Method for purifying long chain polypeptide

A method for purifying a long chain polypeptide, comprising the following steps:

• 1) purification step:
• connecting an upstream chromatographic column and a downstream chromatographic column in series to separate a crude product;
• wherein
• a packing in the upstream chromatographic column and the downstream chromatographic column in step 1) is at least one selected from the group consisting of C18 silica gel packing, C8 silica gel packing, C4 silica gel packing, a polymer packing, and a packing with a same alkyl length and a different pore size;
• a length of the upstream chromatographic column is 8-20 cm;
• a length of the downstream chromatographic column is 8-20 cm;
• mobile phases in step 1):
• A1 phase is a buffered salt solution with a pH value of 2-3;
• and the buffered salt solution is at least one selected from the group consisting of ammonium sulfate, potassium dihydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate;
• B phase is a first organic phase, and the first organic phase is at least one selected from the group consisting of acetonitrile, methanol, isopropanol and ethanol;
• a molar concentration of the buffered salt solution is 20 mM-150 mM, and a detection wavelength of step 1) is 230 nm;
• step 1) comprises a first gradient elution:
• A1 phase %:
• 95%-55%, B phase %:
• 5%-45%, and an elution time is 30-120 min;
• 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 2) of a salt conversion:
• step 2):
• using the upstream chromatographic column in step 1) for the salt conversion, wherein, A2 phase is an acetic acid aqueous solution with a volume ratio of 0.05%-0.2%;
• B phase is a second organic phase, and the second organic phase is acetonitrile;
• and a detection wavelength of step 2) is 230 nm;
• step 2) comprises:
• loading the target peak product obtained in step 1) and rinsed the target peak product with 95% of the A2 phase and 5% of the B phase for 15-30 min for a desalination;
• then performing a second gradient elution for 10-30 min for the salt conversion to collect a target product;
• A2 phase %:
• 95/6-55%, B phase %:
• 5%-45%.