to improve the safety of drugs, high purity is required for existing polypeptide drugs. Most drugs are required to have a purity greater than <NUM>%, and single impurity is also required to be controlled to be less than <NUM>%. The traditional purification process generally needs two steps of purification and one step of salt conversion to meet this standard. However, the yield is particularly low, and labor cost, environmental cost and product cost are high.

The purification of polypeptides is mainly carried out by reversed phase chromatography, and the stationary phases generally include C18, C8, C4, C1, etc. Additionally, polymer packings and even other reversed-phase packings may be used for purification, but the purification process remains essentially unchanged regardless of the type of packings used. There are two basic processes. One process is to fill a column with one kind of packings under a high pressure. The length of the column is generally about <NUM> and the particle size is mostly <NUM>. After purification under different chromatographic conditions, the unqualified parts are recovered and finally qualified products are obtained.

The other process is to fill a column with different types of packings under a high pressure. The length of the column is about <NUM> and the particle size is generally <NUM>. After purification under different chromatographic conditions, the unqualified parts are recovered and purified, and finally qualified products are obtained. For peptides with a short peptide sequence, the above two processes can be completed by one step of purification, while for peptides with a peptide sequence larger than <NUM> amino acids, two steps of purification are needed, along with desalination or salt conversion, it takes three steps to complete.

The disadvantages of the two processes are that purifying a large-scale product requires a substantial period of time, and the unqualified intermediates need to be recycled many times to get qualified products. Because of the need for recovery and purification, the cycle is prolonged, the amount of organic solvent used and the discharge of waste liquid increase, which adds to cost, compromises quality and increases the organic solvent risk coefficient.

The present invention provides a new purification method, as defined in the claims, which can improve the purity of a product, so that the purity of the product is more than <NUM>%, the single impurity content is less than <NUM>%, and the cost and environmental protection concerns can be greatly reduced.

The present invention provides a new purification method, as defined in the claims, which is different from the traditional purification method and addresses the disadvantages of the traditional purification method, such as high cost, long cycle and large discharge of waste liquid caused by multiple recovery, thus greatly improving the yield and being easy to increase production.

One aspect of the present invention provides a method for purifying a long chain polypeptide, including the following steps:.

In the technical solution of the present invention, in step <NUM>) the packing in the upstream chromatographic column is the C18 silica gel packing having a particle size of <NUM>, and the length of the upstream chromatographic column is <NUM>-<NUM>; and-the packing in the downstream chromatographic column is the C18 silica gel packing having a particle size of <NUM>, and the length of the downstream chromatographic column is <NUM>-<NUM>.

In the technical solution of the present invention, step <NUM>) comprises the first gradient elution: the A1 phase%: <NUM>%-<NUM>%, the B phase%: <NUM>%-<NUM>%, and the elution time is <NUM>-<NUM>.

In the technical solution of the present invention, the real-time dilution is as follows: before the target peak product enters the downstream chromatographic column, <NUM>% purified water is input through the chromatographic pump to reduce a ratio of the first organic phase.

In the technical solution of the present invention, the pH value of the A1 phase is <NUM>-<NUM>.

In the technical solution of the present invention, the A2 phase is an ammonium acetate solution with a volume ratio of <NUM> %-<NUM>%.

In the technical solution of the present invention, in step <NUM>), the desalination is performed with <NUM>% of the A2 phase and <NUM>% of the B phase for <NUM>-<NUM>.

In the technical solution of the present invention, in step <NUM>), the second gradient elution is performed for <NUM>-<NUM> for the salt conversion to collect the target product; the A2 phase%: <NUM>%-<NUM>%, the B phase%: <NUM>%-<NUM>%.

A new purification method, wherein two different types of chromatographic columns are arranged simultaneously. The first column is <NUM> C18, and the second column is <NUM> C18. The lengths of the chromatographic columns are both <NUM>-<NUM> considering the column pressure, column effect and the comprehensive cost of packings, and then the chromatographic columns are connected in series. For the purification of ularitide, the first column with larger particle size is mounted in front of the second column with smaller particle size. Then, an oxidized ularitide liquid is purified by the columns. After purification and salt conversion, a purified ularitide is obtained.

A new method for purifying ularitide, wherein two columns are respectively filled with two different types of packings, and then the two columns are connected in series. The method includes a first step of purification and a second step of salt conversion. In the first step, a buffered salt solution with a predetermined concentration and a predetermined pH is used as A1 phase, and acetonitrile is used as B phase. In the second step, acetic acid with a predetermined concentration is used as A2 phase and acetonitrile is used as B phase. The salt conversion is performed by high performance liquid chromatography (HPLC) with a gradient elution. A solution is collected and lyophilized to obtain ularitide acetate.

The peptide chain of ularitide is long, and there are numerous impurities in the synthesis, and it contains amino acids such as Ser which are easily isomerized during the synthesis, resulting in isomeric impurities in the crude peptides. By the new purification method of the present invention, two different types of packings are connected in series for the purification, and the two different types of packings have different separation capabilities. After the purification is performed in the first column, the target peak product does not flow out of the first column, and before entering the downstream chromatographic column, <NUM>% purified water is input by the third pump to reduce the ratio of the organic phase, and then the target peak product enters the second column for the second separation.

The traditional purification process generally needs two steps of purification, which can be completed by only one step of purification in the present invention. Furthermore, the purification method of the present invention reduces the risk of affecting product quality such as intermediate processing, precipitation and denaturation caused by intermediate storage, which can save time and effort.

The purification method of the present invention can separate and remove the isomeric impurities and other impurities that are difficult to separate in the crude peptides, and then uses the reversed-phase HPLC method to convert into acetate, and finally improves the yield and purity of the product.

Meanwhile, the present invention overcomes the shortcomings that traditional purification methods are time consuming, labor intensive and pollutant producing. The present invention provides a new method of purification that is easy to operate, which is beneficial to achieving large-scale preparation.

As an optimization, the molar concentration of the buffered salt in the mobile phase A1 of the HPLC method of the present invention is <NUM>-<NUM>, and the volume ratio of the acetic acid in the mobile phase A2 is <NUM>%-<NUM>%.

As an optimization, a range of the pH value of the mobile phase A1 of the HPLC method of the present invention is <NUM>-<NUM>.

As an optimization, the buffered salt is at least one selected from the group consisting of ammonium sulfate, potassium dihydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate.

As an optimization, the mobile phase B of the HPLC method is acetonitrile.

As an optimization, the stationary phases of the HPLC method are octadecyl, and the particle sizes are <NUM> and <NUM>.

Connecting columns in series for purification utilizes two kinds of packings with different separation capabilities to purify. Two separations are realized without changing column length, reducing the recovery times, shortening the cycle, and reducing the use amount in the organic phase. Moreover, the operation is simple and easily scalable. A major advantage over prior methods is time-saving and cost-saving, especially for polypeptides with a peptide chain length larger than <NUM> because such polypeptides require multiple steps of purification and recovery. The effect will be more prominent. The main reason is that the longer the peptide chain is, the more hydrophobic it is, the more organic phase is used when eluting, and coupled with multiple recovery, so the amount of waste liquid is particularly large.

Examples are as follows:
Purification is performed by chromatographic columns of the following specifications: <NUM> × <NUM> (column diameter × column length), <NUM> × <NUM>, <NUM> × <NUM>.

<NUM> of linear crude ularitide is dissolved and filtered, and a filtrate is collected for use.

Step <NUM>: mobile phases: A1 phase: the pH value of a potassium dihydrogen phosphate solution (<NUM> mmol/L) is adjusted to <NUM> with phosphoric acid; B phase: chromatographic grade acetonitrile; the flow rate is <NUM>-<NUM>/min, and the detection wavelength is <NUM>.

A linear crude ularitide solution is loaded and eluted for <NUM>-<NUM> with the following gradient: A1%: <NUM>%-<NUM>%, B%: <NUM>%-<NUM>%. In the elution process, the waste liquid is discarded if an impurity peak appears during the separation through the chromatographic column <NUM>. When a target product peak comes out, the target product is subjected to a real-time dilution by a third pump connected to a three-way mixer and then enters into the column <NUM> for a secondary separation.

The mobile phase of the real-time dilution is purified water, and the flow rate is <NUM>-<NUM>/min.

The target product obtained by a cyclic purification in step <NUM>, which meets the quality requirements, enters to step <NUM>.

Step <NUM>: mobile phases: A2 phase: a <NUM>-<NUM>% ammonium acetate solution, the pH value is <NUM>-<NUM>, B phase: chromatographic grade acetonitrile, the flow rate is <NUM>-<NUM>/min, and the detection wavelength is <NUM>.

After the column <NUM> is rinsed with a more than <NUM>% acetonitrile solution, the product obtained in step <NUM> is loaded and rinsed with <NUM>% A2 and <NUM>% B for <NUM>-<NUM> for a desalination. Then a gradient elution is performed for <NUM> for salt conversion to collect the target peak product, A2% is <NUM>%-<NUM>% and B% is <NUM>%-<NUM>%. A collected target peptide solution is rotary evaporated under reduced pressure in a water bath having a water temperature of no more than <NUM> and concentrated to about <NUM>-<NUM>/mL and then transferred to a suitable-sized vial. After freeze-drying, the qualified ularitide with a purity more than <NUM>% can be obtained.

<NUM> of white powder solid purified peptides is obtained after the freeze-drying. The purity is <NUM>%, and the single impurity is less than <NUM>%. The yield after purification is <NUM>% (calculated based on the content of ularitide in the crude product), and the total yield is <NUM>%. During the purification process, there is no need to recycle and purify the intermediate. According to the calculation, compared with examples <NUM>-<NUM>, the waste liquid discharge is reduced by approximately <NUM>% when purifying per unit mass of the crude ularitide due to the reduction of the cycle number in step <NUM>.

<NUM> of crude ularitide is dissolved and filtered, and a filtrate is collected for use.

The product obtained by a cyclic purification in step <NUM>, which meets the quality requirements, enters to step <NUM>.

After the column <NUM> is rinsed with a more than <NUM>% acetonitrile solution, the product obtained in step <NUM> is loaded and rinsed with <NUM>% A2 and <NUM>% B for <NUM>-<NUM> for a desalination. Then, a gradient elution is performed for <NUM> for salt conversion to collect the target peak product, A2% is <NUM>%-<NUM>% and B% is <NUM>%-<NUM>%. A collected target peptide solution is rotary evaporated under reduced pressure in a water bath having a water temperature of no more than <NUM> and concentrated to about <NUM>-<NUM>/mL and then transferred to a suitable-sized vial. After freeze-drying, the qualified ularitide with a purity more than <NUM>% can be obtained.

<NUM> of white powder solid purified peptides is obtained after the freeze-drying. The purity is <NUM>%, and the single impurity is less than <NUM>%. The yield after purification is <NUM>% (calculated based on the content of ularitide in the crude product), and the total yield is <NUM>%. According to the calculation, compared with Examples <NUM>-<NUM>, the waste liquid discharge is reduced by approximately <NUM>% when purifying per unit mass of the crude ularitide due to the reduction of the cycle number in step <NUM>.

Step <NUM>: mobile phases: A1 phase: the pH value of an ammonium sulfate solution (<NUM> mmol/L) is adjusted to <NUM> with phosphoric acid; B phase: chromatographic grade acetonitrile; the flow rate is <NUM>-<NUM>/min, and the detection wavelength is <NUM>.

<NUM> of white powder solid purified peptides is obtained after the freeze-drying. The purity is <NUM>%, and the single impurity is less than <NUM>%. The yield after purification is <NUM>% (calculated based on the content of ularitide in the crude product), and the total yield is <NUM>%. According to the calculation, compared with examples <NUM>-<NUM>, the waste liquid discharge is reduced by approximately <NUM>% when purifying per unit mass of the crude ularitide due to the reduction of the cycle number in step <NUM>.

A linear crude ularitide solution is loaded and eluted for <NUM>-<NUM> with the following gradient: A1%: <NUM>%-<NUM>%, B%: <NUM>%-<NUM>%.

<NUM> of white powder solid purified peptides is obtained after the freeze-drying. The purity is <NUM>%, and the single impurity is less than <NUM>%. The yield after purification is <NUM>% (calculated based on the content of ularitide in the crude product), and the total yield is <NUM>%. According to the calculation, when <NUM> of the crude peptides is purified, unqualified fractions need to be recovered and purified at least three times to reach the same result as that obtained in example <NUM>. After the production is enlarged, recovery times of the unqualified fractions increases by at least <NUM>%-<NUM>%, the amount of acetonitrile used increases by <NUM>%-<NUM>%, the amount of waste liquid discharge increases by about <NUM>%, and the cycle increases by <NUM>%.

<NUM> of white powder solid purified peptides is obtained after the freeze-drying. The purity is <NUM>%, and the single impurity is less than <NUM>%. The yield after purification is <NUM>% (calculated based on the content of ularitide in the crude product), and the total yield is <NUM>%. According to the calculation, the amount of acetonitrile used increases by <NUM>%, the amount of waste liquid discharge increases by about <NUM>%, and the cycle increases by <NUM>%. However, in the preparation process, the distillate is precipitated in the storage process, and the dissolution is difficult. Moreover, during preparation, the column pressure is high, which is close to the upper limit of the preparation system. <NUM> reversed-phase packing is not recommended for use and its cost is also high.

<NUM> of white powder solid purified peptides is obtained after the freeze-drying. The purity is <NUM>%, and the single impurity is less than <NUM>%. The yield after purification is <NUM>% (calculated based on the content of ularitide in the crude product), and the total yield is <NUM>%. According to the calculation, compared with example <NUM>, the waste liquid discharge is reduced by approximately <NUM>% when purifying per unit mass of the crude ularitide due to the reduction of the cycle in step <NUM> by about <NUM>%. When the particle size is large, the advantages of connecting columns with the same packing in series are not significant.

<NUM> of white powder solid purified peptides is obtained after the freeze-drying. The purity is <NUM>%, and the single impurity is less than <NUM>%. The yield after purification is <NUM>% (calculated based on the content of ularitide in the crude product), and the total yield is <NUM>%. According to the calculation, compared with Example <NUM>, using the same packing with small particle size, the removal effect of some impurities is better, while the removal effect of some other impurities is weaker. Overall, the waste liquid discharge is reduced by approximately <NUM>% when purifying per unit mass of the crude ularitide due to the reduction of the cycle number in step <NUM> by about <NUM>%. However, the cost of packings increases by <NUM>%, and overall, the advantages are not significant.

<NUM> of crude semaglutide is dissolved and filtered, and a filtrate is collected for use.

Step <NUM>: mobile phases: A1 phase: the pH value of an ammonium bicarbonate solution (<NUM> mmol/L) is adjusted to <NUM> with tetramethyl ammonium hydroxide; B phase: chromatographic grade acetonitrile: isopropanol = <NUM>:<NUM>; the flow rate is <NUM>-<NUM>/min, and the detection wavelength is <NUM>.

A crude semaglutide solution is loaded and eluted for <NUM>-<NUM> with the following gradient: A1%: <NUM>%-<NUM>%, B%: <NUM>%-<NUM>%. In the elution process, the waste liquid is discarded if an impurity peak appears during the separation through the chromatographic column <NUM>. When a target product peak comes out, the target product is subjected to a real-time dilution by a third pump connected to a three-way mixer and then enters into the column <NUM> for a secondary separation.

After the column <NUM> is rinsed with a more than <NUM>% acetonitrile solution, the product is loaded and rinsed with the <NUM>-<NUM>% ammonium acetate solution (pH <NUM>-<NUM>) containing <NUM>% acetonitrile for <NUM>-<NUM>. Then a gradient elution is performed for <NUM> to collect the target peak product, the gradient of acetonitrile: B% is <NUM>%-<NUM>%. A collected target peptide solution is rotary evaporated under reduced pressure in a water bath having a water temperature of no more than <NUM> and concentrated to about <NUM>-<NUM>/mL and then transferred to a suitable-sized vial. After freeze-drying, the qualified semaglutide with a purity more than <NUM>% can be obtained.

<NUM> of white powder solid purified peptides is obtained after the freeze-drying. The purity is <NUM>%, and the single impurity is less than <NUM>%. The yield after purification is <NUM>% (calculated based on the content of semaglutide in the crude product), and the total yield is <NUM>%. After being connected in series, the waste liquid discharge is reduced by <NUM>% and the cycle is reduced by <NUM>%.

<NUM> of crude liraglutide is dissolved and filtered, and a filtrate is collected for use.

Step <NUM>: mobile phases: A1 phase: the pH value of an ammonium bicarbonate solution (<NUM> mmol/L) is adjusted to <NUM> with ammonium hydroxide; B phase: chromatographic grade acetonitrile: isopropanol = <NUM>:<NUM>, the flow rate is <NUM>-<NUM>/min, and the detection wavelength is <NUM>.

A crude liraglutide solution is loaded and eluted for <NUM>-<NUM> with the following gradient: A1%: <NUM>%-<NUM>%, B%: <NUM>%-<NUM>%. In the elution process, the waste liquid is discarded if an impurity peak appears during the separation through the chromatographic column <NUM>. When a target product peak comes out, the target product is subjected to a real-time dilution by a third pump connected to a three-way mixer and then enters into the column <NUM> for a secondary separation.

After the column <NUM> is rinsed with a more than <NUM>% acetonitrile solution, the product is loaded and rinsed with the <NUM>-<NUM>% ammonium acetate solution containing <NUM>% acetonitrile for <NUM>-<NUM>. Then a gradient elution is performed for <NUM> to collect the target peak product, the gradient of acetonitrile: B% is <NUM>%-<NUM>%. A collected target peptide solution is rotary evaporated under reduced pressure in a water bath having a water temperature of no more than <NUM> and concentrated to about <NUM>/mL and then transferred to a suitable-sized vial. After freeze-drying, the qualified liraglutide with a purity more than <NUM>% can be obtained.

Claim 1:
A method for purifying a long chain polypeptide, comprising the following steps:
step <NUM>) purification step: connecting an upstream chromatographic column and a downstream chromatographic column in series to separate a crude product; wherein
the packing in the upstream chromatographic column and the downstream chromatographic column in step <NUM>) is selected from C18 silica gel packing; the length of the upstream chromatographic column is <NUM>-<NUM>; the length of the downstream chromatographic column is <NUM>-<NUM>;
mobile phases in step <NUM>): A1 phase is a buffered salt solution with a pH value of <NUM>-<NUM>; and the buffered salt solution is at least one selected from ammonium sulfate or potassium dihydrogen phosphate; B phase is a first organic phase, and the first organic phase is acetonitrile; a molar concentration of the buffered salt solution is <NUM>-<NUM>, and a detection wavelength of step <NUM>) is <NUM>;
step <NUM>) comprises a first gradient elution: A1 phase%: <NUM>%-<NUM>%, B phase%: <NUM>%-<NUM>%, and an elution time is <NUM>-<NUM>; in the first gradient elution, when an outflow peak of the upstream chromatographic column is an impurity peak, a corresponding mobile phase is discarded; when the outflow peak of the upstream chromatographic column is a target peak, a chromatographic pump connected to a three-way mixer arranged in a middle of the upstream chromatographic column and the downstream chromatographic column is opened, purified water is input to perform a real-time dilution, and then a target peak product enters into the downstream chromatographic column after the real-time dilution;
the method for purifying the long chain polypeptide further comprises step <NUM>) of a salt conversion:
step <NUM>): using the upstream chromatographic column in step <NUM>) for the salt conversion, wherein, A2 phase is an acetic acid aqueous solution with a volume ratio of <NUM>%-<NUM>%; B phase is a second organic phase, and the second organic phase is acetonitrile; and a detection wavelength of step <NUM>) is <NUM>;
step <NUM>) comprises: loading the target peak product obtained in step <NUM>) and rinsed the target peak product with <NUM>% of the A2 phase and <NUM>% of the B phase for <NUM>-<NUM> for a desalination;
then performing a second gradient elution for <NUM>-<NUM> for the salt conversion to collect a target product; A2 phase%: <NUM>%-<NUM>%, B phase%: <NUM>%-<NUM>%,
wherein the long chain polypeptide is ularitide.