WO2024119609A1 - Polypeptide derivative and preparation method therefor - Google Patents

Polypeptide derivative and preparation method therefor Download PDF

Info

Publication number
WO2024119609A1
WO2024119609A1 PCT/CN2023/075022 CN2023075022W WO2024119609A1 WO 2024119609 A1 WO2024119609 A1 WO 2024119609A1 CN 2023075022 W CN2023075022 W CN 2023075022W WO 2024119609 A1 WO2024119609 A1 WO 2024119609A1
Authority
WO
WIPO (PCT)
Prior art keywords
solution
acid
hours
preparation
polypeptide derivative
Prior art date
Application number
PCT/CN2023/075022
Other languages
French (fr)
Chinese (zh)
Inventor
吴玲
李恒
刘悦玲
李岩
潘海
Original Assignee
杭州先为达生物科技股份有限公司
北京先为达生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 杭州先为达生物科技股份有限公司, 北京先为达生物科技有限公司 filed Critical 杭州先为达生物科技股份有限公司
Publication of WO2024119609A1 publication Critical patent/WO2024119609A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present application belongs to the field of polypeptide technology. Specifically, the present application relates to polypeptide derivatives and preparation methods thereof. In addition, the present application also relates to drugs containing the polypeptide derivatives and uses thereof in preparing drugs.
  • US 6184309 discloses a method for removing protecting groups from polymers using acid catalysis, which indicates that many strong and weak organic and inorganic acids can be used as deprotection agents.
  • US 2003/0212249 reports the synthesis of cyclosporin analogs, in which the trimethylsilyl protecting group can be removed with acetic acid or citric acid.
  • US 5135683 relates to the preparation of deprotected polyhydroxy compounds, which indicates that phosphoric acid can be used as a reagent for removing cyclic ketal protecting groups.
  • an octadecane fatty acid containing a protective group is coupled to a polypeptide (such as GLP-1) via an amide bond.
  • a deprotection operation is required under a strong acid environment.
  • the conditions for evaluating the success of the deprotection operation are mainly The concentration of protection and the content of impurities in the deprotected product. During the deprotection process, the concentration of the deprotected protein is greatly limited.
  • a method for preparing a polypeptide derivative comprising:
  • polypeptide derivative intermediate wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group
  • the intermediate of the polypeptide derivative after freeze-drying is deprotected to obtain the polypeptide derivative.
  • the protecting group is selected from any one of C1-C21 alkyl and aryl groups.
  • the protecting group is methyl, ethyl, tert-butyl or benzyl.
  • the carboxylic acid ester is a fatty acid ester.
  • the fatty acid is one or more of octanoic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanedioic acid, palmitic acid, heptadecanedioic acid, stearic acid, nonadecanedioic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosapentaenoic acid or docosahexaenoic acid, 1,8-octanedioic acid, 1,7-heptanedicarboxylic acid, 1,10-decanedioic acid, undecanedioic acid, dodecane
  • the freezing treatment comprises the following steps:
  • An optional secondary drying process may further be included.
  • the pre-freezing temperature of the pre-freezing treatment is 2 to 8° C., and the pre-freezing time is greater than or equal to 2.0 hours; or
  • the freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing treatment time is more than 3 hours; or
  • the temperature of the primary drying process is -10°C to 0°C, the vacuum degree of the primary drying process is 0.05 to 2.0 mbar, and the drying time of the primary drying process is greater than or equal to 20 hours;
  • the temperature of the secondary drying process is 20° C. to 65° C.
  • the vacuum degree of the secondary drying process is 0.05 to 2.0 mbar
  • the drying time of the secondary drying process is greater than 0 hours and less than 8 hours.
  • the polypeptide is selected from one or more of insulin, glucagon, incretin, GLP-1, parathyroid hormone (PTH), GLP-2, oxyntomodulin, amylin, enteroglucagon, somatostatin, leptin, YY peptide, desmopressin, osteocalcin, human growth hormone, glycopeptide antibiotics, non-ribosomal peptide antibiotics, corticotropin, calcitonin, oxytocin, interferon, fibrinolytic, antidiuretic hormone, interleukin, urokinase, thyrotropin-releasing hormone, methionine enkephalin, leucine enkephalin, prostaglandin F2 ⁇ receptor modulator, obestatin, secretin, nesfatin, glucose-dependent insulinotropic peptide (GIP) and analogs or fragments of the above polypeptides.
  • GIP glucose-dependent insulinotropic peptide
  • the polypeptide is selected from GLP-1, a pharmaceutically acceptable salt of GLP-1, a GLP-1 analog, and a pharmaceutically acceptable salt of a GLP-1 analog.
  • the intermediate of the polypeptide derivative after freeze-drying is deprotected under the condition of an acid catalyst solution to obtain a deprotection reaction solution;
  • the deprotection reaction solution is added to a stop solution, and the pH value is adjusted to stop the reaction to obtain a polypeptide derivative.
  • the pH value is adjusted to 6.5-10.0 to terminate the reaction and obtain a polypeptide derivative.
  • the pH value is adjusted to 7.5-9.5 to terminate the reaction and obtain a polypeptide derivative.
  • the stop solution comprises a buffered salt solution and an alkaline solution.
  • the alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, and ammonium bicarbonate solution.
  • the buffer salt solution is selected from one or more of a phosphate buffer solution, a Tris buffer solution, an organic acid buffer solution, a borate buffer solution, and an amino acid buffer solution.
  • the buffered saline solution is selected from one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution (PBS), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate- NaOH buffer solution, sodium barbital-HCl buffer solution, NH4HCO3 buffer solution, sodium carbonate-sodium bicarbonate buffer solution, NaHCO3 buffer solution, Tris-HCl, glycine-NaOH buffer solution, boric acid- borax buffer solution, and Na2B7O4 buffer solution.
  • PBS phosphate buffer solution
  • PB disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution
  • the alkaline solution is a sodium hydroxide solution.
  • the concentration of the buffer salt in the buffer salt solution is 0.001M to 0.6M, preferably 0.01M to 0.6M, and more preferably 0.01M to 0.5M.
  • the concentration of the alkaline solution is 2-10M, preferably 3-8M, and more preferably 4-6M.
  • the volume ratio of the buffered salt solution to the alkaline solution is (0.1-100):1, preferably (0.1-70):1, and more preferably (1-50):1.
  • the acid catalyst is selected from p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
  • One or more acids are selected from p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
  • One or more acids are selected from p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
  • One or more acids are selected from p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
  • polypeptide derivative intermediate wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group
  • the intermediate of the polypeptide derivative is deprotected under acid catalyst solution conditions to obtain a deprotection reaction solution
  • the deprotection reaction solution is added to a stop solution, and the pH value is adjusted to stop the reaction to obtain a polypeptide derivative.
  • the pH value is adjusted to 6.5-10.0 to terminate the reaction and obtain a polypeptide derivative.
  • the pH value is adjusted to 7.5-9.5 to terminate the reaction and obtain a polypeptide derivative.
  • the stop solution comprises a buffered salt solution and an alkaline solution.
  • the alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, and ammonium bicarbonate solution.
  • the buffer salt solution is selected from one or more of a phosphate buffer solution, a Tris buffer solution, an organic acid buffer solution, a borate buffer solution, and an amino acid buffer solution.
  • the buffered saline solution is selected from one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution (PBS), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate- NaOH buffer solution, sodium barbital-HCl buffer solution, NH4HCO3 buffer solution, sodium carbonate-sodium bicarbonate buffer solution, NaHCO3 buffer solution, Tris-HCl, glycine-NaOH buffer solution, boric acid- borax buffer solution, and Na2B7O4 buffer solution.
  • PBS phosphate buffer solution
  • PB disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution
  • the alkaline solution is a sodium hydroxide solution.
  • the concentration of the buffer salt in the buffer salt solution is 0.001M to 0.6M, preferably 0.01M to 0.6M, and more preferably 0.01M to 0.5M.
  • the concentration of the alkaline solution is 2-10M, preferably 3-8M, and more preferably 4-6M.
  • the volume ratio of the buffered salt solution to the alkaline solution is (0.1-100):1, preferably (0.1-70):1, and more preferably (1-50):1.
  • the acid catalyst is selected from one or more of p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
  • the intermediate of the polypeptide derivative after freeze-drying is deprotected to obtain the polypeptide derivative.
  • the protecting group is selected from any one of a C1-C21 alkyl group and an aryl group.
  • the protecting group is methyl, ethyl, tert-butyl or benzyl.
  • the carboxylic acid ester is a fatty acid ester.
  • the fatty acid is one or more of octanoic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanedioic acid, palmitic acid, heptadecanedioic acid, stearic acid, nonadecanedioic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosapentaenoic acid or docosahexaenoic acid, 1,8-octanedioic acid, 1,7-heptanedicarboxylic acid, 1,10-decanedioic acid, undecanedioic acid, dodecane
  • the freezing treatment comprises the following steps:
  • An optional secondary drying process may further be included.
  • the pre-freezing temperature of the pre-freezing treatment is 2 to 8° C., and the pre-freezing time is greater than or equal to 2.0 hours; or
  • the freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing treatment time is more than 3 hours; or
  • the temperature of the primary drying process is -10°C to 0°C, the vacuum degree of the primary drying process is 0.05-2.0 mbar, and the drying time of the primary drying process is greater than or equal to 20 hours;
  • the temperature of the secondary drying treatment is 20° C. to 65° C.
  • the vacuum degree of the secondary drying treatment is 0.05-2.0 mbar
  • the drying time of the secondary drying treatment is greater than 0 hours and less than 8 hours.
  • the polypeptide is selected from one or more of insulin, glucagon, incretin, GLP-1, parathyroid hormone (PTH), GLP-2, oxyntomodulin, amylin, enteroglucagon, somatostatin, leptin, YY peptide, desmopressin, osteocalcin, human growth hormone, glycopeptide antibiotics, non-ribosomal peptide antibiotics, corticotropin, calcitonin, oxytocin, interferon, fibrinolytic, antidiuretic hormone, interleukin, urokinase, thyrotropin-releasing hormone, methionine enkephalin, leucine enkephalin, prostaglandin F2 ⁇ receptor modulator, obestatin, secretin, nesfatin, glucose-dependent insulinotropic peptide (GIP) and analogs or fragments of the above polypeptides.
  • GIP glucose-dependent insulinotropic peptide
  • the polypeptide is selected from GLP-1, a pharmaceutically acceptable salt of GLP-1, a GLP-1 analog, and a pharmaceutically acceptable salt of a GLP-1 analog.
  • a method for preparing a polypeptide derivative comprising:
  • polypeptide derivative intermediate wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group; subjecting the polypeptide derivative intermediate to freeze-thaw treatment to obtain a polypeptide derivative intermediate after freeze-thaw treatment;
  • the intermediate of the polypeptide derivative after the freeze-dried tube is deprotected under the condition of an acid catalyst solution to obtain a deprotection reaction solution;
  • the deprotection reaction solution is added to the stop solution, and the pH value is adjusted to stop the reaction to obtain a plurality of Peptide derivatives.
  • the protecting group is selected from any one of C1-C21 alkyl and aryl groups.
  • the protecting group is methyl, ethyl, tert-butyl or benzyl.
  • the carboxylic acid ester is a fatty acid ester.
  • the fatty acid is one or more of octanoic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanedioic acid, palmitic acid, heptadecanedioic acid, stearic acid, nonadecanedioic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosapentaenoic acid or docosahexaenoic acid, 1,8-octanedioic acid, 1,7-heptanedicarboxylic acid, 1,10-decanedioic acid, undecanedioic acid, dodecane
  • the freezing treatment comprises the following steps:
  • An optional secondary drying process may further be included.
  • the pre-freezing temperature of the pre-freezing treatment is 2 to 8° C., and the pre-freezing time is greater than or equal to 2.0 hours; or
  • the freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing treatment time is more than 3 hours; or
  • the temperature of the primary drying process is -10°C to 0°C, the vacuum degree of the primary drying process is 0.05 to 2.0 mbar, and the drying time of the primary drying process is greater than or equal to 20 hours;
  • the temperature of the secondary drying process is 20° C. to 65° C.
  • the vacuum degree of the secondary drying process is 0.05 to 2.0 mbar
  • the drying time of the secondary drying process is greater than 0 hours and less than 8 hours.
  • the polypeptide is selected from insulin, glucagon, incretin, GLP-1, parathyroid hormone (PTH), GLP-2, oxyntomodulin, amylin, enteroglucagon, somatostatin, leptin, YY peptide, desmopressin, osteocalcin, human growth hormone, glycopeptide antibiotics, non-ribosomal peptide antibiotics, corticotropin, calcitonin, oxytocin, interferon, fibrinolytic agent, antidiuretic hormone, interleukin, urokinase, thyrotropin-releasing hormone, methionine enkephalin, leucine enkephalin, prostaglandin F2 ⁇ receptor modulator, obestatin, secretin, nesfatin, glucose-dependent insulinotropic peptide (GIP) and one or more of the analogs or fragments of the above polypeptides.
  • GIP glucose-dependent insulinotropic peptide
  • the polypeptide is selected from GLP-1, a pharmaceutically acceptable salt of GLP-1, a GLP-1 analog, and a pharmaceutically acceptable salt of a GLP-1 analog.
  • the pH value is adjusted to 6.5-10.0 to terminate the reaction and obtain a polypeptide derivative.
  • the pH value is adjusted to 7.5-9.5 to terminate the reaction and obtain a polypeptide derivative.
  • the stop solution comprises a buffered salt solution and an alkaline solution.
  • the alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, and ammonium bicarbonate solution.
  • the buffer salt solution is selected from one or more of a phosphate buffer solution, a Tris buffer solution, an organic acid buffer solution, a borate buffer solution, and an amino acid buffer solution.
  • the buffered saline solution is selected from one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution (PBS), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate- NaOH buffer solution, sodium barbital-HCl buffer solution, NH4HCO3 buffer solution, sodium carbonate-sodium bicarbonate buffer solution, NaHCO3 buffer solution, Tris-HCl, glycine-NaOH buffer solution, boric acid- borax buffer solution, and Na2B7O4 buffer solution.
  • PBS phosphate buffer solution
  • PB disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution
  • the alkaline solution is a sodium hydroxide solution.
  • the concentration of the buffer salt in the buffer salt solution is 0.001M to 0.6M, preferably 0.01M to 0.6M, and more preferably 0.01M to 0.5M.
  • the concentration of the alkaline solution is 2-10M, preferably 3-8M, and more preferably 4-6M.
  • the volume ratio of the buffered salt solution to the alkaline solution is (0.1-100):1, preferably (0.1-70):1, and more preferably (1-50):1.
  • the acid catalyst is selected from one or more of p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
  • a polypeptide derivative, wherein the polypeptide derivative is prepared by the method described in any one of items 1 to 64.
  • the present application provides a new deprotection process for polypeptide derivatives.
  • the upper limit of the deprotection protein concentration is effectively increased, greatly improving the benefits and efficiency of the deprotection process in protein production;
  • the conventional addition of the deprotection solution to the deprotection reaction solution is adjusted to the addition of the reaction solution to the deprotection solution, which greatly reduces the isomer impurity content in the deprotection process and improves the stability of the operation method.
  • the deprotection effect and stability are further improved.
  • the technical bottleneck in the deprotection process of polypeptide molecules is overcome, and it has obvious practicality for industrial large-scale production.
  • polypeptide or “peptide” or “protein” may be used interchangeably herein.
  • a “polypeptide” or “peptide” or “protein” is any chain of two or more amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), including naturally occurring or non-naturally occurring amino acids or Amino acid analogs, wherein the amino acids in any chain are covalently linked by peptide bonds.
  • they can be naturally occurring proteins, or proteins produced by non-recombinant cells or genetically engineered or recombinant cells, and include molecules having the amino acid sequence of a natural protein, or molecules having one or more amino acids deleted, added and/or substituted from the natural sequence.
  • polypeptide or “peptide” or “protein” of the present application may include abnormal connections, crosslinks and end caps, non-peptide bonds or other modifying groups. These modifying groups are also within the scope of the present application.
  • modifying group refers to structures directly attached to the peptide structure, as well as those indirectly attached to the peptide structure.
  • the modifying group can be coupled to the amino terminal or carboxyl terminal of the peptide structure, or coupled to the peptide flanking the core domain.
  • the modifying group can be connected to the side chain of at least one amino acid residue of the peptide structure, or coupled to the peptide or peptide-mimicking region flanking the core domain (for example, through the epsilon amino group of a lysinyl residue, through the carboxyl group of an aspartic acid residue or a glutamic acid residue, through the hydroxyl group of a tyrosinyl residue, a serine residue or a threonine residue, or other suitable reactive groups on the amino acid side chain).
  • the modifying group covalently coupled to the peptide structure can be combined with a connecting chemical structure by means and methods well known in the art, including, for example, amide, alkylamino, carbamate or urea bonds.
  • the polypeptide is selected from insulin, glucagon, incretin, GLP-1, parathyroid hormone (PTH), GLP-2, oxyntomodulin, amylin, enteroglucagon, somatostatin, leptin, YY peptide, desmopressin, osteocalcin, human growth hormone, glycopeptide antibiotics, non-ribosomal peptide antibiotics, corticotropin, calcitonin, oxytocin, interferon, fibrinolytic, antidiuretic hormone, interleukin, urokinase, thyrotropin-releasing hormone, methionine enkephalin, leucine enkephalin, prostaglandin F2 ⁇ receptor modulator, obestatin, secretin, nesfatin, glucose-dependent insulinotropic peptide (GIP) and one or more of the analogs or fragments of the above polypeptides.
  • PTH parathyroid hormone
  • GLP-2 oxyntomodulin
  • the polypeptide is selected from GLP-1, a pharmaceutically acceptable salt of GLP-1, a GLP-1 analog, and a pharmaceutically acceptable salt of a GLP-1 analog.
  • polypeptide derivatives refer to products obtained by modifying the functional groups (such as amino acid residues) of polypeptides by certain compounds (such as small molecule compounds). The modifications include but are not limited to acylation, amidation, esterification, and thioesterification.
  • the polypeptide derivative intermediate is a polypeptide modified with a carboxylate containing a protecting group.
  • the carboxyl group in the polypeptide derivative needs to be protected, and the position of the carboxyl group is not limited.
  • the carboxyl group at this time can be a carboxyl group at any position in the polypeptide derivative molecule, for example, the carboxyl group can be a carboxylic acid derived from the amino acid backbone in the polypeptide derivative molecule, or a carboxylic acid derived from the side chain in the polypeptide derivative molecule.
  • the type of the carboxyl protecting group used is not important, as long as the derived carboxylic acid protecting group (such as a carboxylate containing a protecting group) is stable to the subsequent reaction conditions and can be removed at an appropriate time without destroying the rest of the molecule.
  • carboxylate refers to -C(O)OR, wherein R is alkyl, aryl, aralkyl and alicyclic, and all carboxylates may be optionally substituted.
  • alkyl represents a divalent alkyl group or an alkylene group
  • aryl represents an aryl having 5-14 ring atoms and at least one ring having a conjugated ⁇ electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl, all of which groups may be optionally substituted, and carbocyclic aryl is a group in which the ring atoms on the aromatic ring are carbon atoms.
  • Carbocyclic aryl includes monocyclic carbocyclic aryl and polycyclic or fused compounds, such as optionally substituted naphthyl.
  • Heterocyclic aryl or heteroaryl is a group having 1-4 heteroatoms as the ring atoms of the aromatic ring and the remaining ring atoms are carbon atoms.
  • Suitable heteroatoms include oxygen, sulfur, nitrogen and selenium.
  • Suitable heteroaryl includes furanyl, thienyl, pyridyl, pyrrolyl, N-low alkyl pyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl, imidazolyl, etc., and all heteroaryl may be optionally substituted.
  • biaryl refers to an aryl group containing more than one aromatic ring, including fused ring systems and aryl groups substituted with other aryl groups, such groups being optionally substituted.
  • alicyclic refers to a compound that has both aliphatic and cyclic properties.
  • Such cyclic compounds include (but are not limited to) aromatic, cycloalkyl and bridged cycloalkyl compounds.
  • Cyclic compounds include heterocycles. Such groups may be optionally substituted.
  • polypeptide analogs refer to molecules that are substantially similar in function to a natural peptide or protein or a fragment thereof.
  • a GLP-1 analog refers to one or more that will be selected to maintain the in vitro or in vivo activity of the natural substrate. In vitro and in vivo activity can be measured using any protocol that is suitable for GLP-1 analogs and available to those of ordinary skill in the art.
  • Exemplary functional activities that can be measured to determine whether a GLP-1 analog maintains the same or similar functional activity include the ability of the analog to bind to its receptor(s) in a cell-based assay or a cell-free assay, the analog to induce changes in cells that respond to the GLP-1R (e.g., proliferation, differentiation, survival, growth, migration, etc.), the ability of an analog to modulate the expression of one or more other genes or proteins in a cell that are responsive to GLP-1.
  • GLP-1 analogs have substantially similar activity to native GLP-1 or a fragment thereof (e.g., activity is about 80%, 90%, 100%, 110% or 120% of native GLP-1). In some embodiments, the analog is less active than native GLP-1 (e.g., activity is about 50%, 60%, 70% or 75% of the native polypeptide).
  • analogs with slightly less activity may be useful, for example in vivo or in cell culture, if the reduced activity still provides the ability to provide sufficient local concentrations of the analog for a sufficient period of time. Thus, an increase in half-life, such as through protease resistance, may compensate for reduced activity resulting from the construction of the analog.
  • the analog is more active than native GLP-1 (e.g., activity is about 130%, 150%, 175%, 200%, 300%, 500%, 800% or even 1000% of native GLP-1).
  • activity refers to one or more functions of native GLP-1.
  • the activity (e.g., biological function) of an analog can be receptor binding, the ability to act as a transcriptional activator or repressor, the ability to participate in a particular signal transduction pathway, or the ability to affect cell behavior (e.g., proliferation, differentiation, survival, or migration).
  • the polypeptide derivative is coupled to the polypeptide via a fatty acid in the form of an amide bond.
  • the fatty acid is a C1 to C25 fatty acid, such as formic acid, acetic acid, C3 fatty acid, C4 fatty acid, C5 fatty acid, C6 fatty acid, C7 fatty acid, C8 fatty acid, C9 fatty acid, C10 fatty acid, C11 fatty acid, C12 fatty acid, C13 fatty acid, C14 fatty acid, C15 fatty acid, C16 fatty acid, C17 fatty acid, C18 fatty acid, C19 fatty acid, C20 fatty acid, C21 fatty acid, C22 fatty acid, C23 fatty acid, C24 fatty acid or C25 fatty acid.
  • C1 to C25 fatty acid such as formic acid, acetic acid, C3 fatty acid, C4 fatty acid, C5 fatty acid, C6 fatty acid, C7 fatty acid, C8 fatty acid, C9 fatty acid, C10 fatty acid, C11 fatty acid, C12 fatty acid, C13 fatty acid, C14
  • the C1-C21 fatty acids include straight-chain fatty acids and branched-chain fatty acids.
  • the C1-C21 fatty acids include saturated fatty acids and unsaturated fatty acids.
  • the fatty acid is one of caprylic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanedioic acid, palmitic acid, heptadecanedioic acid, stearic acid, nonadecanedioic acid, arachidic acid, heneicosanoic acid, hexadecanoic acid, hexadecanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosapentaenoic acid or docosahexaenoic acid, 1,8-octanedioic acid, 1,7-heptanedicarboxylic acid, 1,10-decanedioic acid, undecanoic acid, dodecanedioic acid, tridecanoic acid, myristic acid
  • the term "pharmaceutically acceptable salt” refers to a salt that retains the biological effectiveness and properties of the derivatives of the polypeptide analogs of the present application, and is generally not undesirable biologically or otherwise.
  • the derivatives of the polypeptide analogs of the present application can form acid and/or base salts through the presence of amino and/or carboxyl groups or groups similar thereto.
  • Pharmaceutically acceptable acid addition salts can be formed using inorganic and organic acids, for example, acetate, aspartate, benzoate, benzenesulfonate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlortheophyllonate, citrate, edisylate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthoate, naphthylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogenphosphat
  • Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, etc.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic bases and organic bases.
  • Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from Groups I to XII of the periodic table.
  • the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like.
  • Certain organic amines include isopropylamine, choline salts, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.
  • the pharmaceutically acceptable salts of the present application can be synthesized from the parent compound, alkaline or acidic part by conventional chemical methods.
  • these salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base (e.g., hydroxide, carbonate, bicarbonate, etc. of Na or K), or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid.
  • a suitable base e.g., hydroxide, carbonate, bicarbonate, etc. of Na or K
  • These reactions are usually carried out in water or an organic solvent, or a mixture of the two.
  • a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile.
  • the present application provides a method for preparing a polypeptide derivative, wherein a polypeptide derivative intermediate is obtained, wherein the polypeptide derivative intermediate is a carboxylate-modified polypeptide containing a protecting group; the polypeptide derivative intermediate is subjected to freeze-thaw treatment to obtain a freeze-thaw-treated polypeptide derivative intermediate; and the freeze-thaw-treated polypeptide derivative intermediate is deprotected to obtain a polypeptide derivative.
  • the polypeptide derivative intermediate can be a wet solid, that is, a wet solid of a polypeptide derivative intermediate is obtained, wherein the polypeptide derivative intermediate is a carboxylate-modified polypeptide containing a protecting group; the wet solid of the polypeptide derivative intermediate is subjected to freeze-thaw treatment to obtain a freeze-thaw-treated polypeptide derivative intermediate; and the freeze-thaw-treated polypeptide derivative intermediate is deprotected to obtain a polypeptide derivative.
  • the protecting group is selected from any one of a C1-C21 alkyl group and an aryl group.
  • the protecting group is selected from any one of a C1-C10 alkyl group and an aryl group.
  • the protecting group is methyl, ethyl, tert-butyl or benzyl.
  • the carboxylic acid ester is a fatty acid ester.
  • the fatty acid is one or more of caprylic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanedioic acid, stearic acid, nonadecanedioic acid, arachidic acid, heneicosanoic acid, hexadecanoic acid, hexadecanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosapentaenoic acid or docosahexaenoic acid, 1,8-octanedioic acid, 1,7-heptanedicarboxylic acid, 1,10-decanedioic acid, undecanoic acid, dodecanedioic acid, tridecane
  • the present application effectively increases the upper limit of the deprotected protein concentration by adding a deprotected protein freezing intervention treatment step before the traditional acid catalyst deprotection reaction, thereby greatly improving the benefit and efficiency of the deprotection process in protein production.
  • the pre-freezing treatment includes: a pre-freezing treatment; a freezing treatment; a primary drying treatment; and may further include an optional secondary drying treatment.
  • the pre-freezing treatment includes: pre-freezing treatment, freezing treatment and primary drying treatment.
  • the pre-freezing treatment includes: pre-freezing treatment, freezing treatment, primary drying treatment and secondary drying treatment.
  • the pre-freezing temperature of the pre-freezing treatment is 2 to 8°C, and the pre-freezing time is greater than or equal to 2.0 hours; or the freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing time is more than 3 hours; or the temperature of the primary drying treatment is -10°C to 0°C, the vacuum degree of the primary drying treatment is 0.05 to 2.0 mbar, and the drying time of the primary drying treatment is greater than or equal to 20 hours; the pre-freezing treatment may further include a secondary drying treatment, the temperature of the secondary drying treatment is 20°C to 65°C, the vacuum degree of the secondary drying treatment is 0.05 to 2.0 mbar, and the drying time of the secondary drying treatment is greater than 0 hours and less than 8 hours;
  • the pre-freezing temperature of the pre-freezing treatment may be 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C or any range therebetween;
  • the time of pre-freezing treatment can be 2.0 hours, 2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3.0 hours, 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, 4.0 hours, 4.1 hours, 4.2 hours, 4.3 hours, 4.4 hours, 4.5 hours, 4.6 hours, 4.7 hours, 4.8 hours, 4.9 hours, 5.0 hours, 5.1 hours, 5.2 hours, 5.3 hours, 5.4 hours, 5.5 hours, 5.6 hours, 5.7 hours, 5.8 hours, 5.9 hours, 6.0 hours Hours, 6.1 hours, 6.2 hours, 6.3 hours, 6.4 hours, 6.5 hours, 6.6 hours, 6.7 hours, 6.8 hours, 6.9 hours, 7.0 hours, 7.1 hours, 7.2 hours, 7.3 hours, 7.4 hours, 7.5 hours, 7.6 hours, 7.7 hours, 7.8 hours, 7.9 hours, 8.0 hours,
  • the freezing temperature of the freezing treatment can be -30°C, -31°C, -32°C, -33°C, -34°C, -35°C, -36°C, -37°C, -38°C, -39°C, -40°C, -41°C, -42°C, -43°C, -44°C, -45°C, -46°C, -47°C, -48°C, -49°C, -50°C, -51°C, -52°C, -53°C, -54°C, -55°C, -56°C, -57°C, -58°C, -59°C, -60°C or any range therebetween;
  • the freezing treatment time can be 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, 4.0 hours, 4.1 hours, 4.2 hours, 4.3 hours, 4.4 hours, 4.5 hours, 4.6 hours, 4.7 hours, 4.8 hours, 4.9 hours, 5.0 hours, 5.1 hours, 5.2 hours, 5.3 hours, 5.4 hours, 5.5 hours, 5.6 hours, 5.7 hours Hours, 5.8 hours, 5.9 hours, 6.0 hours, 6.1 hours, 6.2 hours, 6.3 hours, 6.4 hours, 6.5 hours, 6.6 hours, 6.7 hours, 6.8 hours, 6.9 hours, 7.0 hours, 7.1 hours, 7.2 hours, 7.3 hours, 7.4 hours, 7.5 hours, 7.6 hours, 7.7 hours, 7.8 hours, 7.9 hours, 8.0 hours, 8.1 hours, 8.2 hours, 8.3 hours, 8.4 hours, 8.5 hours, 8.6 hours, 8.7 hours, 8.8 hours, 8.9 hours, 9.0 hours, 9.1 hours, 9.2
  • the temperature of the primary drying treatment may be -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C, -1°C, 0°C or any range therebetween;
  • the vacuum degree of the primary drying process can be 0.05mbar, 0.1mbar, 0.15mbar, 0.2mbar, 0.25mbar, 0.3mbar, 0.35mbar, 0.4mbar, 0.45mbar, 0.5mbar, 0.55mbar, 0.6mbar, 0.65mbar, 0.7mbar, 0.75mbar, 0.8mbar, 0.85mbar, 0.9mbar, 0.95mbar, 1.0mbar r, 1.05mbar, 1.1mbar, 1.15mbar, 1.2mbar, 1.25mbar, 1.3mbar, 1.35mbar, 1.4mbar, 1.45mbar, 1.5mbar, 1.55mbar, 1.6mbar, 1.65mbar, 1.7mbar, 1.75mbar, 1.8mbar, 1.85mbar, 1.9mbar, 1.95mbar, 2.0mbar or any range therebetween;
  • the drying time of the primary drying treatment can be 20 hours, 20.1 hours, 20.2 hours, 20.3 hours, 20.4 hours, 20.5 hours, 20.6 hours, 20.7 hours, 20.8 hours, 20.9 hours, 21.0 hours, 21.1 hours, 21.2 hours, 21.3 hours, 21.4 hours, 21.5 hours, 21.6 hours, 21.7 hours, 21.8 hours, 21.9 hours, 22.0 hours, 22.1 hours, 22.2 hours, 22.3 hours, 22.4 hours, 22.5 hours, 22.6 hours, 22.7 hours, 22.8 hours, 22.9 hours, 23.0 hours, 23.1 hours, 23.2 hours, 23.3 hours, 23.4 hours, 23.5 hours, 23.6 hours, 23.7 hours, 23.8 hours, 23.9 hours, 24.0 hours, 24.1 hours, 24.2 hours, 24.3 hours, 24.4 hours, 24.5 hours, 24.6 hours, 24.7 hours, 24.8 hours, 24.9 hours, 25 .4 hours, 22.5 hours, 22.6 hours, 22.7 hours, 22.8 hours, 22.9 hours, 23.0 hours, 23.1 hours, 23.2 hours, 23.3 hours, 2
  • the temperature of the secondary drying treatment can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C or any range therebetween;
  • the vacuum degree of the secondary drying process can be 0.05mbar, 0.1mbar, 0.15mbar, 0.2mbar, 0.25mbar, 0.3mbar, 0.35mbar, 0.4mbar, 0.45mbar, 0.5mbar, 0.55mbar, 0.6mbar, 0.65mbar, 0.7mbar, 0.75mbar, 0.8mbar, 0.85mbar, 0.9mbar, 0.95mbar, 1.0mbar r, 1.05mbar, 1.1mbar, 1.15mbar, 1.2mbar, 1.25mbar, 1.3mbar, 1.35mbar, 1.4mbar, 1.45mbar, 1.5mbar, 1.55mbar, 1.6mbar, 1.65mbar, 1.7mbar, 1.75mbar, 1.8mbar, 1.85mbar, 1.9mbar, 1.95mbar, 2.0mbar or any range therebetween;
  • the drying time of the secondary drying treatment can be 0.01 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours or any range therebetween.
  • the prefreezing treatment includes: pre-freezing treatment, freezing treatment and primary drying treatment; wherein the pre-freezing temperature of the pre-freezing treatment is 2 to 8°C, and the pre-freezing time is greater than or equal to 2.0 hours; the freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing time is more than 3 hours; the temperature of the primary drying treatment is -10°C to 0°C, the vacuum degree of the primary drying treatment is 0.05 to 2.0 mbar, and the drying time of the primary drying treatment is greater than or equal to 20 hours.
  • the pre-freezing treatment includes: pre-freezing treatment, freezing treatment, primary drying treatment and secondary drying treatment, wherein the pre-freezing temperature of the pre-freezing treatment is 2-8°C, and the pre-freezing time is greater than or equal to 2.0 hours; the freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing time is more than 3 hours; the temperature of the primary drying treatment is -10°C to 0°C, the vacuum degree of the primary drying treatment is 0.05-2.0 mbar, and the drying time of the primary drying treatment is greater than or equal to 20 hours; the temperature of the secondary drying treatment is 20°C to 65°C, the vacuum degree of the secondary drying treatment is 0.05-2.0 mbar, and the drying time of the secondary drying treatment is greater than 0 hours and less than 8 hours.
  • the intermediate of the polypeptide derivative after freeze-drying is deprotected under acid catalyst solution conditions to obtain a deprotection reaction solution; the deprotection reaction solution is added to a stop solution, and the pH value is adjusted to stop the reaction to obtain a polypeptide derivative.
  • the pH value is adjusted to 6.5-10.0 to terminate the reaction and obtain a polypeptide derivative.
  • the pH value is adjusted to 7.5-9.5 to terminate the reaction and obtain a polypeptide derivative.
  • the pH value can be adjusted to 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 or any range therebetween.
  • the stop solution comprises a buffered saline solution and an alkaline solution.
  • the alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, and ammonium bicarbonate solution.
  • the buffered salt solution is selected from one or more of a phosphate buffer solution, a Tris buffer solution, an organic acid buffer solution, a borate buffer solution, and an amino acid buffer solution.
  • the buffered saline solution is selected from one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution (PBS), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate-NaOH buffer solution, sodium barbital-HCl buffer solution, NH4HCO3 buffer solution, sodium carbonate-sodium bicarbonate buffer solution, NaHCO3 buffer solution, Tris-HCl, glycine-NaOH buffer solution, boric acid - borax buffer solution, and Na2B7O4 buffer solution.
  • PBS phosphate buffer solution
  • PB disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution
  • the alkaline solution is a sodium hydroxide solution.
  • the concentration of the buffer salt in the buffer salt solution is 0.001M to 0.6M, preferably 0.01M to 0.6M, more preferably 0.01M to 0.5M;
  • the concentration of the buffer salt in the buffer salt solution can be 0.001M, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.11M, 0.12M, 0.13M, 0.14M, 0.15M, 0.16M, 0.17M, 0.18M, 0.19M, 0.2M, 0.21M, 0.22M, 0.23M, 0.24M, 0.25M, 0.26M, 0.27M, 0.28M, 0. .54M, 0.55M, 0.56M, 0.57M, 0.58M, 0.59M, 0.6M or any range therebetween.
  • the concentration of the alkaline solution is 2 to 10 M, preferably 3 to 8 M, and more preferably 4 to 6 M;
  • the concentration of the alkaline solution can be 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3.0M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4.0M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5.0M, 5.1M, 5.2M, 5.3M, 5.4M, 5.5M, 5.6M, 5.7M, 5.8M, 5.9M, 6.0M, 6.1M, 6.2M, 6.3M, 6.4M, 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7.0 .9M, 6.0M, 6.1M, 6.2M, 6.3M, 6.4M, 6.5M, 6.6M, 6.7M, 6.8M,
  • the concentration of sodium hydroxide in the sodium hydroxide solution is 2 to 10 M, preferably 3 to 8 M, and more preferably 4 to 6 M;
  • the concentration of sodium hydroxide in the sodium hydroxide solution can be 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3.0M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4.0M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5.0M, 5.1M, 5.2M, 5.3M, 5.4M, 5.5M, 5.6M, 5.7M, 5.8M, M, 5.9M, 6.0M, 6.1M, 6.2M, 6.3M, 6.4M, 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7.0M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.
  • the volume ratio of the buffered saline solution to the alkaline solution is (0.1-100):1;
  • the volume ratio of the buffered saline solution to the alkaline solution can be 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1 ⁇ 28:1 ⁇ 29:1 ⁇ 30:1 ⁇ 31:1 ⁇ 32:1 ⁇ 33:1 ⁇ 34:1 ⁇ 35:1 ⁇ 36:1 ⁇ 37:1 ⁇ 38:1 ⁇ 39:1 ⁇ 40:1 ⁇ 41:1 ⁇ 42:1 ⁇ 43:1 ⁇ 44:1 ⁇ 45:1 ⁇ 46:1 ⁇ 47:1 ⁇ 48:1 ⁇ 49:1 ⁇ 50:1 ⁇ 51:1 ⁇ 52:1 ⁇ 53:1 ⁇ 54:1 ⁇ 55:1 ⁇ 56:1 ⁇ 57
  • the acid catalyst is selected from one or more of p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
  • the acid catalyst is trifluoroacetic acid
  • the stop solution comprises a buffered salt solution and an alkaline solution
  • the pH value is adjusted to 6.5 to 10.0 to terminate the reaction and obtain a polypeptide derivative.
  • the present application provides a method for preparing a polypeptide derivative, wherein a polypeptide derivative intermediate is obtained, wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group; the polypeptide derivative intermediate is added to an acid catalyst solution for deprotection to obtain a deprotection reaction liquid; the deprotection reaction liquid is added to a stop solution, the pH value is adjusted to stop the reaction, and the polypeptide derivative is obtained.
  • the polypeptide derivative is subjected to freeze-thaw treatment to obtain an intermediate of the polypeptide derivative after freeze-thaw treatment; and the intermediate of the polypeptide derivative after freeze-thaw treatment is deprotected to obtain the polypeptide derivative.
  • the present application provides a method for preparing a polypeptide derivative, wherein a wet solid of a polypeptide derivative intermediate is obtained, wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group; the wet solid of the polypeptide derivative intermediate is added to an acid catalyst solution for deprotection to obtain a deprotection reaction liquid; the deprotection reaction liquid is added to a stop solution, the pH value is adjusted to stop the reaction, and the polypeptide derivative is obtained.
  • the wet solid is freeze-dried to obtain an intermediate of the freeze-dried polypeptide derivative; and the freeze-dried intermediate of the polypeptide derivative is deprotected to obtain a polypeptide derivative.
  • pH value please refer to the above description of pH value in deprotection.
  • the description of the stop solution refers to the description of the stop solution in deprotection above.
  • the description of the acid catalyst refers to the description of the acid catalyst in the deprotection process described above.
  • the description of the protecting group refers to the above description of the protecting group.
  • fatty acids and carboxylates refer to the above descriptions of fatty acids and carboxylates, respectively.
  • the description of the pre-freezing treatment refers to the description of the pre-freezing treatment mentioned above.
  • the present application also provides a method for preparing a polypeptide derivative, comprising: obtaining a polypeptide derivative intermediate, wherein the polypeptide derivative intermediate is a carboxylate-modified polypeptide containing a protecting group; subjecting the polypeptide derivative to a freeze-thaw treatment to obtain a freeze-thaw treated polypeptide derivative intermediate; adding the freeze-thaw treated polypeptide derivative intermediate to an acid catalyst solution for deprotection to obtain a deprotection reaction solution; adding the deprotection reaction solution to a stop solution, adjusting the pH value to stop the reaction, and obtaining a polypeptide derivative.
  • the present application also provides a method for preparing a polypeptide derivative, comprising: obtaining a wet solid of a polypeptide derivative intermediate, wherein the polypeptide derivative intermediate is a polypeptide modified with a carboxylate containing a protecting group; subjecting the wet solid to a freeze-dried treatment to obtain a polypeptide derivative intermediate after freeze-dried treatment; adding the polypeptide derivative intermediate after freeze-dried treatment to an acid catalyst solution for deprotection to obtain a deprotection reaction solution; adding the deprotection reaction solution to a stop solution, adjusting the pH value to stop the reaction, and obtaining a polypeptide derivative.
  • the description of the pH value refers to the above description of the pH value in deprotection.
  • the description of the stop solution refers to the description of the stop solution in deprotection above.
  • the description of the acid catalyst refers to the description of the acid catalyst in the deprotection process described above.
  • the description of the protecting group refers to the above description of the protecting group.
  • fatty acids and carboxylates refer to the above descriptions of fatty acids and carboxylates, respectively.
  • the description of the pre-freezing treatment refers to the description of the pre-freezing treatment mentioned above.
  • the present application provides a polypeptide derivative, wherein the polypeptide derivative is prepared by the above method.
  • the present application provides a method for preparing a polypeptide derivative by adding a deprotected protein freeze intervention treatment step before the traditional acid catalyst deprotection reaction, which effectively improves the deprotected protein concentration.
  • the limit is to greatly improve the benefits and efficiency of the deprotection process in protein production.
  • the present application provides a method for preparing a polypeptide derivative.
  • the conventional method of adding the deprotection termination solution to the deprotection reaction solution is changed to adding the reaction solution to the deprotection termination solution. This greatly reduces the content of isomer impurities in the deprotection process and improves the stability of the operation method.
  • the termination method by optimizing the termination method by adding a buffer solution and adjusting the volume of the buffer solution components, the deprotection effect and stability are further improved.
  • Mobile phase B 70% ACN + 30% H 2 O + 0.2% TFA;
  • the purified GLP-1 reconstituted sample was obtained.
  • the wet solid of the GLP-1 derivative intermediate prepared in Example 1 was taken and lyophilized on a plate.
  • the lyophilization conditions were as follows: pre-freezing the sample: maintaining the temperature at 4°C for 2 hours, then cooling to -45°C and freezing, maintaining the freezing temperature for 3 hours; primary drying: raising the temperature to -5°C after pre-freezing, and lyophilizing for 40 hours under an air pressure of 0.2 mbar to complete the primary drying. After drying, the intermediate of the polypeptide derivative treated with lyophilization was obtained.
  • the post-freezing reaction solution obtained by the above method was subjected to RP-HPLC to detect the product after deprotection termination.
  • the RP-HPLC determination conditions were as follows:
  • Mobile phase B 70% ACN + 30% H 2 O + 0.2% TFA;
  • the non-freezing treatment post-reaction solution was used to detect the deprotection termination product by RP-HPLC.
  • the RP-HPLC determination conditions were as follows:
  • Mobile phase B 70% ACN + 30% H 2 O + 0.2% TFA;
  • the deprotected protein concentration can only be maintained at a low level of 5 mg/mL, and the content of the incompletely deprotected sample is 0.957%.
  • the deprotection treatment concentration cannot be further increased.
  • the deprotected protein treatment concentration can reach 250 mg/mL, and the content of the incompletely deprotected sample is still lower than the content of the incompletely deprotected sample with a protein concentration of 7.5 mg/mL under conventional operation, especially in the concentration range of 200 mg/mL.
  • the content of the incompletely deprotected sample is maintained at a low level, that is, the deprotection efficiency after freeze-drying has been greatly improved, which has obvious practicality for industrial large-scale production.
  • Mobile phase B 70% ACN + 30% H 2 O + 0.2% TFA;

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Endocrinology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Provided are a polypeptide derivative and a preparation method therefor. The preparation method comprises: obtaining a polypeptide derivative intermediate, performing freeze-drying pretreatment, and performing deprotection to obtain the polypeptide derivative. The freeze-drying pretreatment improves the upper limit of concentration of a deprotected protein, adjusts a termination mode and condition in deprotection, reduces the impurity content of an isomer, improves the stability of an operating method, and improves the content of a protein of interest.

Description

一种多肽衍生物及其制备方法A polypeptide derivative and preparation method thereof 技术领域Technical Field
本申请属于多肽技术领域。具体而言，本申请涉及多肽衍生物及其制备方法。另外，本申请还涉及含该多肽衍生物的药物以及在制备药物中的用途等。The present application belongs to the field of polypeptide technology. Specifically, the present application relates to polypeptide derivatives and preparation methods thereof. In addition, the present application also relates to drugs containing the polypeptide derivatives and uses thereof in preparing drugs.
背景技术Background technique
在医药开发和生产过程中，为获得具有药学活性的目标产物，往往需要经历复杂且多个生产步骤，各个生产环节的条件环境也不相同，而各反应步骤中经常仅需要针对一个反应中心进行转换，为达到该目的，研发者需要精心挑选具有特定选择性的反应试剂和反应条件。同时，为达到该目的的另一种方式是，对当下步骤中不希望发生反应的位置进行暂时性的结构修饰(即保护)，以达到在该步骤下目标位置发生反应时，该结构修饰位置官能团不受影响，在完成整个反应后，可通过特定步骤将保护官能团恢复为修饰前的结构(即脱保护)。如醇、羧酸、胺类、醛、硫醇等都是常见的需要进行保护和脱保护的基团。In the process of pharmaceutical development and production, in order to obtain a target product with pharmaceutical activity, it is often necessary to go through complex and multiple production steps, and the conditions and environments of each production link are also different. In each reaction step, it is often necessary to convert only one reaction center. To achieve this goal, researchers need to carefully select reaction reagents and reaction conditions with specific selectivity. At the same time, another way to achieve this goal is to temporarily modify the structure (i.e., protect) the position where the reaction is not desired in the current step, so that when the target position reacts in this step, the functional group at the modified position is not affected. After the entire reaction is completed, the protected functional group can be restored to the structure before modification (i.e., deprotection) through specific steps. For example, alcohols, carboxylic acids, amines, aldehydes, thiols, etc. are all common groups that need to be protected and deprotected.
在实际应用中，脱保护方式也各有不同。US 6184309公开了一种使用酸催化从聚合物中除去保护基团的方法，其指出许多强和弱有机和无机酸能作为脱保护剂。US 2003/0212249报导了环孢菌素类似物的合成，其中三甲代甲硅烷基保护基团可以用乙酸或柠檬酸除去。US 5135683涉及脱保护的多羟基化合物的制备，其指出磷酸可作为除去环状缩酮保护基团的试剂。In practical applications, the deprotection methods are also different. US 6184309 discloses a method for removing protecting groups from polymers using acid catalysis, which indicates that many strong and weak organic and inorganic acids can be used as deprotection agents. US 2003/0212249 reports the synthesis of cyclosporin analogs, in which the trimethylsilyl protecting group can be removed with acetic acid or citric acid. US 5135683 relates to the preparation of deprotected polyhydroxy compounds, which indicates that phosphoric acid can be used as a reagent for removing cyclic ketal protecting groups.
发明内容Summary of the invention
然而，针对脱保护反应，仍然存在脱保护反应中存在异构体杂质、脱保护不完全、脱保护底物浓度受限等多种问题，对于化合物工业生产、制备与纯化带来巨大挑战。However, there are still many problems in the deprotection reaction, such as the presence of isomeric impurities, incomplete deprotection, and limited concentration of deprotected substrates, which pose great challenges to the industrial production, preparation and purification of compounds.
本申请中通过酰胺键将含有保护基团的十八烷烃脂肪酸与多肽(如GLP-1)进行偶联，为去除脂肪酸上的叔丁基保护基团，需要在强酸环境下对其进行脱保护操作。通常而言，考察脱保护操作成功与否的条件主要为脱 保护的浓度以及脱保护产物中的杂质体含量等。脱保护过程中，脱保护蛋白浓度受到极大限制，当脱保护蛋白超过特定浓度，蛋白脱保护将不完全，导致脱保护不完全的样品含量急剧增加，严重影响蛋白质生产及纯化效益和效率。同时，常规强碱性溶液终止脱保护过程中，会产生难以分离去除的消旋异构体，不同批次生产时的异构体含量差异较大，尤其是在较大体系中尤为显著。为解决上述问题亟待改善相关工艺，寻找适宜高浓度蛋白脱保护，且杂质含量低，方法稳定性高的脱保护方法。In the present application, an octadecane fatty acid containing a protective group is coupled to a polypeptide (such as GLP-1) via an amide bond. In order to remove the tert-butyl protective group on the fatty acid, a deprotection operation is required under a strong acid environment. Generally speaking, the conditions for evaluating the success of the deprotection operation are mainly The concentration of protection and the content of impurities in the deprotected product. During the deprotection process, the concentration of the deprotected protein is greatly limited. When the deprotected protein exceeds a certain concentration, the protein deprotection will be incomplete, resulting in a sharp increase in the content of incompletely deprotected samples, seriously affecting the benefits and efficiency of protein production and purification. At the same time, during the deprotection process terminated by conventional strong alkaline solutions, racemic isomers that are difficult to separate and remove will be produced. The isomer content varies greatly in different batches of production, especially in larger systems. In order to solve the above problems, it is urgent to improve the relevant processes and find a deprotection method that is suitable for high-concentration protein deprotection, has low impurity content, and has high method stability.
本申请技术方案如下：The technical solution of this application is as follows:
1.一种多肽衍生物的制备方法，包括：1. A method for preparing a polypeptide derivative, comprising:
得到多肽衍生物中间体，所述多肽衍生物中间体为经含有保护基的羧酸酯修饰的多肽；Obtaining a polypeptide derivative intermediate, wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group;
对所述多肽衍生物中间体进行冻干预处理，得到冻干预处理后的多肽衍生物的中间体；Performing freeze-thaw treatment on the polypeptide derivative intermediate to obtain a freeze-thaw treated polypeptide derivative intermediate;
对所述冻干预处理后的多肽衍生物的中间体进行脱保护，得到多肽衍生物。The intermediate of the polypeptide derivative after freeze-drying is deprotected to obtain the polypeptide derivative.
2.根据项1所述的制备方法，2. The preparation method according to item 1,
所述保护基选自C1～C21烷基、芳基中的任一种。The protecting group is selected from any one of C1-C21 alkyl and aryl groups.
3.根据项2所述的制备方法，3. The preparation method according to item 2,
所述保护基为甲基、乙基、叔丁基或苄基。The protecting group is methyl, ethyl, tert-butyl or benzyl.
4.根据项1所述的制备方法，4. The preparation method according to item 1,
所述羧酸酯为脂肪酸酯。The carboxylic acid ester is a fatty acid ester.
5.根据项4所述的制备方法，5. The preparation method according to item 4,
所述脂肪酸为辛酸、壬酸、癸酸、十一酸、月桂酸、十三酸、肉豆蔻酸、十五酸、棕榈酸、十七酸、硬脂酸、十九酸、花生酸、二十一酸、二十二酸、二十三酸、二十四酸、棕榈油酸、油酸、亚麻油酸、蓖麻油酸、异柠檬酸、二十碳五烯酸或二十二碳六烯酸、1,8-辛二酸、1,7-庚二甲酸、1,10-癸二酸、十一烷二酸、十二烷二酸、十三烷二酸、十四烷二酸、十五烷二酸、十六烷二酸、十七烷二酸或十八烷二酸中的一种或两种以上。The fatty acid is one or more of octanoic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanedioic acid, palmitic acid, heptadecanedioic acid, stearic acid, nonadecanedioic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosapentaenoic acid or docosahexaenoic acid, 1,8-octanedioic acid, 1,7-heptanedicarboxylic acid, 1,10-decanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid or octadecanedioic acid.
6.根据项1所述的制备方法，6. The preparation method according to item 1,
所述冻干预处理包括如下步骤：The freezing treatment comprises the following steps:
预冻处理； Pre-freezing treatment;
冷冻处理；Freezing treatment;
一次干燥处理；以及A primary drying process; and
进一步可以包括任选二次干燥处理。An optional secondary drying process may further be included.
7.根据项6所述的制备方法，7. The preparation method according to item 6,
所述预冻处理的预冻温度为2～8℃，预冻处理的时间为大于等于2.0小时；或者The pre-freezing temperature of the pre-freezing treatment is 2 to 8° C., and the pre-freezing time is greater than or equal to 2.0 hours; or
所述冷冻处理的冷冻温度为-30℃～-60℃，冷冻处理的时间为3小时以上；或者The freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing treatment time is more than 3 hours; or
所述一次干燥处理的温度为-10℃～0℃，一次干燥处理的真空度为0.05～2.0mbar，一次干燥处理的干燥时间为大于等于20小时；The temperature of the primary drying process is -10°C to 0°C, the vacuum degree of the primary drying process is 0.05 to 2.0 mbar, and the drying time of the primary drying process is greater than or equal to 20 hours;
所述二次干燥处理的温度为20℃～65℃，二次干燥处理的真空度为0.05～2.0mbar，二次干燥处理的干燥时间为大于0小时且在8小时以下。The temperature of the secondary drying process is 20° C. to 65° C., the vacuum degree of the secondary drying process is 0.05 to 2.0 mbar, and the drying time of the secondary drying process is greater than 0 hours and less than 8 hours.
8.根据项1所述的制备方法，8. The preparation method according to item 1,
多肽选自胰岛素、胰高血糖素、肠促胰岛素、GLP-1、甲状旁腺素(PTH)、GLP-2、胃泌酸调节素、淀粉不溶素(Amylin)、肠高血糖素、生长激素抑制素、瘦素、YY肽、去氨加压素、骨钙蛋白、人生长激素、糖肽抗生素、非核糖体肽抗生素、促皮质素、降钙素、催产素、干扰素、纤维蛋白溶媒、抗利尿激素、白细胞介素、尿激酶、促甲状腺激素释放激素、甲硫氨酸脑啡肽、亮氨酸脑啡肽、前列腺素F2α受体调节剂、肥胖抑制素、分泌素、nesfatin、葡萄糖依赖性促胰岛素肽(GIP)及上述多肽的类似物或片段中的一种或两种以上。The polypeptide is selected from one or more of insulin, glucagon, incretin, GLP-1, parathyroid hormone (PTH), GLP-2, oxyntomodulin, amylin, enteroglucagon, somatostatin, leptin, YY peptide, desmopressin, osteocalcin, human growth hormone, glycopeptide antibiotics, non-ribosomal peptide antibiotics, corticotropin, calcitonin, oxytocin, interferon, fibrinolytic, antidiuretic hormone, interleukin, urokinase, thyrotropin-releasing hormone, methionine enkephalin, leucine enkephalin, prostaglandin F2α receptor modulator, obestatin, secretin, nesfatin, glucose-dependent insulinotropic peptide (GIP) and analogs or fragments of the above polypeptides.
9.根据项8所述的制备方法，9. The preparation method according to item 8,
所述多肽选自GLP-1、GLP-1的药学上可接受的盐、GLP-1类似物、GLP-1类似物的药学上可接受的盐。The polypeptide is selected from GLP-1, a pharmaceutically acceptable salt of GLP-1, a GLP-1 analog, and a pharmaceutically acceptable salt of a GLP-1 analog.
10.根据项1所述的制备方法，10. The preparation method according to item 1,
在所述脱保护步骤中：In the deprotection step:
冻干预处理后的多肽衍生物的中间体在酸催化剂溶液条件下进行脱保护，得到脱保护反应液；The intermediate of the polypeptide derivative after freeze-drying is deprotected under the condition of an acid catalyst solution to obtain a deprotection reaction solution;
将所述脱保护反应液加入到终止液中，调节pH值以终止反应，得到多肽衍生物。The deprotection reaction solution is added to a stop solution, and the pH value is adjusted to stop the reaction to obtain a polypeptide derivative.
11.根据项10所述的制备方法， 11. The preparation method according to item 10,
调节pH值至6.5～10.0以终止反应，得到多肽衍生物。The pH value is adjusted to 6.5-10.0 to terminate the reaction and obtain a polypeptide derivative.
12.根据项11所述的制备方法，12. The preparation method according to item 11,
调节pH值至7.5～9.5以终止反应，得到多肽衍生物。The pH value is adjusted to 7.5-9.5 to terminate the reaction and obtain a polypeptide derivative.
13.根据项10所述的制备方法，13. The preparation method according to item 10,
所述终止液包含缓冲盐溶液和碱性溶液。The stop solution comprises a buffered salt solution and an alkaline solution.
14.根据项13所述的制备方法，14. The preparation method according to item 13,
所述碱性溶液选自氢氧化钠溶液、氢氧化钾溶液、碳酸钠溶液、碳酸钾溶液、碳酸氢钠溶液、碳酸氢钾溶液、碳酸氢铵溶液中的一种或两种以上。The alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, and ammonium bicarbonate solution.
15.根据项13所述的制备方法，15. The preparation method according to item 13,
所述缓冲盐溶液选自磷酸盐类缓冲溶液、Tris类缓冲溶液、有机酸类缓冲溶液、硼酸盐类缓冲溶液、氨基酸类缓冲溶液中的一种或两种以上。The buffer salt solution is selected from one or more of a phosphate buffer solution, a Tris buffer solution, an organic acid buffer solution, a borate buffer solution, and an amino acid buffer solution.
16.根据项15所述的制备方法，16. The preparation method according to item 15,
所述缓冲盐溶液选自磷酸氢二钠-柠檬酸缓冲溶液、柠檬酸-NaOH-HCl缓冲溶液、柠檬酸-柠檬酸钠缓冲溶液、乙酸-乙酸钠缓冲溶液、磷酸盐缓冲溶液(PBS)、磷酸氢二钠-磷酸二氢钠缓冲溶液(PB)、磷酸氢二钠-磷酸二氢钾缓冲溶液、磷酸二氢钾-NaOH缓冲溶液、巴比妥钠-HCl缓冲溶液、NH4HCO3缓冲溶液、碳酸钠-碳酸氢钠缓冲溶液、NaHCO3缓冲溶液、Tris-HCl、甘氨酸-NaOH缓冲溶液、硼酸-硼砂缓冲溶液、Na2B7O4缓冲溶液中的一种或两种以上。The buffered saline solution is selected from one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution (PBS), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate- NaOH buffer solution, sodium barbital-HCl buffer solution, NH4HCO3 buffer solution, sodium carbonate-sodium bicarbonate buffer solution, NaHCO3 buffer solution, Tris-HCl, glycine-NaOH buffer solution, boric acid- borax buffer solution, and Na2B7O4 buffer solution.
17.根据项14所述的制备方法，17. The preparation method according to item 14,
所述碱性溶液为氢氧化钠溶液。The alkaline solution is a sodium hydroxide solution.
18.根据项13所述的制备方法，18. The preparation method according to item 13,
所述缓冲盐溶液中缓冲盐的浓度为0.001M～0.6M，优选为0.01M～0.6M,更优选为0.01M～0.5M。The concentration of the buffer salt in the buffer salt solution is 0.001M to 0.6M, preferably 0.01M to 0.6M, and more preferably 0.01M to 0.5M.
19.根据项14或17所述的制备方法，19. The preparation method according to item 14 or 17,
所述碱性溶液的浓度为2～10M，优选为3～8M，更优选为4～6M。The concentration of the alkaline solution is 2-10M, preferably 3-8M, and more preferably 4-6M.
20.根据项13所述的制备方法，20. The preparation method according to item 13,
所述缓冲盐溶液和所述碱性溶液的体积比为(0.1～100)：1，优选为(0.1～70)：1，更优选为(1～50)：1。The volume ratio of the buffered salt solution to the alkaline solution is (0.1-100):1, preferably (0.1-70):1, and more preferably (1-50):1.
21.根据项10所述的制备方法，21. The preparation method according to item 10,
酸催化剂选自对甲苯磺酸、三氟乙酸、乙酸、甲磺酸、盐酸、磷酸和磺 酸中的一种或两种以上。The acid catalyst is selected from p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid. One or more acids.
22.一种多肽衍生物的制备方法，22. A method for preparing a polypeptide derivative,
得到多肽衍生物中间体，所述多肽衍生物中间体为含有保护基的羧酸酯修饰的多肽；Obtaining a polypeptide derivative intermediate, wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group;
所述多肽衍生物的中间体在酸催化剂溶液条件下进行脱保护，得到脱保护反应液；The intermediate of the polypeptide derivative is deprotected under acid catalyst solution conditions to obtain a deprotection reaction solution;
将所述脱保护反应液加入到终止液中，调节pH值以终止反应，得到多肽衍生物。The deprotection reaction solution is added to a stop solution, and the pH value is adjusted to stop the reaction to obtain a polypeptide derivative.
23.根据项22所述的制备方法，23. The preparation method according to item 22,
调节pH值至6.5～10.0以终止反应，得到多肽衍生物。The pH value is adjusted to 6.5-10.0 to terminate the reaction and obtain a polypeptide derivative.
24.根据项23所述的制备方法，24. The preparation method according to item 23,
调节pH值至7.5～9.5以终止反应，得到多肽衍生物。The pH value is adjusted to 7.5-9.5 to terminate the reaction and obtain a polypeptide derivative.
25.根据项22所述的制备方法，25. The preparation method according to item 22,
所述终止液包含缓冲盐溶液和碱性溶液。The stop solution comprises a buffered salt solution and an alkaline solution.
26.根据项25所述的制备方法，26. The preparation method according to item 25,
所述碱性溶液选自氢氧化钠溶液、氢氧化钾溶液、碳酸钠溶液、碳酸钾溶液、碳酸氢钠溶液、碳酸氢钾溶液、碳酸氢铵溶液中的一种或两种以上。The alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, and ammonium bicarbonate solution.
27.根据项25所述的制备方法，27. The preparation method according to item 25,
所述缓冲盐溶液选自磷酸盐类缓冲溶液、Tris类缓冲溶液、有机酸类缓冲溶液、硼酸盐类缓冲溶液、氨基酸类缓冲溶液中的一种或两种以上。The buffer salt solution is selected from one or more of a phosphate buffer solution, a Tris buffer solution, an organic acid buffer solution, a borate buffer solution, and an amino acid buffer solution.
28.根据项27所述的制备方法，28. The preparation method according to item 27,
所述缓冲盐溶液选自磷酸氢二钠-柠檬酸缓冲溶液、柠檬酸-NaOH-HCl缓冲溶液、柠檬酸-柠檬酸钠缓冲溶液、乙酸-乙酸钠缓冲溶液、磷酸盐缓冲溶液(PBS)、磷酸氢二钠-磷酸二氢钠缓冲溶液(PB)、磷酸氢二钠-磷酸二氢钾缓冲溶液、磷酸二氢钾-NaOH缓冲溶液、巴比妥钠-HCl缓冲溶液、NH4HCO3缓冲溶液、碳酸钠-碳酸氢钠缓冲溶液、NaHCO3缓冲溶液、Tris-HCl、甘氨酸-NaOH缓冲溶液、硼酸-硼砂缓冲溶液、Na2B7O4缓冲溶液中的一种或两种以上。The buffered saline solution is selected from one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution (PBS), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate- NaOH buffer solution, sodium barbital-HCl buffer solution, NH4HCO3 buffer solution, sodium carbonate-sodium bicarbonate buffer solution, NaHCO3 buffer solution, Tris-HCl, glycine-NaOH buffer solution, boric acid- borax buffer solution, and Na2B7O4 buffer solution.
29.根据项26所述的制备方法，29. The preparation method according to item 26,
所述碱性溶液为氢氧化钠溶液。The alkaline solution is a sodium hydroxide solution.
30.根据项25所述的制备方法， 30. The preparation method according to item 25,
所述缓冲盐溶液中缓冲盐的浓度为0.001M～0.6M，优选为0.01M～0.6M,更优选为0.01M～0.5M。The concentration of the buffer salt in the buffer salt solution is 0.001M to 0.6M, preferably 0.01M to 0.6M, and more preferably 0.01M to 0.5M.
31.根据项25或26所述的制备方法，31. The preparation method according to item 25 or 26,
所述碱性溶液的浓度为2～10M，优选为3～8M，更优选为4～6M。The concentration of the alkaline solution is 2-10M, preferably 3-8M, and more preferably 4-6M.
32.根据项25所述的制备方法，32. The preparation method according to item 25,
所述缓冲盐溶液和所述碱性溶液的体积比为(0.1～100)：1，优选为(0.1～70)：1，更优选为(1～50)：1。The volume ratio of the buffered salt solution to the alkaline solution is (0.1-100):1, preferably (0.1-70):1, and more preferably (1-50):1.
33.根据项22所述的制备方法，33. The preparation method according to item 22,
酸催化剂选自对甲苯磺酸、三氟乙酸、乙酸、甲磺酸、盐酸、磷酸和磺酸中的一种或两种以上。The acid catalyst is selected from one or more of p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
34.根据项22所述的制备方法，34. The preparation method according to item 22,
对所述多肽衍生物的中间体进行冻干预处理，得到冻干预处理后的多肽衍生物的中间体；Performing freeze-thaw treatment on the intermediate of the polypeptide derivative to obtain the freeze-thaw treated intermediate of the polypeptide derivative;
对所述冻干预处理后的多肽衍生物的中间体进行脱保护，得到多肽衍生物。The intermediate of the polypeptide derivative after freeze-drying is deprotected to obtain the polypeptide derivative.
35.根据项22所述的制备方法，35. The preparation method according to item 22,
所述保护基选自C1-C21烷基、芳基中的任一种。The protecting group is selected from any one of a C1-C21 alkyl group and an aryl group.
36根据项35所述的制备方法，36. The preparation method according to item 35,
所述保护基为甲基、乙基、叔丁基或苄基。The protecting group is methyl, ethyl, tert-butyl or benzyl.
37.根据项22所述的制备方法，37. The preparation method according to item 22,
所述羧酸酯为脂肪酸酯。The carboxylic acid ester is a fatty acid ester.
38.根据项37所述的制备方法，38. The preparation method according to item 37,
所述脂肪酸为辛酸、壬酸、癸酸、十一酸、月桂酸、十三酸、肉豆蔻酸、十五酸、棕榈酸、十七酸、硬脂酸、十九酸、花生酸、二十一酸、二十二酸、二十三酸、二十四酸、棕榈油酸、油酸、亚麻油酸、蓖麻油酸、异柠檬酸、二十碳五烯酸或二十二碳六烯酸、1,8-辛二酸、1,7-庚二甲酸、1,10-癸二酸、十一烷二酸、十二烷二酸、十三烷二酸、十四烷二酸、十五烷二酸、十六烷二酸、十七烷二酸或十八烷二酸中的一种或两种以上。The fatty acid is one or more of octanoic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanedioic acid, palmitic acid, heptadecanedioic acid, stearic acid, nonadecanedioic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosapentaenoic acid or docosahexaenoic acid, 1,8-octanedioic acid, 1,7-heptanedicarboxylic acid, 1,10-decanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid or octadecanedioic acid.
39.根据项34所述的制备方法，39. The preparation method according to item 34,
所述冻干预处理包括如下步骤：The freezing treatment comprises the following steps:
预冻处理； Pre-freezing treatment;
冷冻处理；Freezing treatment;
一次干燥处理；以及A primary drying process; and
进一步可以包括任选二次干燥处理。An optional secondary drying process may further be included.
40.根据项39所述的制备方法，40. The preparation method according to item 39,
所述预冻处理的预冻温度为2～8℃，预冻处理的时间为大于等于2.0小时；或者The pre-freezing temperature of the pre-freezing treatment is 2 to 8° C., and the pre-freezing time is greater than or equal to 2.0 hours; or
所述冷冻处理的冷冻温度为-30℃～-60℃，冷冻处理的时间为3小时以上；或者The freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing treatment time is more than 3 hours; or
所述一次干燥处理的温度为-10℃～0℃，一次干燥处理的真空度为0.05-2.0mbar，一次干燥处理的干燥时间为大于等于20小时；The temperature of the primary drying process is -10°C to 0°C, the vacuum degree of the primary drying process is 0.05-2.0 mbar, and the drying time of the primary drying process is greater than or equal to 20 hours;
所述二次干燥处理的温度为20℃～65℃，二次干燥处理的真空度为0.05-2.0mbar，二次干燥处理的干燥时间为大于0小时且在8小时以下。The temperature of the secondary drying treatment is 20° C. to 65° C., the vacuum degree of the secondary drying treatment is 0.05-2.0 mbar, and the drying time of the secondary drying treatment is greater than 0 hours and less than 8 hours.
41.根据项22所述的制备方法，41. The preparation method according to item 22,
多肽选自胰岛素、胰高血糖素、肠促胰岛素、GLP-1、甲状旁腺素(PTH)、GLP-2、胃泌酸调节素、淀粉不溶素(Amylin)、肠高血糖素、生长激素抑制素、瘦素、YY肽、去氨加压素、骨钙蛋白、人生长激素、糖肽抗生素、非核糖体肽抗生素、促皮质素、降钙素、催产素、干扰素、纤维蛋白溶媒、抗利尿激素、白细胞介素、尿激酶、促甲状腺激素释放激素、甲硫氨酸脑啡肽、亮氨酸脑啡肽、前列腺素F2α受体调节剂、肥胖抑制素、分泌素、nesfatin、葡萄糖依赖性促胰岛素肽(GIP)及上述多肽的类似物或片段中的一种或两种以上。The polypeptide is selected from one or more of insulin, glucagon, incretin, GLP-1, parathyroid hormone (PTH), GLP-2, oxyntomodulin, amylin, enteroglucagon, somatostatin, leptin, YY peptide, desmopressin, osteocalcin, human growth hormone, glycopeptide antibiotics, non-ribosomal peptide antibiotics, corticotropin, calcitonin, oxytocin, interferon, fibrinolytic, antidiuretic hormone, interleukin, urokinase, thyrotropin-releasing hormone, methionine enkephalin, leucine enkephalin, prostaglandin F2α receptor modulator, obestatin, secretin, nesfatin, glucose-dependent insulinotropic peptide (GIP) and analogs or fragments of the above polypeptides.
42.根据项41所述的制备方法，42. The preparation method according to item 41,
所述多肽选自GLP-1、GLP-1的药学上可接受的盐、GLP-1类似物、GLP-1类似物的药学上可接受的盐。The polypeptide is selected from GLP-1, a pharmaceutically acceptable salt of GLP-1, a GLP-1 analog, and a pharmaceutically acceptable salt of a GLP-1 analog.
43.一种多肽衍生物的制备方法，包括：43. A method for preparing a polypeptide derivative, comprising:
得到多肽衍生物中间体，所述多肽衍生物中间体为含有保护基的羧酸酯修饰的多肽；对所述多肽衍生物的中间体进行冻干预处理，得到冻干预处理后的多肽衍生物的中间体；Obtaining a polypeptide derivative intermediate, wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group; subjecting the polypeptide derivative intermediate to freeze-thaw treatment to obtain a polypeptide derivative intermediate after freeze-thaw treatment;
所述冻干预处理后的多肽衍生物的中间体在酸催化剂溶液条件下进行脱保护，得到脱保护反应液；The intermediate of the polypeptide derivative after the freeze-dried tube is deprotected under the condition of an acid catalyst solution to obtain a deprotection reaction solution;
将所述脱保护反应液加入到终止液中，调节pH值以终止反应，得到多 肽衍生物。The deprotection reaction solution is added to the stop solution, and the pH value is adjusted to stop the reaction to obtain a plurality of Peptide derivatives.
44.根据项43所述的制备方法，44. The preparation method according to item 43,
所述保护基选自C1～C21烷基、芳基中的任一种。The protecting group is selected from any one of C1-C21 alkyl and aryl groups.
45.根据项44所述的制备方法，45. The preparation method according to item 44,
所述保护基为甲基、乙基、叔丁基或苄基。The protecting group is methyl, ethyl, tert-butyl or benzyl.
46.根据项43所述的制备方法，46. The preparation method according to item 43,
所述羧酸酯为脂肪酸酯。The carboxylic acid ester is a fatty acid ester.
47.根据项46所述的制备方法，47. The preparation method according to item 46,
所述脂肪酸为辛酸、壬酸、癸酸、十一酸、月桂酸、十三酸、肉豆蔻酸、十五酸、棕榈酸、十七酸、硬脂酸、十九酸、花生酸、二十一酸、二十二酸、二十三酸、二十四酸、棕榈油酸、油酸、亚麻油酸、蓖麻油酸、异柠檬酸、二十碳五烯酸或二十二碳六烯酸、1,8-辛二酸、1,7-庚二甲酸、1,10-癸二酸、十一烷二酸、十二烷二酸、十三烷二酸、十四烷二酸、十五烷二酸、十六烷二酸、十七烷二酸或十八烷二酸中的一种或两种以上。The fatty acid is one or more of octanoic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanedioic acid, palmitic acid, heptadecanedioic acid, stearic acid, nonadecanedioic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosapentaenoic acid or docosahexaenoic acid, 1,8-octanedioic acid, 1,7-heptanedicarboxylic acid, 1,10-decanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid or octadecanedioic acid.
48.根据项43所述的制备方法，48. The preparation method according to item 43,
所述冻干预处理包括如下步骤：The freezing treatment comprises the following steps:
预冻处理；Pre-freezing treatment;
冷冻处理；Freezing treatment;
一次干燥处理；以及A primary drying process; and
进一步可以包括任选二次干燥处理。An optional secondary drying process may further be included.
49.根据项48所述的制备方法，49. The preparation method according to item 48,
所述预冻处理的预冻温度为2～8℃，预冻处理的时间为大于等于2.0小时；或者The pre-freezing temperature of the pre-freezing treatment is 2 to 8° C., and the pre-freezing time is greater than or equal to 2.0 hours; or
所述冷冻处理的冷冻温度为-30℃～-60℃，冷冻处理的时间为3小时以上；或者The freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing treatment time is more than 3 hours; or
所述一次干燥处理的温度为-10℃～0℃，一次干燥处理的真空度为0.05～2.0mbar，一次干燥处理的干燥时间为大于等于20小时；The temperature of the primary drying process is -10°C to 0°C, the vacuum degree of the primary drying process is 0.05 to 2.0 mbar, and the drying time of the primary drying process is greater than or equal to 20 hours;
所述二次干燥处理的温度为20℃～65℃，二次干燥处理的真空度为0.05～2.0mbar，二次干燥处理的干燥时间为大于0小时且在8小时以下。The temperature of the secondary drying process is 20° C. to 65° C., the vacuum degree of the secondary drying process is 0.05 to 2.0 mbar, and the drying time of the secondary drying process is greater than 0 hours and less than 8 hours.
50.根据项43所述的制备方法，50. The preparation method according to item 43,
多肽选自胰岛素、胰高血糖素、肠促胰岛素、GLP-1、甲状旁腺素(PTH)、 GLP-2、胃泌酸调节素、淀粉不溶素(Amylin)、肠高血糖素、生长激素抑制素、瘦素、YY肽、去氨加压素、骨钙蛋白、人生长激素、糖肽抗生素、非核糖体肽抗生素、促皮质素、降钙素、催产素、干扰素、纤维蛋白溶媒、抗利尿激素、白细胞介素、尿激酶、促甲状腺激素释放激素、甲硫氨酸脑啡肽、亮氨酸脑啡肽、前列腺素F2α受体调节剂、肥胖抑制素、分泌素、nesfatin、葡萄糖依赖性促胰岛素肽(GIP)及上述多肽的类似物或片段中的一种或两种以上。The polypeptide is selected from insulin, glucagon, incretin, GLP-1, parathyroid hormone (PTH), GLP-2, oxyntomodulin, amylin, enteroglucagon, somatostatin, leptin, YY peptide, desmopressin, osteocalcin, human growth hormone, glycopeptide antibiotics, non-ribosomal peptide antibiotics, corticotropin, calcitonin, oxytocin, interferon, fibrinolytic agent, antidiuretic hormone, interleukin, urokinase, thyrotropin-releasing hormone, methionine enkephalin, leucine enkephalin, prostaglandin F2α receptor modulator, obestatin, secretin, nesfatin, glucose-dependent insulinotropic peptide (GIP) and one or more of the analogs or fragments of the above polypeptides.
51.根据项50所述的制备方法，51. The preparation method according to item 50,
所述多肽选自GLP-1、GLP-1的药学上可接受的盐、GLP-1类似物、GLP-1类似物的药学上可接受的盐。The polypeptide is selected from GLP-1, a pharmaceutically acceptable salt of GLP-1, a GLP-1 analog, and a pharmaceutically acceptable salt of a GLP-1 analog.
52.根据项43所述的制备方法，52. The preparation method according to item 43,
调节pH值至6.5～10.0以终止反应，得到多肽衍生物。The pH value is adjusted to 6.5-10.0 to terminate the reaction and obtain a polypeptide derivative.
53.根据项52所述的制备方法，53. The preparation method according to item 52,
调节pH值至7.5～9.5以终止反应，得到多肽衍生物。The pH value is adjusted to 7.5-9.5 to terminate the reaction and obtain a polypeptide derivative.
54.根据项43所述的制备方法，54. The preparation method according to item 43,
所述终止液包含缓冲盐溶液和碱性溶液。The stop solution comprises a buffered salt solution and an alkaline solution.
55.根据项54所述的制备方法，55. The preparation method according to item 54,
所述碱性溶液选自氢氧化钠溶液、氢氧化钾溶液、碳酸钠溶液、碳酸钾溶液、碳酸氢钠溶液、碳酸氢钾溶液、碳酸氢铵溶液中的一种或两种以上。The alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, and ammonium bicarbonate solution.
56.根据项54所述的制备方法，56. The preparation method according to item 54,
所述缓冲盐溶液选自磷酸盐类缓冲溶液、Tris类缓冲溶液、有机酸类缓冲溶液、硼酸盐类缓冲溶液、氨基酸类缓冲溶液中的一种或两种以上，The buffer salt solution is selected from one or more of a phosphate buffer solution, a Tris buffer solution, an organic acid buffer solution, a borate buffer solution, and an amino acid buffer solution.
57.根据项56所述的制备方法，57. The preparation method according to item 56,
所述缓冲盐溶液选自磷酸氢二钠-柠檬酸缓冲溶液、柠檬酸-NaOH-HCl缓冲溶液、柠檬酸-柠檬酸钠缓冲溶液、乙酸-乙酸钠缓冲溶液、磷酸盐缓冲溶液(PBS)、磷酸氢二钠-磷酸二氢钠缓冲溶液(PB)、磷酸氢二钠-磷酸二氢钾缓冲溶液、磷酸二氢钾-NaOH缓冲溶液、巴比妥钠-HCl缓冲溶液、NH4HCO3缓冲溶液、碳酸钠-碳酸氢钠缓冲溶液、NaHCO3缓冲溶液、Tris-HCl、甘氨酸-NaOH缓冲溶液、硼酸-硼砂缓冲溶液、Na2B7O4缓冲溶液中的一种或两种以上。The buffered saline solution is selected from one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution (PBS), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate- NaOH buffer solution, sodium barbital-HCl buffer solution, NH4HCO3 buffer solution, sodium carbonate-sodium bicarbonate buffer solution, NaHCO3 buffer solution, Tris-HCl, glycine-NaOH buffer solution, boric acid- borax buffer solution, and Na2B7O4 buffer solution.
58.根据项54所述的制备方法， 58. The preparation method according to item 54,
所述碱性溶液溶液为氢氧化钠溶液。The alkaline solution is a sodium hydroxide solution.
59.根据项54所述的制备方法，59. The preparation method according to item 54,
所述缓冲盐溶液中缓冲盐的浓度为0.001M～0.6M，优选为0.01M～0.6M,更优选为0.01M～0.5M。The concentration of the buffer salt in the buffer salt solution is 0.001M to 0.6M, preferably 0.01M to 0.6M, and more preferably 0.01M to 0.5M.
60.根据项54或58所述的制备方法，60. The preparation method according to item 54 or 58,
所述碱性溶液的浓度为2～10M，优选为3～8M，更优选为4～6M。The concentration of the alkaline solution is 2-10M, preferably 3-8M, and more preferably 4-6M.
61.根据项54所述的制备方法，61. The preparation method according to item 54,
所述缓冲盐溶液和所述碱性溶液的体积比为(0.1～100)：1，优选为(0.1～70)：1，更优选为(1～50)：1。The volume ratio of the buffered salt solution to the alkaline solution is (0.1-100):1, preferably (0.1-70):1, and more preferably (1-50):1.
62.根据项43所述的制备方法，62. The preparation method according to item 43,
酸催化剂选自对甲苯磺酸、三氟乙酸、乙酸、甲磺酸、盐酸、磷酸和磺酸中的一种或两种以上。The acid catalyst is selected from one or more of p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
63.一种多肽衍生物，其中，所述多肽衍生物经项1～64任一项所述方法制备而得。63. A polypeptide derivative, wherein the polypeptide derivative is prepared by the method described in any one of items 1 to 64.
与现有技术相比，本申请的有益效果为：Compared with the prior art, the beneficial effects of this application are:
本申请提供了一种新的多肽衍生物脱保护工艺，一方面，通过在传统酸催化剂作用下进行脱保护反应进行前增加对脱保护蛋白冻干预处理环节，有效提高了脱保护蛋白浓度上限，极大提高蛋白质生产中脱保护工艺的效益和效率；另一方面，通过调整脱保护终止液添加方式，由常规的将脱保护液加入脱保护反应液中调整为将反应液加入脱保护液中，极大降低了脱保护过程中异构体杂质含量且提高了操作方法稳定性，进一步的，通过对该终止方式中增加缓冲溶液、调整缓冲溶液成分体积等优化，进一步提高了脱保护效果和稳定性。克服了多肽分子脱保护过程中的技术瓶颈，具备明显的工业化大规模生产实用性。The present application provides a new deprotection process for polypeptide derivatives. On the one hand, by adding a deprotection protein freeze-intervention treatment link before the deprotection reaction under the action of a traditional acid catalyst, the upper limit of the deprotection protein concentration is effectively increased, greatly improving the benefits and efficiency of the deprotection process in protein production; on the other hand, by adjusting the deprotection termination solution addition method, the conventional addition of the deprotection solution to the deprotection reaction solution is adjusted to the addition of the reaction solution to the deprotection solution, which greatly reduces the isomer impurity content in the deprotection process and improves the stability of the operation method. Further, by optimizing the addition of a buffer solution to the termination method, adjusting the volume of the buffer solution components, etc., the deprotection effect and stability are further improved. The technical bottleneck in the deprotection process of polypeptide molecules is overcome, and it has obvious practicality for industrial large-scale production.
具体实施方式Detailed ways
下面所给出的定义是为了更清楚地说明它们在说明书和权利要求书中使用的含义和范围。The definitions set forth below are intended to more clearly illustrate their meaning and scope as they are used in the specification and claims.
本申请的“多肽”或“肽”或“蛋白质”可以交替使用。“多肽”或“肽”或“蛋白质”是包含两种或两种以上的氨基酸的任意链，无论翻译后修饰(例如，糖基化或磷酸化)的情况如何，包括天然发生或非天然发生的氨基酸或 氨基酸类似物，其中任意链中的氨基酸通过肽键共价连接。在一些实施方式中可以为天然存在的蛋白质、或者是非重组细胞或基因工程或重组细胞产生的蛋白质，并且包含具有天然蛋白质的氨基酸序列的分子，或具有天然序列的一个或多个氨基酸的缺失、添加和/或取代的分子。The term "polypeptide" or "peptide" or "protein" may be used interchangeably herein. A "polypeptide" or "peptide" or "protein" is any chain of two or more amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), including naturally occurring or non-naturally occurring amino acids or Amino acid analogs, wherein the amino acids in any chain are covalently linked by peptide bonds. In some embodiments, they can be naturally occurring proteins, or proteins produced by non-recombinant cells or genetically engineered or recombinant cells, and include molecules having the amino acid sequence of a natural protein, or molecules having one or more amino acids deleted, added and/or substituted from the natural sequence.
在本领域中，众所周知可以在多肽的结构中进行诸如取代、添加或替换的某些修饰和变化而基本上不改变该多肽的生物学功能，从而得到生物学等同的多肽。It is well known in the art that certain modifications and changes such as substitutions, additions or replacements can be made in the structure of a polypeptide without substantially changing the biological function of the polypeptide, thereby obtaining a biologically equivalent polypeptide.
本申请的“多肽”或“肽”或“蛋白质”可包含有异常连接、交联和末端帽子，非肽键或其它修饰基团。这些修饰基团也在本申请范围之内。术语“修饰基团”是指包括直接附着于所述肽结构的结构，以及那些间接附着于所述肽结构的结构。例如，所述修饰基团可以偶联于肽结构的氨基末端或羧基末端，或偶联于所述核心结构域侧翼的肽。或者，所述修饰基团可以连接于肽结构的至少一个氨基酸残基的侧链，或偶联于所述核心结构域侧翼的肽或肽模拟区域(例如，通过赖氨酰残基的ε氨基，通过天冬氨酸残基或谷氨酸残基的羧基，通过酪氨酰残基、丝氨酸残基或苏氨酸残基的羟基，或在氨基酸侧链上的其它适合的反应基团)。共价偶联于所述肽结构的修饰基团可通过本领域中公知的方式和方法结合于连接性化学结构，包括，例如酰胺，烷氨基，氨基甲酸盐或尿素键。The "polypeptide" or "peptide" or "protein" of the present application may include abnormal connections, crosslinks and end caps, non-peptide bonds or other modifying groups. These modifying groups are also within the scope of the present application. The term "modifying group" refers to structures directly attached to the peptide structure, as well as those indirectly attached to the peptide structure. For example, the modifying group can be coupled to the amino terminal or carboxyl terminal of the peptide structure, or coupled to the peptide flanking the core domain. Alternatively, the modifying group can be connected to the side chain of at least one amino acid residue of the peptide structure, or coupled to the peptide or peptide-mimicking region flanking the core domain (for example, through the epsilon amino group of a lysinyl residue, through the carboxyl group of an aspartic acid residue or a glutamic acid residue, through the hydroxyl group of a tyrosinyl residue, a serine residue or a threonine residue, or other suitable reactive groups on the amino acid side chain). The modifying group covalently coupled to the peptide structure can be combined with a connecting chemical structure by means and methods well known in the art, including, for example, amide, alkylamino, carbamate or urea bonds.
在本申请的一些实施方式中，多肽选自胰岛素、胰高血糖素、肠促胰岛素、GLP-1、甲状旁腺素(PTH)、GLP-2、胃泌酸调节素、淀粉不溶素(Amylin)、肠高血糖素、生长激素抑制素、瘦素、YY肽、去氨加压素、骨钙蛋白、人生长激素、糖肽抗生素、非核糖体肽抗生素、促皮质素、降钙素、催产素、干扰素、纤维蛋白溶媒、抗利尿激素、白细胞介素、尿激酶、促甲状腺激素释放激素、甲硫氨酸脑啡肽、亮氨酸脑啡肽、前列腺素F2α受体调节剂、肥胖抑制素、分泌素、nesfatin、葡萄糖依赖性促胰岛素肽(GIP)及上述多肽的类似物或片段中的一种或两种以上。In some embodiments of the present application, the polypeptide is selected from insulin, glucagon, incretin, GLP-1, parathyroid hormone (PTH), GLP-2, oxyntomodulin, amylin, enteroglucagon, somatostatin, leptin, YY peptide, desmopressin, osteocalcin, human growth hormone, glycopeptide antibiotics, non-ribosomal peptide antibiotics, corticotropin, calcitonin, oxytocin, interferon, fibrinolytic, antidiuretic hormone, interleukin, urokinase, thyrotropin-releasing hormone, methionine enkephalin, leucine enkephalin, prostaglandin F2α receptor modulator, obestatin, secretin, nesfatin, glucose-dependent insulinotropic peptide (GIP) and one or more of the analogs or fragments of the above polypeptides.
在本申请的一些实施方式中，所述多肽选自GLP-1、GLP-1的药学上可接受的盐、GLP-1类似物、GLP-1类似物的药学上可接受的盐。In some embodiments of the present application, the polypeptide is selected from GLP-1, a pharmaceutically acceptable salt of GLP-1, a GLP-1 analog, and a pharmaceutically acceptable salt of a GLP-1 analog.
在本申请中，“多肽衍生物”是指多肽的功能基团(如氨基酸残基)被某些化合物(如小分子化合物)修饰后所得到的产物。所述修饰包括但不限于酰基化、酰胺化、酯化、硫酯化。 In this application, "polypeptide derivatives" refer to products obtained by modifying the functional groups (such as amino acid residues) of polypeptides by certain compounds (such as small molecule compounds). The modifications include but are not limited to acylation, amidation, esterification, and thioesterification.
在本申请中，多肽衍生物中间体为经含有保护基的羧酸酯修饰的多肽。In the present application, the polypeptide derivative intermediate is a polypeptide modified with a carboxylate containing a protecting group.
在本申请中，需要对多肽衍生物中的羧基进行保护，不对羧基的位置进行限定，此时的羧基可以是多肽衍生物分子中任何位置的羧基，例如，羧基可以为来源于多肽衍生物分子中的氨基酸骨架上的羧酸，也可以来源于多肽衍生物分子中的侧链上的羧酸。所采用的羧基的保护基团的种类并不重要，只要衍生的保护羧酸的基团(如含有保护基的羧酸酯)对随后的反应条件稳定，且可以在不破坏分子的其余部分的情况下，在适宜的时机去除即可。In the present application, the carboxyl group in the polypeptide derivative needs to be protected, and the position of the carboxyl group is not limited. The carboxyl group at this time can be a carboxyl group at any position in the polypeptide derivative molecule, for example, the carboxyl group can be a carboxylic acid derived from the amino acid backbone in the polypeptide derivative molecule, or a carboxylic acid derived from the side chain in the polypeptide derivative molecule. The type of the carboxyl protecting group used is not important, as long as the derived carboxylic acid protecting group (such as a carboxylate containing a protecting group) is stable to the subsequent reaction conditions and can be removed at an appropriate time without destroying the rest of the molecule.
在本申请中，羧酸酯指的是-C(O)OR，其中R为烷基、芳基、芳烷基和脂环，所有羧酸酯可任选取代。In the present application, carboxylate refers to -C(O)OR, wherein R is alkyl, aryl, aralkyl and alicyclic, and all carboxylates may be optionally substituted.
在本申请的一些实施方式中，烷基表示二价烷基或亚烷基；In some embodiments of the present application, alkyl represents a divalent alkyl group or an alkylene group;
在本申请的一些实施方式中，芳基表示具有5-14个环原子和至少一个具有共轭π电子系统的环的芳基并包括碳环芳基、杂环芳基和联芳基，所有这些基团可任选取代，碳环芳基为其中芳族环上的环原子是碳原子的基团。碳环芳基包括单环碳环芳基和多环或稠合化合物，例如任选取代的萘基。杂环芳基或杂芳基为具有1-4个杂原子作为芳族环的环原子且其余的环原子是碳原子的基团。适宜的杂原子包括氧、硫、氮和硒。适宜的杂芳基包括呋喃基、噻吩基、吡啶基、吡咯基、N-低级烷基吡咯基、吡啶基-N-氧化物、嘧啶基、吡嗪基、咪唑基等，所有杂芳基可任选被取代。In some embodiments of the present application, aryl represents an aryl having 5-14 ring atoms and at least one ring having a conjugated π electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl, all of which groups may be optionally substituted, and carbocyclic aryl is a group in which the ring atoms on the aromatic ring are carbon atoms. Carbocyclic aryl includes monocyclic carbocyclic aryl and polycyclic or fused compounds, such as optionally substituted naphthyl. Heterocyclic aryl or heteroaryl is a group having 1-4 heteroatoms as the ring atoms of the aromatic ring and the remaining ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, nitrogen and selenium. Suitable heteroaryl includes furanyl, thienyl, pyridyl, pyrrolyl, N-low alkyl pyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl, imidazolyl, etc., and all heteroaryl may be optionally substituted.
在本申请的一些实施方式中，联芳基表示包含多于一个芳族环的芳基，包括稠合环系统和用其它的芳基取代的芳基，这样的基团可任选取代。In some embodiments of the present application, biaryl refers to an aryl group containing more than one aromatic ring, including fused ring systems and aryl groups substituted with other aryl groups, such groups being optionally substituted.
在本申请的一些实施方式中，脂环表示兼有脂族和环状化合物性质的化合物。这样的环状化合物包括(但不限于)芳族、环烷基和桥接环烷基化合物。环状化合物包括杂环。这样的基团可任选取代。In some embodiments of the present application, alicyclic refers to a compound that has both aliphatic and cyclic properties. Such cyclic compounds include (but are not limited to) aromatic, cycloalkyl and bridged cycloalkyl compounds. Cyclic compounds include heterocycles. Such groups may be optionally substituted.
在本申请的一些实施方式中，“多肽类似物”是指在功能上基本上类似于天然肽或蛋白或其片段的分子。In some embodiments of the present application, "polypeptide analogs" refer to molecules that are substantially similar in function to a natural peptide or protein or a fragment thereof.
在本申请的一些实施方式中，GLP-1类似物是指将被选择，以保持天然底物的体外或体内活性的一种或更多种。体外和体内活性可采用适于GLP-1类似物的、本领域普通技术人员可利用的任何方案加以测量。可被测量以确定GLP-1类似物是否保持相同或相似功能活性的示例性功能活性包括类似物在基于细胞的测定或无细胞测定中结合其受体(一种或多种)的能力、类似物诱导应答于GLP-1R的细胞变化(例如，增殖、分化、存活、生长、迁移 等等)、类似物调节在细胞中应答于GLP-1的一个或更多个其它基因或蛋白质的表达的能力。In some embodiments of the present application, a GLP-1 analog refers to one or more that will be selected to maintain the in vitro or in vivo activity of the natural substrate. In vitro and in vivo activity can be measured using any protocol that is suitable for GLP-1 analogs and available to those of ordinary skill in the art. Exemplary functional activities that can be measured to determine whether a GLP-1 analog maintains the same or similar functional activity include the ability of the analog to bind to its receptor(s) in a cell-based assay or a cell-free assay, the analog to induce changes in cells that respond to the GLP-1R (e.g., proliferation, differentiation, survival, growth, migration, etc.), the ability of an analog to modulate the expression of one or more other genes or proteins in a cell that are responsive to GLP-1.
GLP-1类似物具有与天然GLP-1或其片段基本上相似的活性(例如，活性为天然GLP-1的大约80％、90％、100％、110％或120％)。在一些实施方式中，类似物活性较天然GLP-1低(例如，活性为该天然多肽的大约50％、60％、70％或75％)。我们注意到，如果活性降低仍提供在足够的时间期间内提供足够的类似物局部浓度的能力，活性稍微低一些的类似物可能是有用的，例如在体内或在细胞培养物中。因此，例如通过蛋白酶抗性得到的半衰期增加可补偿类似物的构建所导致的活性降低。在另外的实施方式中，类似物比天然GLP-1活性更强(例如，活性为天然GLP-1的大约130％、150％、175％、200％、300％、500％、800％或甚至1000％)。在前述的任一种中，“活性”指天然GLP-1的一种或多种功能。例如，类似物的活性(例如，生物功能)可以是受体结合、作为转录激活子或抑制子起作用的能力、参与特定的信号转导通路的能力、或影响细胞行为(例如，增殖、分化、存活或迁移)的能力。GLP-1 analogs have substantially similar activity to native GLP-1 or a fragment thereof (e.g., activity is about 80%, 90%, 100%, 110% or 120% of native GLP-1). In some embodiments, the analog is less active than native GLP-1 (e.g., activity is about 50%, 60%, 70% or 75% of the native polypeptide). We note that analogs with slightly less activity may be useful, for example in vivo or in cell culture, if the reduced activity still provides the ability to provide sufficient local concentrations of the analog for a sufficient period of time. Thus, an increase in half-life, such as through protease resistance, may compensate for reduced activity resulting from the construction of the analog. In other embodiments, the analog is more active than native GLP-1 (e.g., activity is about 130%, 150%, 175%, 200%, 300%, 500%, 800% or even 1000% of native GLP-1). In any of the foregoing, "activity" refers to one or more functions of native GLP-1. For example, the activity (e.g., biological function) of an analog can be receptor binding, the ability to act as a transcriptional activator or repressor, the ability to participate in a particular signal transduction pathway, or the ability to affect cell behavior (e.g., proliferation, differentiation, survival, or migration).
在本申请的一些实施方式中，所述多肽衍生物通过脂肪酸以酰胺键的形式对多肽进行偶联。In some embodiments of the present application, the polypeptide derivative is coupled to the polypeptide via a fatty acid in the form of an amide bond.
在本申请的一些实施方式中，脂肪酸为C1～C25脂肪酸。例如甲酸、乙酸、C3脂肪酸、C4脂肪酸、C5脂肪酸、C6脂肪酸、C7脂肪酸、C8脂肪酸、C9脂肪酸、C10脂肪酸、C11脂肪酸、C12脂肪酸、C13脂肪酸、C14脂肪酸、C15脂肪酸、C16脂肪酸、C17脂肪酸、C18脂肪酸、C19脂肪酸、C20脂肪酸、C21脂肪酸、C22脂肪酸、C23脂肪酸、C24脂肪酸或C25脂肪酸。In some embodiments of the present application, the fatty acid is a C1 to C25 fatty acid, such as formic acid, acetic acid, C3 fatty acid, C4 fatty acid, C5 fatty acid, C6 fatty acid, C7 fatty acid, C8 fatty acid, C9 fatty acid, C10 fatty acid, C11 fatty acid, C12 fatty acid, C13 fatty acid, C14 fatty acid, C15 fatty acid, C16 fatty acid, C17 fatty acid, C18 fatty acid, C19 fatty acid, C20 fatty acid, C21 fatty acid, C22 fatty acid, C23 fatty acid, C24 fatty acid or C25 fatty acid.
本申请中，所述的C1～C21脂肪酸包括直链脂肪酸和支链脂肪酸。In the present application, the C1-C21 fatty acids include straight-chain fatty acids and branched-chain fatty acids.
本申请中，所述的C1～C21脂肪酸包括饱和脂肪酸和不饱和脂肪酸。In the present application, the C1-C21 fatty acids include saturated fatty acids and unsaturated fatty acids.
在本申请的一些实施方式中，所述脂肪酸为辛酸、壬酸、癸酸、十一酸、月桂酸、十三酸、肉豆蔻酸、十五酸、棕榈酸、十七酸、硬脂酸、十九酸、花生酸、二十一酸、二十二酸、二十三酸、二十四酸、棕榈油酸、油酸、亚麻油酸、蓖麻油酸、异柠檬酸、二十碳五烯酸或二十二碳六烯酸、1,8-辛二酸、1,7-庚二甲酸、1,10-癸二酸、十一烷二酸、十二烷二酸、十三烷二酸、十四烷二酸、十五烷二酸、十六烷二酸、十七烷二酸或十八烷二酸中的一种 或两种以上。In some embodiments of the present application, the fatty acid is one of caprylic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanedioic acid, palmitic acid, heptadecanedioic acid, stearic acid, nonadecanedioic acid, arachidic acid, heneicosanoic acid, hexadecanoic acid, hexadecanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosapentaenoic acid or docosahexaenoic acid, 1,8-octanedioic acid, 1,7-heptanedicarboxylic acid, 1,10-decanedioic acid, undecanoic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid or octadecanedioic acid. Or two or more.
在本申请中，术语“药学上可接受的盐”是指保持了本申请的多肽类似物的衍生物的生物有效性和性质的盐，并且其通常在生物上或在其它方面不是不期望的。在许多情形下，本申请的多肽类似物的衍生物通过氨基和/或羧基或与其类似的基团的存在，可形成酸和/或碱盐。In the present application, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the derivatives of the polypeptide analogs of the present application, and is generally not undesirable biologically or otherwise. In many cases, the derivatives of the polypeptide analogs of the present application can form acid and/or base salts through the presence of amino and/or carboxyl groups or groups similar thereto.
可使用无机酸及有机酸来形成药学上可接受的酸加成盐，例如，乙酸盐、天冬氨酸盐、苯甲酸盐、苯磺酸盐、溴化物/氢溴酸盐、碳酸氢盐/碳酸盐、硫酸氢盐/硫酸盐、樟脑磺酸盐、氯化物/盐酸盐、chlortheophyllonate、柠檬酸盐、乙二磺酸盐、富马酸盐、葡庚糖酸盐、葡萄糖酸盐、葡糖醛酸盐、马尿酸盐、氢碘酸盐/碘化物、羟乙磺酸盐、乳酸盐、乳糖酸盐、月桂基硫酸盐、苹果酸盐、马来酸盐、丙二酸盐、扁桃酸盐、甲磺酸盐、甲基硫酸盐、萘酸盐、萘磺酸盐、烟酸盐、硝酸盐、十八烷酸盐、油酸盐、草酸盐、棕榈酸盐、巴莫酸盐、磷酸盐/磷酸氢盐/磷酸二氢盐、聚半乳糖醛酸盐、丙酸盐、硬脂酸盐、琥珀酸盐、磺基水杨酸盐、酒石酸盐、甲苯磺酸盐及三氟乙酸盐。Pharmaceutically acceptable acid addition salts can be formed using inorganic and organic acids, for example, acetate, aspartate, benzoate, benzenesulfonate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlortheophyllonate, citrate, edisylate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthoate, naphthylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogenphosphate/dihydrogenphosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, toluenesulfonate, and trifluoroacetate.
可由其衍生盐的无机酸包括，例如，盐酸、氢溴酸、硫酸、硝酸、磷酸等。Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
可由其衍生盐的有机酸包括，例如，乙酸、丙酸、羟乙酸、草酸、马来酸、丙二酸、琥珀酸、富马酸、酒石酸、柠檬酸、苯甲酸、扁桃酸、甲烷磺酸、乙烷磺酸、甲苯磺酸、磺基水杨酸等。可与无机碱及有机碱来形成药学上可接受的碱加成盐。Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, etc. Pharmaceutically acceptable base addition salts can be formed with inorganic bases and organic bases.
可由其衍生盐的无机碱包括，例如，铵盐及周期表第I族至第XII族的金属。在某些实施方案中，所述盐衍生自钠、钾、铵、钙、镁、铁、银、锌及铜；尤其适宜的盐包括铵盐、钾盐、钠盐、钙盐及镁盐。Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from Groups I to XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.
可由其衍生盐的有机碱可包括，例如，伯、仲和叔胺、取代胺包括天然存在的取代胺、环状胺、碱性离子交换树脂等。某些有机胺包括异丙基胺、胆碱盐、二乙醇胺、二乙胺、赖氨酸、葡甲胺、哌嗪及氨丁三醇。Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, choline salts, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.
本申请的药学上可接受的盐可通过惯用化学方法从母体化合物、碱性或酸性部分来合成。通常，这些盐可通过使这些化合物的游离酸形式与化学计量的适当碱(例如Na或K的氢氧化物、碳酸盐、碳酸氢盐等)进行反应来制备，或通过使这些化合物的游离碱形式与化学计量的适当酸进行反应来制备。这些反应通常是在水或有机溶剂、或二者的混合物中进行。通常，在可行的 情况下，使用非水性介质，如醚、乙酸乙酯、乙醇、异丙醇或乙腈是合乎需要的。The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound, alkaline or acidic part by conventional chemical methods. Generally, these salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base (e.g., hydroxide, carbonate, bicarbonate, etc. of Na or K), or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. These reactions are usually carried out in water or an organic solvent, or a mixture of the two. Generally, in a feasible In some cases, it may be desirable to use a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile.
本申请提供了一种多肽衍生物的制备方法，其中，得到多肽衍生物中间体，所述多肽衍生物中间体是含有保护基的羧酸酯修饰的多肽；对所述多肽衍生物中间体进行冻干预处理，得到冻干预处理后的多肽衍生物的中间体；对所述冻干预处理后的多肽衍生物的中间体进行脱保护，得到多肽衍生物。The present application provides a method for preparing a polypeptide derivative, wherein a polypeptide derivative intermediate is obtained, wherein the polypeptide derivative intermediate is a carboxylate-modified polypeptide containing a protecting group; the polypeptide derivative intermediate is subjected to freeze-thaw treatment to obtain a freeze-thaw-treated polypeptide derivative intermediate; and the freeze-thaw-treated polypeptide derivative intermediate is deprotected to obtain a polypeptide derivative.
在本申请中，多肽衍生物中间体可以为湿固体，即得到多肽衍生物中间体的湿固体，所述多肽衍生物中间体是含有保护基的羧酸酯修饰的多肽；对所述多肽衍生物中间体的湿固体进行冻干预处理，得到冻干预处理后的多肽衍生物的中间体；对所述冻干预处理后的多肽衍生物的中间体进行脱保护，得到多肽衍生物。In the present application, the polypeptide derivative intermediate can be a wet solid, that is, a wet solid of a polypeptide derivative intermediate is obtained, wherein the polypeptide derivative intermediate is a carboxylate-modified polypeptide containing a protecting group; the wet solid of the polypeptide derivative intermediate is subjected to freeze-thaw treatment to obtain a freeze-thaw-treated polypeptide derivative intermediate; and the freeze-thaw-treated polypeptide derivative intermediate is deprotected to obtain a polypeptide derivative.
在本申请的一些实施方式中，所述保护基选自C1-C21烷基、芳基中的任一种。In some embodiments of the present application, the protecting group is selected from any one of a C1-C21 alkyl group and an aryl group.
在本申请的一些实施方式中，所述保护基选自C1-C10烷基、芳基中的任一种。In some embodiments of the present application, the protecting group is selected from any one of a C1-C10 alkyl group and an aryl group.
在本申请的一些实施方式中，所述保护基为甲基、乙基、叔丁基或苄基。In some embodiments of the present application, the protecting group is methyl, ethyl, tert-butyl or benzyl.
在本申请的一些实施方式中，所述羧酸酯为脂肪酸酯。In some embodiments of the present application, the carboxylic acid ester is a fatty acid ester.
在本申请的一些实施方式中，所述脂肪酸为辛酸、壬酸、癸酸、十一酸、月桂酸、十三酸、肉豆蔻酸、十五酸、棕榈酸、十七酸、硬脂酸、十九酸、花生酸、二十一酸、二十二酸、二十三酸、二十四酸、棕榈油酸、油酸、亚麻油酸、蓖麻油酸、异柠檬酸、二十碳五烯酸或二十二碳六烯酸、1,8-辛二酸、1,7-庚二甲酸、1,10-癸二酸、十一烷二酸、十二烷二酸、十三烷二酸、十四烷二酸、十五烷二酸、十六烷二酸、十七烷二酸或十八烷二酸中的一种或两种以上。In some embodiments of the present application, the fatty acid is one or more of caprylic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanedioic acid, stearic acid, nonadecanedioic acid, arachidic acid, heneicosanoic acid, hexadecanoic acid, hexadecanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosapentaenoic acid or docosahexaenoic acid, 1,8-octanedioic acid, 1,7-heptanedicarboxylic acid, 1,10-decanedioic acid, undecanoic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid or octadecanedioic acid.
本申请通过在传统酸催化剂脱保护反应进行前增加对脱保护蛋白冻干预处理环节，有效提高了脱保护蛋白浓度上限，极大提高蛋白质生产中脱保护工艺的效益和效率。The present application effectively increases the upper limit of the deprotected protein concentration by adding a deprotected protein freezing intervention treatment step before the traditional acid catalyst deprotection reaction, thereby greatly improving the benefit and efficiency of the deprotection process in protein production.
在本申请的一些实施方式中，所述冻干预处理包括：预冻处理；冷冻处理；一次干燥处理；以及进一步可以包括任选二次干燥处理。In some embodiments of the present application, the pre-freezing treatment includes: a pre-freezing treatment; a freezing treatment; a primary drying treatment; and may further include an optional secondary drying treatment.
在本申请的一些实施方式中，所述冻干预处理包括：预冻处理、冷冻处理及一次干燥处理。 In some embodiments of the present application, the pre-freezing treatment includes: pre-freezing treatment, freezing treatment and primary drying treatment.
在本申请的一些实施方式中，所述冻干预处理包括：预冻处理、冷冻处理、一次干燥处理和二次干燥处理。In some embodiments of the present application, the pre-freezing treatment includes: pre-freezing treatment, freezing treatment, primary drying treatment and secondary drying treatment.
在本申请的一些实施方式中，所述预冻处理的预冻温度为2～8℃，预冻处理的时间大于等于2.0小时；或者所述冷冻处理的冷冻温度为-30℃～-60℃，冷冻处理的时间为3小时以上；或者所述一次干燥处理的温度为-10℃～0℃，一次干燥处理的真空度为0.05～2.0mbar，一次干燥处理的干燥时间大于等于20小时；所述冻干预处理可进一步包括二次干燥处理，二次干燥处理的温度为20℃～65℃，二次干燥处理的真空度为0.05～2.0mbar，二次干燥处理的干燥时间为大于0小时且在8小时以下；In some embodiments of the present application, the pre-freezing temperature of the pre-freezing treatment is 2 to 8°C, and the pre-freezing time is greater than or equal to 2.0 hours; or the freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing time is more than 3 hours; or the temperature of the primary drying treatment is -10°C to 0°C, the vacuum degree of the primary drying treatment is 0.05 to 2.0 mbar, and the drying time of the primary drying treatment is greater than or equal to 20 hours; the pre-freezing treatment may further include a secondary drying treatment, the temperature of the secondary drying treatment is 20°C to 65°C, the vacuum degree of the secondary drying treatment is 0.05 to 2.0 mbar, and the drying time of the secondary drying treatment is greater than 0 hours and less than 8 hours;
例如，所述预冻处理的预冻温度可以为2℃、3℃、4℃、5℃、6℃、7℃、8℃或其之间的任意范围；For example, the pre-freezing temperature of the pre-freezing treatment may be 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C or any range therebetween;
预冻处理的时间可以为2.0小时、2.1小时、2.2小时、2.3小时、2.4小时、2.5小时、2.6小时、2.7小时、2.8小时、2.9小时、3.0小时、3.1小时、3.2小时、3.3小时、3.4小时、3.5小时、3.6小时、3.7小时、3.8小时、3.9小时、4.0小时、4.1小时、4.2小时、4.3小时、4.4小时、4.5小时、4.6小时、4.7小时、4.8小时、4.9小时、5.0小时、5.1小时、5.2小时、5.3小时、5.4小时、5.5小时、5.6小时、5.7小时、5.8小时、5.9小时、6.0小时、6.1小时、6.2小时、6.3小时、6.4小时、6.5小时、6.6小时、6.7小时、6.8小时、6.9小时、7.0小时、7.1小时、7.2小时、7.3小时、7.4小时、7.5小时、7.6小时、7.7小时、7.8小时、7.9小时、8.0小时、8.1小时、8.2小时、8.3小时、8.4小时、8.5小时、8.6小时、8.7小时、8.8小时、8.9小时、9.0小时、9.1小时、9.2小时、9.3小时、9.4小时、9.5小时、9.6小时、9.7小时、9.8小时、9.9小时、10.0小时及以上或其之间的任意范围；The time of pre-freezing treatment can be 2.0 hours, 2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3.0 hours, 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, 4.0 hours, 4.1 hours, 4.2 hours, 4.3 hours, 4.4 hours, 4.5 hours, 4.6 hours, 4.7 hours, 4.8 hours, 4.9 hours, 5.0 hours, 5.1 hours, 5.2 hours, 5.3 hours, 5.4 hours, 5.5 hours, 5.6 hours, 5.7 hours, 5.8 hours, 5.9 hours, 6.0 hours Hours, 6.1 hours, 6.2 hours, 6.3 hours, 6.4 hours, 6.5 hours, 6.6 hours, 6.7 hours, 6.8 hours, 6.9 hours, 7.0 hours, 7.1 hours, 7.2 hours, 7.3 hours, 7.4 hours, 7.5 hours, 7.6 hours, 7.7 hours, 7.8 hours, 7.9 hours, 8.0 hours, 8.1 hours, 8.2 hours, 8.3 hours, 8.4 hours, 8.5 hours, 8.6 hours, 8.7 hours, 8.8 hours, 8.9 hours, 9.0 hours, 9.1 hours, 9.2 hours, 9.3 hours, 9.4 hours, 9.5 hours, 9.6 hours, 9.7 hours, 9.8 hours, 9.9 hours, 10.0 hours and above or any range therebetween;
所述冷冻处理的冷冻温度可以为-30℃、-31℃、-32℃、-33℃、-34℃、-35℃、-36℃、-37℃、-38℃、-39℃、-40℃、-41℃、-42℃、-43℃、-44℃、-45℃、-46℃、-47℃、-48℃、-49℃、-50℃、-51℃、-52℃、-53℃、-54℃、-55℃、-56℃、-57℃、-58℃、-59℃、-60℃或其之间的任意范围；The freezing temperature of the freezing treatment can be -30°C, -31°C, -32°C, -33°C, -34°C, -35°C, -36°C, -37°C, -38°C, -39°C, -40°C, -41°C, -42°C, -43°C, -44°C, -45°C, -46°C, -47°C, -48°C, -49°C, -50°C, -51°C, -52°C, -53°C, -54°C, -55°C, -56°C, -57°C, -58°C, -59°C, -60°C or any range therebetween;
冷冻处理的时间可以为3.1小时、3.2小时、3.3小时、3.4小时、3.5小时、3.6小时、3.7小时、3.8小时、3.9小时、4.0小时、4.1小时、4.2小时、4.3小时、4.4小时、4.5小时、4.6小时、4.7小时、4.8小时、4.9小时、5.0小时、5.1小时、5.2小时、5.3小时、5.4小时、5.5小时、5.6小时、5.7小 时、5.8小时、5.9小时、6.0小时、6.1小时、6.2小时、6.3小时、6.4小时、6.5小时、6.6小时、6.7小时、6.8小时、6.9小时、7.0小时、7.1小时、7.2小时、7.3小时、7.4小时、7.5小时、7.6小时、7.7小时、7.8小时、7.9小时、8.0小时、8.1小时、8.2小时、8.3小时、8.4小时、8.5小时、8.6小时、8.7小时、8.8小时、8.9小时、9.0小时、9.1小时、9.2小时、9.3小时、9.4小时、9.5小时、9.6小时、9.7小时、9.8小时、9.9小时、10.0小时及以上或其之间的任意范围；The freezing treatment time can be 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, 4.0 hours, 4.1 hours, 4.2 hours, 4.3 hours, 4.4 hours, 4.5 hours, 4.6 hours, 4.7 hours, 4.8 hours, 4.9 hours, 5.0 hours, 5.1 hours, 5.2 hours, 5.3 hours, 5.4 hours, 5.5 hours, 5.6 hours, 5.7 hours Hours, 5.8 hours, 5.9 hours, 6.0 hours, 6.1 hours, 6.2 hours, 6.3 hours, 6.4 hours, 6.5 hours, 6.6 hours, 6.7 hours, 6.8 hours, 6.9 hours, 7.0 hours, 7.1 hours, 7.2 hours, 7.3 hours, 7.4 hours, 7.5 hours, 7.6 hours, 7.7 hours, 7.8 hours, 7.9 hours, 8.0 hours, 8.1 hours, 8.2 hours, 8.3 hours, 8.4 hours, 8.5 hours, 8.6 hours, 8.7 hours, 8.8 hours, 8.9 hours, 9.0 hours, 9.1 hours, 9.2 hours, 9.3 hours, 9.4 hours, 9.5 hours, 9.6 hours, 9.7 hours, 9.8 hours, 9.9 hours, 10.0 hours and any range therebetween;
一次干燥处理的温度可以为-10℃、-9℃、-8℃、-7℃、-6℃、-5℃、-4℃、-3℃、-2℃、-1℃、0℃或其之间的任意范围；The temperature of the primary drying treatment may be -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C, -1°C, 0°C or any range therebetween;
一次干燥处理的真空度可以为0.05mbar、0.1mbar、0.15mbar、0.2mbar、0.25mbar、0.3mbar、0.35mbar、0.4mbar、0.45mbar、0.5mbar、0.55mbar、0.6mbar、0.65mbar、0.7mbar、0.75mbar、0.8mbar、0.85mbar、0.9mbar、0.95mbar、1.0mbar、1.05mbar、1.1mbar、1.15mbar、1.2mbar、1.25mbar、1.3mbar、1.35mbar、1.4mbar、1.45mbar、1.5mbar、1.55mbar、1.6mbar、1.65mbar、1.7mbar、1.75mbar、1.8mbar、1.85mbar、1.9mbar、1.95mbar、2.0mbar或其之间的任意范围；The vacuum degree of the primary drying process can be 0.05mbar, 0.1mbar, 0.15mbar, 0.2mbar, 0.25mbar, 0.3mbar, 0.35mbar, 0.4mbar, 0.45mbar, 0.5mbar, 0.55mbar, 0.6mbar, 0.65mbar, 0.7mbar, 0.75mbar, 0.8mbar, 0.85mbar, 0.9mbar, 0.95mbar, 1.0mbar r, 1.05mbar, 1.1mbar, 1.15mbar, 1.2mbar, 1.25mbar, 1.3mbar, 1.35mbar, 1.4mbar, 1.45mbar, 1.5mbar, 1.55mbar, 1.6mbar, 1.65mbar, 1.7mbar, 1.75mbar, 1.8mbar, 1.85mbar, 1.9mbar, 1.95mbar, 2.0mbar or any range therebetween;
一次干燥处理的干燥时间可以为20小时、20.1小时、20.2小时、20.3小时、20.4小时、20.5小时、20.6小时、20.7小时、20.8小时、20.9小时、21.0小时、21.1小时、21.2小时、21.3小时、21.4小时、21.5小时、21.6小时、21.7小时、21.8小时、21.9小时、22.0小时、22.1小时、22.2小时、22.3小时、22.4小时、22.5小时、22.6小时、22.7小时、22.8小时、22.9小时、23.0小时、23.1小时、23.2小时、23.3小时、23.4小时、23.5小时、23.6小时、23.7小时、23.8小时、23.9小时、24.0小时、24.1小时、24.2小时、24.3小时、24.4小时、24.5小时、24.6小时、24.7小时、24.8小时、24.9小时、25.0小时、25.1小时、25.2小时、25.3小时、25.4小时、25.5小时、25.6小时、25.7小时、25.8小时、25.9小时、26.0小时、26.1小时、26.2小时、26.3小时、26.4小时、26.5小时、26.6小时、26.7小时、26.8小时、26.9小时、27.0小时、27.1小时、27.2小时、27.3小时、27.4小时、27.5小时、27.6小时、27.7小时、27.8小时、27.9小时、28.0小时、28.1小时、28.2小时、28.3小时、28.4小时、28.5小时、28.6小时、28.7小时、28.8小时、28.9小时、29.0小时、29.1小时、29.2小时、29.3小时、29.4小时、29.5小时、29.6小时、29.7小时、29.8小时、29.9小时、30.0小时及以上或其之间的任意范围； The drying time of the primary drying treatment can be 20 hours, 20.1 hours, 20.2 hours, 20.3 hours, 20.4 hours, 20.5 hours, 20.6 hours, 20.7 hours, 20.8 hours, 20.9 hours, 21.0 hours, 21.1 hours, 21.2 hours, 21.3 hours, 21.4 hours, 21.5 hours, 21.6 hours, 21.7 hours, 21.8 hours, 21.9 hours, 22.0 hours, 22.1 hours, 22.2 hours, 22.3 hours, 22.4 hours, 22.5 hours, 22.6 hours, 22.7 hours, 22.8 hours, 22.9 hours, 23.0 hours, 23.1 hours, 23.2 hours, 23.3 hours, 23.4 hours, 23.5 hours, 23.6 hours, 23.7 hours, 23.8 hours, 23.9 hours, 24.0 hours, 24.1 hours, 24.2 hours, 24.3 hours, 24.4 hours, 24.5 hours, 24.6 hours, 24.7 hours, 24.8 hours, 24.9 hours, 25 .4 hours, 22.5 hours, 22.6 hours, 22.7 hours, 22.8 hours, 22.9 hours, 23.0 hours, 23.1 hours, 23.2 hours, 23.3 hours, 23.4 hours, 23.5 hours, 23.6 hours, 23.7 hours, 23.8 hours, 23.9 hours, 24.0 hours, 24.1 hours, 24.2 hours, 24.3 hours, 24.4 hours, 24.5 hours, 24.6 hours, 24.7 hours, 24.8 hours, 24.9 hours, 25. 0 hours, 25.1 hours, 25.2 hours, 25.3 hours, 25.4 hours, 25.5 hours, 25.6 hours, 25.7 hours, 25.8 hours, 25.9 hours, 26.0 hours, 26.1 hours, 26.2 hours, 26.3 hours, 26.4 hours, 26.5 hours, 26.6 hours, 26.7 hours, 26.8 hours, 26.9 hours, 27.0 hours, 27.1 hours, 27.2 hours, 27.3 hours, 27.4 hours, 27.5 hours, 27.6 15.0 hours, 15.7 hours, 15.8 hours, 15.9 hours, 16.0 hours, 17.1 hours, 17.2 hours, 17.3 hours, 17.4 hours, 18.0 hours, 18.1 hours, 18.2 hours, 18.3 hours, 18.4 hours, 18.5 hours, 18.6 hours, 18.7 hours, 18.8 hours, 18.9 hours, 19.0 hours, 19.1 hours, 19.2 hours, 19.3 hours, 19.4 hours, 19.5 hours, 19.6 hours, 19.7 hours, 19.8 hours, 19.9 hours, 20.0 hours and any range therebetween;
二次干燥处理的温度可以为20℃、21℃、22℃、23℃、24℃、25℃、26℃、27℃、28℃、29℃、30℃、31℃、32℃、33℃、34℃、35℃、36℃、37℃、38℃、39℃、40℃、41℃、42℃、43℃、44℃、45℃、46℃、47℃、48℃、49℃、50℃、51℃、52℃、53℃、54℃、55℃、56℃、57℃、58℃、59℃、60℃、61℃、62℃、63℃、64℃、65℃或其之间的任意范围；The temperature of the secondary drying treatment can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C or any range therebetween;
二次干燥处理的真空度可以为0.05mbar、0.1mbar、0.15mbar、0.2mbar、0.25mbar、0.3mbar、0.35mbar、0.4mbar、0.45mbar、0.5mbar、0.55mbar、0.6mbar、0.65mbar、0.7mbar、0.75mbar、0.8mbar、0.85mbar、0.9mbar、0.95mbar、1.0mbar、1.05mbar、1.1mbar、1.15mbar、1.2mbar、1.25mbar、1.3mbar、1.35mbar、1.4mbar、1.45mbar、1.5mbar、1.55mbar、1.6mbar、1.65mbar、1.7mbar、1.75mbar、1.8mbar、1.85mbar、1.9mbar、1.95mbar、2.0mbar或其之间的任意范围；The vacuum degree of the secondary drying process can be 0.05mbar, 0.1mbar, 0.15mbar, 0.2mbar, 0.25mbar, 0.3mbar, 0.35mbar, 0.4mbar, 0.45mbar, 0.5mbar, 0.55mbar, 0.6mbar, 0.65mbar, 0.7mbar, 0.75mbar, 0.8mbar, 0.85mbar, 0.9mbar, 0.95mbar, 1.0mbar r, 1.05mbar, 1.1mbar, 1.15mbar, 1.2mbar, 1.25mbar, 1.3mbar, 1.35mbar, 1.4mbar, 1.45mbar, 1.5mbar, 1.55mbar, 1.6mbar, 1.65mbar, 1.7mbar, 1.75mbar, 1.8mbar, 1.85mbar, 1.9mbar, 1.95mbar, 2.0mbar or any range therebetween;
二次干燥处理的干燥时间可以为0.01小时、1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时或其之间的任意范围。The drying time of the secondary drying treatment can be 0.01 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours or any range therebetween.
在本申请的一些实施方式中，所述冻干预处理包括：预冻处理、冷冻处理及一次干燥处理；其中，所述预冻处理的预冻温度为2～8℃，预冻处理的时间大于等于2.0小时；冷冻处理的冷冻温度为-30℃～-60℃，冷冻处理的时间为3小时以上；一次干燥处理的温度为-10℃～0℃，一次干燥处理的真空度为0.05～2.0mbar，一次干燥处理的干燥时间大于等于20小时。In some embodiments of the present application, the prefreezing treatment includes: pre-freezing treatment, freezing treatment and primary drying treatment; wherein the pre-freezing temperature of the pre-freezing treatment is 2 to 8°C, and the pre-freezing time is greater than or equal to 2.0 hours; the freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing time is more than 3 hours; the temperature of the primary drying treatment is -10°C to 0°C, the vacuum degree of the primary drying treatment is 0.05 to 2.0 mbar, and the drying time of the primary drying treatment is greater than or equal to 20 hours.
在本申请的一些实施方式中，所述冻干预处理包括：预冻处理、冷冻处理、一次干燥处理和二次干燥处理，其中，所述预冻处理的预冻温度为2～8℃，预冻处理的时间大于等于2.0小时；冷冻处理的冷冻温度为-30℃～-60℃，冷冻处理的时间为3小时以上；一次干燥处理的温度为-10℃～0℃，一次干燥处理的真空度为0.05～2.0mbar，一次干燥处理的干燥时间大于等于20小时；二次干燥处理的温度为20℃～65℃，二次干燥处理的真空度为0.05-2.0mbar，二次干燥处理的干燥时间为大于0小时且在8小时以下。In some embodiments of the present application, the pre-freezing treatment includes: pre-freezing treatment, freezing treatment, primary drying treatment and secondary drying treatment, wherein the pre-freezing temperature of the pre-freezing treatment is 2-8°C, and the pre-freezing time is greater than or equal to 2.0 hours; the freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing time is more than 3 hours; the temperature of the primary drying treatment is -10°C to 0°C, the vacuum degree of the primary drying treatment is 0.05-2.0 mbar, and the drying time of the primary drying treatment is greater than or equal to 20 hours; the temperature of the secondary drying treatment is 20°C to 65°C, the vacuum degree of the secondary drying treatment is 0.05-2.0 mbar, and the drying time of the secondary drying treatment is greater than 0 hours and less than 8 hours.
在本申请的一些实施方式中，在所述脱保护步骤中：将冻干预处理后的多肽衍生物的中间体在酸催化剂溶液条件下进行脱保护，得到脱保护反应液；将所述脱保护反应液加入到终止液中，调节pH值以终止反应，得到多肽衍生物。In some embodiments of the present application, in the deprotection step: the intermediate of the polypeptide derivative after freeze-drying is deprotected under acid catalyst solution conditions to obtain a deprotection reaction solution; the deprotection reaction solution is added to a stop solution, and the pH value is adjusted to stop the reaction to obtain a polypeptide derivative.
在本申请的一些实施方式中，调节pH值至6.5～10.0以终止反应，得到多肽衍生物。 In some embodiments of the present application, the pH value is adjusted to 6.5-10.0 to terminate the reaction and obtain a polypeptide derivative.
在本申请的一些实施方式中，调节pH值至7.5～9.5以终止反应，得到多肽衍生物。In some embodiments of the present application, the pH value is adjusted to 7.5-9.5 to terminate the reaction and obtain a polypeptide derivative.
在本申请的一些实施方式中，调节pH值可以至6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9、10.0或其之间的任意范围。In some embodiments of the present application, the pH value can be adjusted to 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 or any range therebetween.
在本申请的一些实施方式中，所述终止液包含缓冲盐溶液和碱性溶液。In some embodiments of the present application, the stop solution comprises a buffered saline solution and an alkaline solution.
在本申请的一些实施方式中，所述碱性溶液选自氢氧化钠溶液、氢氧化钾溶液、碳酸钠溶液、碳酸钾溶液、碳酸氢钠溶液、碳酸氢钾溶液、碳酸氢铵溶液中的一种或两种以上。In some embodiments of the present application, the alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, and ammonium bicarbonate solution.
在本申请的一些实施方式中，所述缓冲盐溶液选自磷酸盐类缓冲溶液、Tris类缓冲溶液、有机酸类缓冲溶液、硼酸盐类缓冲溶液、氨基酸类缓冲溶液中的一种或两种以上。In some embodiments of the present application, the buffered salt solution is selected from one or more of a phosphate buffer solution, a Tris buffer solution, an organic acid buffer solution, a borate buffer solution, and an amino acid buffer solution.
在本申请的一些实施方式中，所述缓冲盐溶液选自磷酸氢二钠-柠檬酸缓冲溶液、柠檬酸-NaOH-HCl缓冲溶液、柠檬酸-柠檬酸钠缓冲溶液、乙酸-乙酸钠缓冲溶液、磷酸盐缓冲溶液(PBS)、磷酸氢二钠-磷酸二氢钠缓冲溶液(PB)、磷酸氢二钠-磷酸二氢钾缓冲溶液、磷酸二氢钾-NaOH缓冲溶液、巴比妥钠-HCl缓冲溶液、NH4HCO3缓冲溶液、碳酸钠-碳酸氢钠缓冲溶液、NaHCO3缓冲溶液、Tris-HCl、甘氨酸-NaOH缓冲溶液、硼酸-硼砂缓冲溶液、Na2B7O4缓冲溶液中的一种或两种以上。In some embodiments of the present application, the buffered saline solution is selected from one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution (PBS), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate-NaOH buffer solution, sodium barbital-HCl buffer solution, NH4HCO3 buffer solution, sodium carbonate-sodium bicarbonate buffer solution, NaHCO3 buffer solution, Tris-HCl, glycine-NaOH buffer solution, boric acid - borax buffer solution, and Na2B7O4 buffer solution.
在本申请的一些实施方式中，所述碱性溶液为氢氧化钠溶液。In some embodiments of the present application, the alkaline solution is a sodium hydroxide solution.
在本申请的一些实施方式中，所述缓冲盐溶液中缓冲盐的浓度为0.001M～0.6M，优选为0.01M～0.6M,更优选为0.01M～0.5M；In some embodiments of the present application, the concentration of the buffer salt in the buffer salt solution is 0.001M to 0.6M, preferably 0.01M to 0.6M, more preferably 0.01M to 0.5M;
例如，所述缓冲盐溶液中缓冲盐的浓度可以为0.001M、0.01M、0.02M、0.03M、0.04M、0.05M、0.06M、0.07M、0.08M、0.09M、0.1M、0.11M、0.12M、0.13M、0.14M、0.15M、0.16M、0.17M、0.18M、0.19M、0.2M、0.21M、0.22M、0.23M、0.24M、0.25M、0.26M、0.27M、0.28M、0.29M、0.3M、0.31M、0.32M、0.33M、0.34M、0.35M、0.36M、0.37M、0.38M、0.39M、0.4M、0.41M、0.42M、0.43M、0.44M、0.45M、0.46M、0.47M、0.48M、0.49M、0.5M、0.51M、0.52M、0.53M、0.54M、0.55M、0.56M、0.57M、0.58M、0.59M、0.6M或其之间的任意范围。 For example, the concentration of the buffer salt in the buffer salt solution can be 0.001M, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.11M, 0.12M, 0.13M, 0.14M, 0.15M, 0.16M, 0.17M, 0.18M, 0.19M, 0.2M, 0.21M, 0.22M, 0.23M, 0.24M, 0.25M, 0.26M, 0.27M, 0.28M, 0. .54M, 0.55M, 0.56M, 0.57M, 0.58M, 0.59M, 0.6M or any range therebetween.
在本申请的一些实施方式中，所述碱性溶液的浓度为2～10M，优选为3～8M，更优选为4～6M；In some embodiments of the present application, the concentration of the alkaline solution is 2 to 10 M, preferably 3 to 8 M, and more preferably 4 to 6 M;
例如，所述碱性溶液的浓度可以为2M、2.1M、2.2M、2.3M、2.4M、2.5M、2.6M、2.7M、2.8M、2.9M、3.0M、3M、3.1M、3.2M、3.3M、3.4M、3.5M、3.6M、3.7M、3.8M、3.9M、4.0M、4.1M、4.2M、4.3M、4.4M、4.5M、4.6M、4.7M、4.8M、4.9M、5.0M、5.1M、5.2M、5.3M、5.4M、5.5M、5.6M、5.7M、5.8M、5.9M、6.0M、6.1M、6.2M、6.3M、6.4M、6.5M、6.6M、6.7M、6.8M、6.9M、7.0M、7.1M、7.2M、7.3M、7.4M、7.5M、7.6M、7.7M、7.8M、7.9M、8M、8.1M、8.2M、8.3M、8.4M、8.5M、8.6M、8.7M、8.8M、8.9M、9M、9.1M、9.2M、9.3M、9.4M、9.5M、9.6M、9.7M、9.8M、9.9M、10M或其之间的任意范围。For example, the concentration of the alkaline solution can be 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3.0M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4.0M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5.0M, 5.1M, 5.2M, 5.3M, 5.4M, 5.5M, 5.6M, 5.7M, 5.8M, 5.9M, 6.0M, 6.1M, 6.2M, 6.3M, 6.4M, 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7.0 .9M, 6.0M, 6.1M, 6.2M, 6.3M, 6.4M, 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7.0M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, 10M or any range therebetween.
在本申请的一些实施方式中，所述氢氧化钠溶液中氢氧化钠的浓度为2～10M，优选为3～8M，更优选为4～6M；In some embodiments of the present application, the concentration of sodium hydroxide in the sodium hydroxide solution is 2 to 10 M, preferably 3 to 8 M, and more preferably 4 to 6 M;
例如，所述氢氧化钠溶液中氢氧化钠的浓度可以为2M、2.1M、2.2M、2.3M、2.4M、2.5M、2.6M、2.7M、2.8M、2.9M、3.0M、3M、3.1M、3.2M、3.3M、3.4M、3.5M、3.6M、3.7M、3.8M、3.9M、4.0M、4.1M、4.2M、4.3M、4.4M、4.5M、4.6M、4.7M、4.8M、4.9M、5.0M、5.1M、5.2M、5.3M、5.4M、5.5M、5.6M、5.7M、5.8M、5.9M、6.0M、6.1M、6.2M、6.3M、6.4M、6.5M、6.6M、6.7M、6.8M、6.9M、7.0M、7.1M、7.2M、7.3M、7.4M、7.5M、7.6M、7.7M、7.8M、7.9M、8M、8.1M、8.2M、8.3M、8.4M、8.5M、8.6M、8.7M、8.8M、8.9M、9M、9.1M、9.2M、9.3M、9.4M、9.5M、9.6M、9.7M、9.8M、9.9M、10M或其之间的任意范围。For example, the concentration of sodium hydroxide in the sodium hydroxide solution can be 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3.0M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4.0M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5.0M, 5.1M, 5.2M, 5.3M, 5.4M, 5.5M, 5.6M, 5.7M, 5.8M, M, 5.9M, 6.0M, 6.1M, 6.2M, 6.3M, 6.4M, 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7.0M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, 10M or any range therebetween.
在本申请的一些实施方式中，所述缓冲盐溶液和所述碱性溶液的体积比为(0.1～100)：1；In some embodiments of the present application, the volume ratio of the buffered saline solution to the alkaline solution is (0.1-100):1;
例如，所述缓冲盐溶液和所述碱性溶液的体积比可以为0.1:1、0.2:1、0.3:1、0.4:1、0.5:1、0.6:1、0.7:1、0.8:1、0.9:1、1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1、11:1、12:1、13:1、14:1、15:1、16:1、17:1、18:1、19:1、20:1、21:1、22:1、23:1、24:1、25:1、26:1、27:1、28:1、29:1、30:1、31:1、32:1、33:1、34:1、35:1、36:1、37:1、38:1、39:1、40:1、41:1、42:1、43:1、44:1、45:1、46:1、47:1、48:1、49:1、50:1、51:1、52:1、53:1、54:1、55:1、56:1、57:1、58:1、59:1、60:1、61:1、62:1、63:1、64:1、65:1、66:1、 67:1、68:1、69:1、70:1、71:1、72:1、73:1、74:1、75:1、76:1、77:1、78:1、79:1、80:1、81:1、82:1、83:1、84:1、85:1、86:1、87:1、88:1、89:1、90:1、91:1、92:1、93:1、94:1、95:1、96:1、97:1、98:1、99:1、100:1、或其之间的任意范围。For example, the volume ratio of the buffered saline solution to the alkaline solution can be 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1、28:1、29:1、30:1、31:1、32:1、33:1、34:1、35:1、36:1、37:1、38:1、39:1、40:1、41:1、42:1、43:1、44:1、45:1、46:1、47:1、48:1、49:1、50:1、51:1、52:1、53:1、54:1、55:1、56:1、57:1、58:1、59:1、60:1、61:1、62:1、63:1、64:1、65:1、66:1、 1 :1, or any range therebetween.
在本申请的一些实施方式中，酸催化剂选自对甲苯磺酸、三氟乙酸、乙酸、甲磺酸、盐酸、磷酸和磺酸中的一种或两种以上。In some embodiments of the present application, the acid catalyst is selected from one or more of p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
在本申请的一些实施方式中，在所述脱保护步骤中，酸催化剂为三氟乙酸，终止液包含缓冲盐溶液和碱性溶液，调节pH值至6.5～10.0以终止反应，得到多肽衍生物。In some embodiments of the present application, in the deprotection step, the acid catalyst is trifluoroacetic acid, the stop solution comprises a buffered salt solution and an alkaline solution, and the pH value is adjusted to 6.5 to 10.0 to terminate the reaction and obtain a polypeptide derivative.
本申请提供了一种多肽衍生物的制备方法，其中，得到多肽衍生物中间体，所述多肽衍生物中间体是含有保护基的羧酸酯修饰的多肽；所述多肽衍生物的中间体中加入到酸催化剂溶液中进行脱保护，得到脱保护反应液；将所述脱保护反应液加入到终止液中，调节pH值以终止反应，得到多肽衍生物。The present application provides a method for preparing a polypeptide derivative, wherein a polypeptide derivative intermediate is obtained, wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group; the polypeptide derivative intermediate is added to an acid catalyst solution for deprotection to obtain a deprotection reaction liquid; the deprotection reaction liquid is added to a stop solution, the pH value is adjusted to stop the reaction, and the polypeptide derivative is obtained.
在本申请的一些实施方式中，对所述多肽衍生物进行冻干预处理，得到冻干预处理后的多肽衍生物的中间体；对所述冻干预处理后的多肽衍生物的中间体进行脱保护，得到多肽衍生物。In some embodiments of the present application, the polypeptide derivative is subjected to freeze-thaw treatment to obtain an intermediate of the polypeptide derivative after freeze-thaw treatment; and the intermediate of the polypeptide derivative after freeze-thaw treatment is deprotected to obtain the polypeptide derivative.
本申请提供了一种多肽衍生物的制备方法，其中，得到多肽衍生物中间体的湿固体，所述多肽衍生物中间体是含有保护基的羧酸酯修饰的多肽；所述多肽衍生物的中间体的湿固体中加入到酸催化剂溶液中进行脱保护，得到脱保护反应液；将所述脱保护反应液加入到终止液中，调节pH值以终止反应，得到多肽衍生物。The present application provides a method for preparing a polypeptide derivative, wherein a wet solid of a polypeptide derivative intermediate is obtained, wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group; the wet solid of the polypeptide derivative intermediate is added to an acid catalyst solution for deprotection to obtain a deprotection reaction liquid; the deprotection reaction liquid is added to a stop solution, the pH value is adjusted to stop the reaction, and the polypeptide derivative is obtained.
在本申请的一些实施方式中，对所述湿固体进行冻干预处理，得到冻干预处理后的多肽衍生物的中间体；对所述冻干预处理后的多肽衍生物的中间体进行脱保护，得到多肽衍生物。In some embodiments of the present application, the wet solid is freeze-dried to obtain an intermediate of the freeze-dried polypeptide derivative; and the freeze-dried intermediate of the polypeptide derivative is deprotected to obtain a polypeptide derivative.
在具体实施方式中，针对pH值的描述参见上述关于脱保护中pH值的描述。In a specific embodiment, for the description of pH value, please refer to the above description of pH value in deprotection.
在具体实施方式中，所述终止液的描述参见上述关于脱保护中终止液的描述。In a specific embodiment, the description of the stop solution refers to the description of the stop solution in deprotection above.
在具体实施方式中，酸催化剂的描述参见上述关于脱保护中酸催化剂的描述。 In a specific embodiment, the description of the acid catalyst refers to the description of the acid catalyst in the deprotection process described above.
在具体实施方式中，保护基的描述参见上述关于保护基的描述。In the specific embodiment, the description of the protecting group refers to the above description of the protecting group.
在具体实施方式中，脂肪酸和羧酸酯的描述分别参见上述关于脂肪酸和羧酸酯的描述。In a specific embodiment, the descriptions of fatty acids and carboxylates refer to the above descriptions of fatty acids and carboxylates, respectively.
在具体实施方式中，所述冻干预处理的描述参见上述关于冻干预处理的描述。In a specific embodiment, the description of the pre-freezing treatment refers to the description of the pre-freezing treatment mentioned above.
本申请还提供了一种多肽衍生物的制备方法，包括：得到多肽衍生物中间体，所述多肽衍生物中间体是含有保护基的羧酸酯修饰的多肽；对所述多肽衍生物进行冻干预处理，得到冻干预处理后的多肽衍生物的中间体；将所述冻干预处理后的多肽衍生物的中间体加入到酸催化剂溶液中进行脱保护，得到脱保护反应液；将所述脱保护反应液加入到终止液中，调节pH值以终止反应，得到多肽衍生物。The present application also provides a method for preparing a polypeptide derivative, comprising: obtaining a polypeptide derivative intermediate, wherein the polypeptide derivative intermediate is a carboxylate-modified polypeptide containing a protecting group; subjecting the polypeptide derivative to a freeze-thaw treatment to obtain a freeze-thaw treated polypeptide derivative intermediate; adding the freeze-thaw treated polypeptide derivative intermediate to an acid catalyst solution for deprotection to obtain a deprotection reaction solution; adding the deprotection reaction solution to a stop solution, adjusting the pH value to stop the reaction, and obtaining a polypeptide derivative.
本申请还提供了一种多肽衍生物的制备方法，包括：得到多肽衍生物中间体的湿固体，所述多肽衍生物中间体是含有保护基的羧酸酯修饰的多肽；对所述湿固体进行冻干预处理，得到冻干预处理后的多肽衍生物的中间体；将所述冻干预处理后的多肽衍生物的中间体加入到酸催化剂溶液中进行脱保护，得到脱保护反应液；将所述脱保护反应液加入到终止液中，调节pH值以终止反应，得到多肽衍生物。在具体实施方式中，针对pH值的描述参见上述关于脱保护中pH值的描述。The present application also provides a method for preparing a polypeptide derivative, comprising: obtaining a wet solid of a polypeptide derivative intermediate, wherein the polypeptide derivative intermediate is a polypeptide modified with a carboxylate containing a protecting group; subjecting the wet solid to a freeze-dried treatment to obtain a polypeptide derivative intermediate after freeze-dried treatment; adding the polypeptide derivative intermediate after freeze-dried treatment to an acid catalyst solution for deprotection to obtain a deprotection reaction solution; adding the deprotection reaction solution to a stop solution, adjusting the pH value to stop the reaction, and obtaining a polypeptide derivative. In a specific embodiment, the description of the pH value refers to the above description of the pH value in deprotection.
在具体实施方式中，所述终止液的描述参见上述关于脱保护中终止液的描述。In a specific embodiment, the description of the stop solution refers to the description of the stop solution in deprotection above.
在具体实施方式中，酸催化剂的描述参见上述关于脱保护中酸催化剂的描述。In a specific embodiment, the description of the acid catalyst refers to the description of the acid catalyst in the deprotection process described above.
在具体实施方式中，保护基的描述参见上述关于保护基的描述。In the specific embodiment, the description of the protecting group refers to the above description of the protecting group.
在具体实施方式中，脂肪酸和羧酸酯的描述分别参见上述关于脂肪酸和羧酸酯的描述。In a specific embodiment, the descriptions of fatty acids and carboxylates refer to the above descriptions of fatty acids and carboxylates, respectively.
在具体实施方式中，所述冻干预处理的描述参见上述关于冻干预处理的描述。In a specific embodiment, the description of the pre-freezing treatment refers to the description of the pre-freezing treatment mentioned above.
本申请提供了一种多肽衍生物，其中，所述多肽衍生物经上述方法制备而得。The present application provides a polypeptide derivative, wherein the polypeptide derivative is prepared by the above method.
本申请提供了一种多肽衍生物制备方法，通过在传统酸催化剂脱保护反应进行前增加对脱保护蛋白冻干预处理环节，有效提高了脱保护蛋白浓度上 限，极大提高蛋白质生产中脱保护工艺的效益和效率。The present application provides a method for preparing a polypeptide derivative by adding a deprotected protein freeze intervention treatment step before the traditional acid catalyst deprotection reaction, which effectively improves the deprotected protein concentration. The limit is to greatly improve the benefits and efficiency of the deprotection process in protein production.
本申请提供了一种多肽衍生物制备方法，通过调整脱保护终止液添加方式，由常规的将脱保护液加入脱保护反应液中调整为将反应液加入脱保护液中，极大降低了脱保护过程中异构体杂质含量且提高了操作方法稳定性，进一步的，通过对该终止方式中增加缓冲溶液、调整缓冲溶液成分体积等优化，进一步提高了脱保护效果和稳定性。The present application provides a method for preparing a polypeptide derivative. By adjusting the method for adding a deprotection termination solution, the conventional method of adding the deprotection termination solution to the deprotection reaction solution is changed to adding the reaction solution to the deprotection termination solution. This greatly reduces the content of isomer impurities in the deprotection process and improves the stability of the operation method. Furthermore, by optimizing the termination method by adding a buffer solution and adjusting the volume of the buffer solution components, the deprotection effect and stability are further improved.
实施例Example
实施例1 GLP-1衍生物中间体的制备Example 1 Preparation of GLP-1 derivative intermediates
1.1 GLP-1多肽的合成、表达与纯化1.1 Synthesis, expression and purification of GLP-1 peptide
按照WO2019201328实施例1-4所述的GLP-1多肽类似物制备方法合成Val8Glu22Lys30Arg26,34-GLP-1(7-37)多肽分子。概括而言，通过发酵工艺得到含有目标融合蛋白的可溶性菌体，之后称取100g菌体加入到破菌缓冲溶液中，搅拌混匀，菌体破碎两次，离心收集上清。通过阴离子层析捕获目标融合蛋白并洗脱，加入20mmol/L PB(pH7.4)的酶切稀释液稀释后酶切过夜，去除标签序列，室温反应24h后酸沉终止。离心收集沉淀并水洗，复溶后通过复合模式层析对目标蛋白进行纯化收集，稀释后酸沉离心水洗，得到GLP-1湿固体沉淀。According to the preparation method of GLP-1 polypeptide analogs described in Examples 1-4 of WO2019201328, Val 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7-37) polypeptide molecules were synthesized. In summary, a soluble bacterial cell containing the target fusion protein was obtained by a fermentation process, and then 100 g of bacterial cells were weighed and added to a bacterial lysis buffer solution, stirred and mixed, the bacterial cells were broken twice, and the supernatant was collected by centrifugation. The target fusion protein was captured and eluted by anion chromatography, and 20 mmol/L PB (pH 7.4) enzyme diluent was added for dilution and enzyme digestion overnight to remove the tag sequence. After 24 hours of reaction at room temperature, acid precipitation was terminated. The precipitate was collected by centrifugation and washed with water. After redissolution, the target protein was purified and collected by composite mode chromatography. After dilution, the acid precipitate was centrifuged and washed with water to obtain a GLP-1 wet solid precipitate.
1.2脂肪酸侧链的合成1.2 Synthesis of fatty acid side chains
称取20mg式I所示的脂肪酸(赛诺邦格生物科技股份有限公司)溶于0.2mL ACN中，向其中依次加入0.6mL的20mg/mL的四氟苯酚、0.6mL的20mg/mL的EDCI、0.8mL的ACN，室温下反应1.5h进行活化，得到活化后的脂肪酸。
20 mg of the fatty acid represented by Formula I (Sinobank Biotech Co., Ltd.) was weighed and dissolved in 0.2 mL of ACN, to which 0.6 mL of 20 mg/mL tetrafluorophenol, 0.6 mL of 20 mg/mL EDCI, and 0.8 mL of ACN were added in sequence, and the mixture was reacted at room temperature for 1.5 h for activation to obtain the activated fatty acid.
1.3 GLP-1衍生物中间体制备1.3 Preparation of GLP-1 derivative intermediates
称取1g GLP-1湿固体沉淀溶于25mL水中，使用1mol/L NaOH调节pH 至7.5～8.5，通过RP-HPLC检测其纯度约为98％，浓度8mg/mL，方法如下：Weigh 1 g of GLP-1 wet solid precipitate and dissolve it in 25 mL of water. Use 1 mol/L NaOH to adjust the pH. To 7.5-8.5, the purity is about 98% by RP-HPLC, the concentration is 8 mg/mL, the method is as follows:
色谱柱：Sepax C8 4.6*250mm 5μm 200A；Chromatographic column: Sepax C8 4.6*250mm 5μm 200A;
流动相A：30％ACN+70％H2O+0.2％TFA，Mobile phase A: 30% ACN + 70% H 2 O + 0.2% TFA,
流动相B：70％ACN+30％H2O+0.2％TFA；Mobile phase B: 70% ACN + 30% H 2 O + 0.2% TFA;
洗脱梯度：
Elution gradient:
流速1.0mL/min；柱温15℃；检测器UV-280nmFlow rate: 1.0 mL/min; column temperature: 15°C; detector: UV-280 nm
得到经纯化的GLP-1复溶后样品。The purified GLP-1 reconstituted sample was obtained.
量取4.75mL复溶后GLP-1样品，之后向其中加入4.75mL ACN以及19uL DIPEA，再加入上述活化后的脂肪酸，室温下反应1～2h后倒入50mL的水中，使用10％盐酸及1mol/L NaOH调节pH至5.1酸沉12～48h，离心水洗，得到GLP-1衍生物中间体湿固体。Measure 4.75 mL of the reconstituted GLP-1 sample, then add 4.75 mL of ACN and 19 uL of DIPEA, and then add the above-mentioned activated fatty acid. After reacting at room temperature for 1 to 2 hours, pour into 50 mL of water, adjust the pH to 5.1 with 10% hydrochloric acid and 1 mol/L NaOH, and precipitate with acid for 12 to 48 hours. Centrifuge and wash with water to obtain a wet solid of the GLP-1 derivative intermediate.
实施例2冻干预处理Example 2: Freezing treatment
取实施例1制备的GLP-1衍生物中间体湿固体，铺盘冻干，冻干条件为样品预冻：温度4℃，维持2h，然后降温至-45℃冷冻，维持冷冻温度3h；一次干燥：预冻后升高温度至-5℃，气压0.2mbar条件下冻干40h，以完成一次干燥，干燥结束后，得到冻干预处理的多肽衍生物的中间体。The wet solid of the GLP-1 derivative intermediate prepared in Example 1 was taken and lyophilized on a plate. The lyophilization conditions were as follows: pre-freezing the sample: maintaining the temperature at 4°C for 2 hours, then cooling to -45°C and freezing, maintaining the freezing temperature for 3 hours; primary drying: raising the temperature to -5°C after pre-freezing, and lyophilizing for 40 hours under an air pressure of 0.2 mbar to complete the primary drying. After drying, the intermediate of the polypeptide derivative treated with lyophilization was obtained.
各取5mg、10mg、20mg、40mg、60mg、80mg、100mg、125mg、150mg、200mg、250mg冻干预处理的多肽衍生物的中间体分别加入1mL含水10％的TFA溶液进行溶解，使其浓度分别为5mg/mL，10mg/mL，20mg/mL，40mg/mL，60mg/mL，80mg/mL，100mg/mL，125mg/mL，150mg/mL，200mg/mL以及250mg/mL，10～30℃下搅拌反应1～2h，之后向反应液中加入5mol/L的NaOH 2.5mL终止反应，并使用1mol/L的NaOH溶液调节pH值至pH7.5～9.5，不断搅拌至溶液澄清，得到经冻干预处理的反应后溶液。5 mg, 10 mg, 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 125 mg, 150 mg, 200 mg and 250 mg of the intermediate of the frozen-intervention polypeptide derivative were added into 1 mL of 10% TFA solution to dissolve them, so that the concentrations were 5 mg/mL, 10 mg/mL, 20 mg/mL, 40 mg/mL, 60 mg/mL, 80 mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 200 mg/mL and 250 mg/mL, respectively. The reaction was stirred at 10-30°C for 1-2 h, and then 2.5 mL of 5 mol/L NaOH was added to the reaction solution to terminate the reaction. The pH value was adjusted to pH 7.5-9.5 with 1 mol/L NaOH solution. The reaction was continued until the solution was clear to obtain a frozen-intervention reaction solution.
将上述方法获得的经冻干预处理的反应后溶液采用RP-HPLC检测脱保护终止后产物，，RP-HPLC测定条件如下：The post-freezing reaction solution obtained by the above method was subjected to RP-HPLC to detect the product after deprotection termination. The RP-HPLC determination conditions were as follows:
色谱柱：Sepax C8 4.6*250mm 5μm 200A；Chromatographic column: Sepax C8 4.6*250mm 5μm 200A;
流动相A：30％ACN+70％H2O+0.2％TFA，Mobile phase A: 30% ACN + 70% H 2 O + 0.2% TFA,
流动相B：70％ACN+30％H2O+0.2％TFA；Mobile phase B: 70% ACN + 30% H 2 O + 0.2% TFA;
洗脱梯度：
Elution gradient:
流速1.0mL/min；柱温15℃；检测器UV-280nmFlow rate: 1.0 mL/min; column temperature: 15°C; detector: UV-280 nm
所述不同脱保护蛋白浓度下经冻干后脱保护考察结果分别如表1所示。The results of the deprotection investigation after lyophilization at different deprotected protein concentrations are shown in Table 1.
表1
Table 1
对比例1Comparative Example 1
各取实施例1制备的15mg GLP-1衍生物中间体湿固体分别加入6mL、3mL、2mL、1.5mL含水10％的TFA溶液进行溶解，使其浓度分别为2.5mg/mL，5mg/mL，7.5mg/mL以及10mg/mL，在10～30℃下搅拌反应1～2h，之后向反应液中加入5mol/L的NaOH 2.5mL终止反应，并使用1mol/L的NaOH溶液调节pH值至pH7.5～9.5不断搅拌至溶液澄清，得到非冻干预处理的反应后溶液。15 mg of the wet solid of the GLP-1 derivative intermediate prepared in Example 1 was added into 6 mL, 3 mL, 2 mL and 1.5 mL of 10% TFA solution for dissolution, respectively, to make the concentrations 2.5 mg/mL, 5 mg/mL, 7.5 mg/mL and 10 mg/mL, and the reaction was stirred at 10-30°C for 1-2 h. Then, 2.5 mL of 5 mol/L NaOH was added to the reaction solution to terminate the reaction, and 1 mol/L NaOH solution was used to adjust the pH value to pH 7.5-9.5 and stirred continuously until the solution became clear to obtain a non-freezing-treated post-reaction solution.
将非冻干预处理的反应后溶液采用RP-HPLC检测脱保护终止后产物，RP-HPLC测定条件如下：The non-freezing treatment post-reaction solution was used to detect the deprotection termination product by RP-HPLC. The RP-HPLC determination conditions were as follows:
色谱柱：Sepax C8 4.6*250mm 5μm 200A；Chromatographic column: Sepax C8 4.6*250mm 5μm 200A;
流动相A：30％ACN+70％H2O+0.2％TFA，Mobile phase A: 30% ACN + 70% H 2 O + 0.2% TFA,
流动相B：70％ACN+30％H2O+0.2％TFA；Mobile phase B: 70% ACN + 30% H 2 O + 0.2% TFA;
洗脱梯度：
Elution gradient:
流速1.0mL/min；柱温15℃；检测器UV-280nmFlow rate: 1.0 mL/min; column temperature: 15°C; detector: UV-280 nm
所述不同脱保护蛋白浓度下非冻干后脱保护考察结果分别如表2所示。The results of the non-lyophilized deprotection investigation at different deprotected protein concentrations are shown in Table 2.
表2
Table 2
由表1和表2可知，若不进行冻干预处理，脱保护的蛋白浓度只能保持在5mg/mL的较低的水平下，脱保护不完全样品的含量为0.957％。随着蛋白浓度升高至7.5mg/mL，脱保护不完全样品含量急剧增加至33.0％。因此，脱保护的处理浓度无法进一步提升。而进行冻干预处理后，可使脱保护蛋白处理浓度达到250mg/mL，其脱保护不完全样品含量仍低于常规操作下7.5mg/mL蛋白浓度的脱保护不完全样品含量，尤其是在200mg/mL浓度范围以下脱保护不完全样品的含量均维持在较低水平，即冻干后脱保护效率得到了极大的提升，对工业大规模生产具有明显的实用性。As shown in Tables 1 and 2, if the freeze-drying treatment is not performed, the deprotected protein concentration can only be maintained at a low level of 5 mg/mL, and the content of the incompletely deprotected sample is 0.957%. As the protein concentration increases to 7.5 mg/mL, the content of the incompletely deprotected sample increases sharply to 33.0%. Therefore, the deprotection treatment concentration cannot be further increased. After the freeze-drying treatment, the deprotected protein treatment concentration can reach 250 mg/mL, and the content of the incompletely deprotected sample is still lower than the content of the incompletely deprotected sample with a protein concentration of 7.5 mg/mL under conventional operation, especially in the concentration range of 200 mg/mL. The content of the incompletely deprotected sample is maintained at a low level, that is, the deprotection efficiency after freeze-drying has been greatly improved, which has obvious practicality for industrial large-scale production.
实施例3脱保护终止液的优化Example 3 Optimization of deprotection termination solution
将脱保护反应液加入到预配好的终止液中，之后调整pH至中性即可。具体如下：如实施例2制备的冻干预处理的多肽衍生物的中间体100mg，加入1mL含水10％的TFA溶液中进行溶解，10～30℃下搅拌反应1～2h，得到脱保护反应液。将脱保护反应液加入到预配好的终止液中(预配好的终止液，具体组成和含量如表3所示)中，之后再使用1mol/L的NaOH溶液调节pH值至pH 7.5～9.5，不断搅拌至溶液澄清。反应终止后采用RP-HPLC方法对样品的纯度及蛋白含量进行检测，RP-HPLC方法如下：Add the deprotection reaction solution to the pre-prepared stop solution, and then adjust the pH to neutral. Specifically as follows: 100 mg of the intermediate of the lyophilized polypeptide derivative prepared in Example 2 is added to 1 mL of a 10% TFA solution for dissolution, and the reaction is stirred at 10 to 30°C for 1 to 2 hours to obtain a deprotection reaction solution. Add the deprotection reaction solution to the pre-prepared stop solution (the pre-prepared stop solution, the specific composition and content are shown in Table 3), and then use 1 mol/L NaOH solution to adjust the pH value to pH 7.5 to 9.5, and continue stirring until the solution is clear. After the reaction is terminated, the purity and protein content of the sample are detected by RP-HPLC method. The RP-HPLC method is as follows:
色谱柱：Sepax C8 4.6*250mm 5μm 200A；Chromatographic column: Sepax C8 4.6*250mm 5μm 200A;
流动相A：30％ACN+70％H2O+0.2％TFA，Mobile phase A: 30% ACN + 70% H 2 O + 0.2% TFA,
流动相B：70％ACN+30％H2O+0.2％TFA； 洗脱梯度：
Mobile phase B: 70% ACN + 30% H 2 O + 0.2% TFA; Elution gradient:
不同实验组具体终止液的组成和浓度见表3.The specific composition and concentration of the stop solution in different experimental groups are shown in Table 3.
表3
table 3
结果如表4所示The results are shown in Table 4
表4

Table 4

对比例2Comparative Example 2
取实施例2制备的冻干预处理的多肽衍生物的中间体10g，加入到100mL含水10％的TFA溶液进行溶解，10～30℃下搅拌反应1～2h，之后向反应液中加入5mol/L的NaOH终止反应，并使用1mol/L的NaOH溶液调节pH值至pH7.5～9.5，不断搅拌至溶液澄清。反应终止后采用RP-HPLC方法对样品的纯度及蛋白含量进行检测，RP-HPLC方法如下：Take 10g of the intermediate of the frozen pre-treated polypeptide derivative prepared in Example 2, add it to 100mL of 10% TFA solution to dissolve it, stir and react at 10-30°C for 1-2h, then add 5mol/L NaOH to the reaction solution to terminate the reaction, and use 1mol/L NaOH solution to adjust the pH value to pH7.5-9.5, and stir continuously until the solution is clear. After the reaction is terminated, the purity and protein content of the sample are detected by RP-HPLC method, and the RP-HPLC method is as follows:
色谱柱：Sepax C8 4.6*250mm 5μm 200A；Chromatographic column: Sepax C8 4.6*250mm 5μm 200A;
流动相A：30％ACN+70％H2O+0.2％TFA，Mobile phase A: 30% ACN + 70% H 2 O + 0.2% TFA,
流动相B：70％ACN+30％H2O+0.2％TFA；Mobile phase B: 70% ACN + 30% H 2 O + 0.2% TFA;
洗脱梯度：
Elution gradient:
流速1.0mL/min；柱温15℃；检测器UV-280nm。Flow rate: 1.0 mL/min; column temperature: 15°C; detector: UV-280 nm.
相同的实验条件重复重复7次，分别记为批次1、批次2、批次3、批次4、批次5、批次6和批次7。The same experimental conditions were repeated 7 times, which were recorded as batch 1, batch 2, batch 3, batch 4, batch 5, batch 6 and batch 7 respectively.
结果见表5。The results are shown in Table 5.
使用上述终止工艺终止后样品结果汇总如表5所示。The sample results after termination using the above termination process are summarized in Table 5.
表5
table 5
由表5可知，将5.0mol/L的氢氧化钠溶液加入到脱保护反应液中并调节pH过程中,一方面终点处的pH值较难控制，易造成溶液pH偏高，尤其体系较大时，pH值达到10以上时，或者局部碱浓度过高，均会产生难以分离去除的消旋异构体，不同批次生产时的异构体含量差异较大。As can be seen from Table 5, when a 5.0 mol/L sodium hydroxide solution is added to the deprotection reaction solution and the pH is adjusted, on the one hand, the pH value at the end point is difficult to control, which can easily cause the solution pH to be too high. Especially when the system is large, when the pH value reaches above 10, or when the local alkali concentration is too high, racemic isomers that are difficult to separate and remove will be produced, and the isomer content in different batches of production varies greatly.
由表4可知，不同的缓冲盐类型(实验M1～M3)，使用不同缓冲体系终止反应至接近中性条件，令人意外的发现，将脱保护反应液分别加入相同浓度和体积的NH4Ac、Tris-Base、Na2HPO4缓冲溶液和NaOH的体系中，各组终止后异构体杂质含量均明显降低。As shown in Table 4, different buffer salt types (experiments M1 to M3) used different buffer systems to terminate the reaction to near neutral conditions. Surprisingly, when the deprotection reaction solution was added to the system of NH 4 Ac, Tris-Base, Na 2 HPO 4 buffer solution and NaOH with the same concentration and volume, the isomer impurity content of each group after termination was significantly reduced.
进一步的，对磷酸盐缓冲溶液的浓度、终止液缓冲盐体积以及氢氧化钠的体积进行考察，结果列于表4中(M4-14)。值得关注的，相同种类缓冲溶液的不同体积(M4～M6、M7～M11、M12～M14组内)、不同浓度(M4～M6、M7～M11、M12～M14组间)，以及相同缓冲溶液下不同NaOH终止体积(M15～M17)均可降低特异性异构体含量至0.2％以下，同时值得注意的是，脱保护目的蛋白含量也显著提高。 Further, the concentration of phosphate buffer solution, the volume of stop solution buffer salt and the volume of sodium hydroxide were investigated, and the results are listed in Table 4 (M4-14). It is worth noting that different volumes of the same type of buffer solution (within M4-M6, M7-M11, M12-M14 groups), different concentrations (between M4-M6, M7-M11, M12-M14 groups), and different NaOH stop volumes (M15-M17) under the same buffer solution can reduce the specific isomer content to less than 0.2%. It is also worth noting that the content of the deprotected target protein is also significantly increased.

Claims (25)

  1. 一种多肽衍生物的制备方法，其中，包括：A method for preparing a polypeptide derivative, comprising:
    得到多肽衍生物中间体，所述多肽衍生物中间体为经含有保护基的羧酸酯修饰的多肽；Obtaining a polypeptide derivative intermediate, wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group;
    对所述多肽衍生物中间体进行冻干预处理，得到冻干预处理后的多肽衍生物的中间体；Performing freeze-thaw treatment on the polypeptide derivative intermediate to obtain a freeze-thaw treated polypeptide derivative intermediate;
    对所述冻干预处理后的多肽衍生物的中间体进行脱保护，得到多肽衍生物。The intermediate of the polypeptide derivative after freeze-drying is deprotected to obtain the polypeptide derivative.
  2. 根据权利要求1所述的制备方法，其中，所述保护基选自C1～C21烷基、芳基中的任一种。The preparation method according to claim 1, wherein the protecting group is selected from any one of a C1-C21 alkyl group and an aryl group.
  3. 根据权利要求2所述的制备方法，其中，所述保护基为甲基、乙基、叔丁基或苄基。The preparation method according to claim 2, wherein the protecting group is methyl, ethyl, tert-butyl or benzyl.
  4. 根据权利要求1所述的制备方法，其中，The preparation method according to claim 1, wherein
    所述羧酸酯为脂肪酸酯。The carboxylic acid ester is a fatty acid ester.
  5. 根据权利要求1所述的制备方法，其中，The preparation method according to claim 1, wherein
    所述冻干预处理包括如下步骤：The freezing treatment comprises the following steps:
    预冻处理；Pre-freezing treatment;
    冷冻处理；Freezing treatment;
    一次干燥处理。One-time drying process.
  6. 根据权利要求5所述的制备方法，其中，The preparation method according to claim 5, wherein
    所述冻干预处理进一步可以包括二次干燥处理。The pre-freezing treatment may further include a secondary drying process.
  7. 根据权利要求5或6所述的制备方法，其中，所述预冻处理的预冻温度为2～8℃，预冻处理的时间为大于等于2.0小时；或者The preparation method according to claim 5 or 6, wherein the pre-freezing temperature of the pre-freezing treatment is 2 to 8°C, and the pre-freezing time is greater than or equal to 2.0 hours; or
    所述冷冻处理的冷冻温度为-30℃～-60℃，冷冻处理的时间为3小时以上；或者The freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing treatment time is more than 3 hours; or
    所述一次干燥处理的温度为-10℃～0℃，一次干燥处理的真空度为0.05～2.0mbar，一次干燥处理的干燥时间为大于等于20小时；The temperature of the primary drying process is -10°C to 0°C, the vacuum degree of the primary drying process is 0.05 to 2.0 mbar, and the drying time of the primary drying process is greater than or equal to 20 hours;
    所述二次干燥处理的温度为20℃～65℃，二次干燥处理的真空度为0.05～2.0mbar，二次干燥处理的干燥时间为大于0小时且在8小时以下。The temperature of the secondary drying process is 20° C. to 65° C., the vacuum degree of the secondary drying process is 0.05 to 2.0 mbar, and the drying time of the secondary drying process is greater than 0 hours and less than 8 hours.
  8. 根据权利要求1所述的制备方法，其中， The preparation method according to claim 1, wherein
    多肽选自胰岛素、胰高血糖素、肠促胰岛素、GLP-1、甲状旁腺素(PTH)、GLP-2、胃泌酸调节素、淀粉不溶素(Amylin)、肠高血糖素、生长激素抑制素、瘦素、YY肽、去氨加压素、骨钙蛋白、人生长激素、糖肽抗生素、非核糖体肽抗生素、促皮质素、降钙素、催产素、干扰素、纤维蛋白溶媒、抗利尿激素、白细胞介素、尿激酶、促甲状腺激素释放激素、甲硫氨酸脑啡肽、亮氨酸脑啡肽、前列腺素F2α受体调节剂、肥胖抑制素、分泌素、nesfatin、葡萄糖依赖性促胰岛素肽(GIP)及上述多肽的类似物或片段中的一种或两种以上。The polypeptide is selected from one or more of insulin, glucagon, incretin, GLP-1, parathyroid hormone (PTH), GLP-2, oxyntomodulin, amylin, enteroglucagon, somatostatin, leptin, YY peptide, desmopressin, osteocalcin, human growth hormone, glycopeptide antibiotics, non-ribosomal peptide antibiotics, corticotropin, calcitonin, oxytocin, interferon, fibrinolytic, antidiuretic hormone, interleukin, urokinase, thyrotropin-releasing hormone, methionine enkephalin, leucine enkephalin, prostaglandin F2α receptor modulator, obestatin, secretin, nesfatin, glucose-dependent insulinotropic peptide (GIP) and analogs or fragments of the above polypeptides.
  9. 根据权利要求1～8任一项所述的制备方法，其中，The preparation method according to any one of claims 1 to 8, wherein
    在所述脱保护步骤中：In the deprotection step:
    冻干预处理后的多肽衍生物的中间体在酸催化剂溶液条件下进行脱保护，得到脱保护反应液；The intermediate of the polypeptide derivative after freeze-drying is deprotected under the condition of an acid catalyst solution to obtain a deprotection reaction solution;
    将所述脱保护反应液加入到终止液中，调节pH值以终止反应，得到多肽衍生物。The deprotection reaction solution is added to a stop solution, and the pH value is adjusted to stop the reaction to obtain a polypeptide derivative.
  10. 根据权利要求9所述的制备方法，其中，The preparation method according to claim 9, wherein
    调节pH值至6.5～10.0以终止反应，得到多肽衍生物，The pH value is adjusted to 6.5-10.0 to terminate the reaction and obtain a polypeptide derivative.
    优选地，调节pH值至7.5～9.5以终止反应，得到多肽衍生物。Preferably, the pH value is adjusted to 7.5-9.5 to terminate the reaction and obtain the polypeptide derivative.
  11. 根据权利要求9所述的制备方法，其中，The preparation method according to claim 9, wherein
    所述终止液包含缓冲盐溶液和碱性溶液，The stop solution comprises a buffered saline solution and an alkaline solution,
    优选地，所述碱性溶液选自氢氧化钠溶液、氢氧化钾溶液、碳酸钠溶液、碳酸钾溶液、碳酸氢钠溶液、碳酸氢钾溶液、碳酸氢铵溶液中的一种或两种以上，优选为氢氧化钠溶液，Preferably, the alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, and ammonium bicarbonate solution, preferably sodium hydroxide solution.
    进一步优选地，所述缓冲盐溶液选自磷酸氢二钠-柠檬酸缓冲溶液、柠檬酸-NaOH-HCl缓冲溶液、柠檬酸-柠檬酸钠缓冲溶液、乙酸-乙酸钠缓冲溶液、磷酸盐缓冲溶液(PBS)、磷酸氢二钠-磷酸二氢钠缓冲溶液(PB)、磷酸氢二钠-磷酸二氢钾缓冲溶液、磷酸二氢钾-NaOH缓冲溶液、巴比妥钠-HCl缓冲溶液、NH4HCO3缓冲溶液、碳酸钠-碳酸氢钠缓冲溶液、NaHCO3缓冲溶液、Tris-HCl、甘氨酸-NaOH缓冲溶液、硼酸-硼砂缓冲溶液、Na2B7O4缓冲溶液中的一种或两种以上。Further preferably, the buffered saline solution is selected from one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution (PBS), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate-NaOH buffer solution, sodium barbital-HCl buffer solution, NH4HCO3 buffer solution, sodium carbonate-sodium bicarbonate buffer solution, NaHCO3 buffer solution, Tris-HCl, glycine-NaOH buffer solution, boric acid- borax buffer solution, and Na2B7O4 buffer solution.
  12. 根据权利要求11所述的制备方法，其中，The preparation method according to claim 11, wherein
    所述缓冲盐溶液中缓冲盐的浓度为0.001M～0.6M，优选为0.01M～0.6M, 更优选为0.01M～0.5M，The concentration of the buffer salt in the buffer salt solution is 0.001M to 0.6M, preferably 0.01M to 0.6M. More preferably, it is 0.01M to 0.5M.
    优选地，所述碱性溶液的浓度为2～10M，优选为3～8M，更优选为4～6M。Preferably, the concentration of the alkaline solution is 2-10M, preferably 3-8M, more preferably 4-6M.
  13. 根据权利要求11所述的制备方法，其中，The preparation method according to claim 11, wherein
    所述缓冲盐溶液和所述碱性溶液的体积比为(0.1～100)：1，优选为(0.1～70)：1，更优选为(1～50)：1。The volume ratio of the buffered salt solution to the alkaline solution is (0.1-100):1, preferably (0.1-70):1, and more preferably (1-50):1.
  14. 根据权利要求9所述的制备方法，其中，The preparation method according to claim 9, wherein
    酸催化剂选自对甲苯磺酸、三氟乙酸、乙酸、甲磺酸、盐酸、磷酸和磺酸中的一种或两种以上。The acid catalyst is selected from one or more of p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
  15. 一种多肽衍生物的制备方法，其中，A method for preparing a polypeptide derivative, wherein:
    得到多肽衍生物中间体，所述多肽衍生物中间体为含有保护基的羧酸酯修饰的多肽；Obtaining a polypeptide derivative intermediate, wherein the polypeptide derivative intermediate is a polypeptide modified by a carboxylate containing a protecting group;
    所述多肽衍生物的中间体在酸催化剂溶液条件下进行脱保护，得到脱保护反应液；The intermediate of the polypeptide derivative is deprotected under acid catalyst solution conditions to obtain a deprotection reaction solution;
    将所述脱保护反应液加入到终止液中，调节pH值以终止反应，得到多肽衍生物。The deprotection reaction solution is added to a stop solution, and the pH value is adjusted to stop the reaction to obtain a polypeptide derivative.
  16. 根据权利要求15所述的制备方法，其中，The preparation method according to claim 15, wherein
    调节pH值至6.5～10.0以终止反应，得到多肽衍生物；adjusting the pH value to 6.5-10.0 to terminate the reaction and obtain a polypeptide derivative;
    优选地，调节pH值至7.5～9.5以终止反应，得到多肽衍生物。Preferably, the pH value is adjusted to 7.5-9.5 to terminate the reaction and obtain the polypeptide derivative.
  17. 根据权利要求15所述的制备方法，其中，The preparation method according to claim 15, wherein
    所述终止液包含缓冲盐溶液和碱性溶液；The stop solution comprises a buffered saline solution and an alkaline solution;
    优选地，所述碱性溶液选自氢氧化钠溶液、氢氧化钾溶液、碳酸钠溶液、碳酸钾溶液、碳酸氢钠溶液、碳酸氢钾溶液、碳酸氢铵溶液中的一种或两种以上，优选为氢氧化钠溶液；Preferably, the alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, and ammonium bicarbonate solution, preferably sodium hydroxide solution;
    进一步优选地，所述缓冲盐溶液选自磷酸氢二钠-柠檬酸缓冲溶液、柠檬酸-NaOH-HCl缓冲溶液、柠檬酸-柠檬酸钠缓冲溶液、乙酸-乙酸钠缓冲溶液、磷酸盐缓冲溶液(PBS)、磷酸氢二钠-磷酸二氢钠缓冲溶液(PB)、磷酸氢二钠-磷酸二氢钾缓冲溶液、磷酸二氢钾-NaOH缓冲溶液、巴比妥钠-HCl缓冲溶液、NH4HCO3缓冲溶液、碳酸钠-碳酸氢钠缓冲溶液、NaHCO3缓冲溶液、Tris-HCl、甘氨酸-NaOH缓冲溶液、硼酸-硼砂缓冲溶液、Na2B7O4缓冲溶液中的一种或两种以上。Further preferably, the buffered saline solution is selected from one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution (PBS), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate-NaOH buffer solution, sodium barbital-HCl buffer solution, NH4HCO3 buffer solution, sodium carbonate-sodium bicarbonate buffer solution, NaHCO3 buffer solution, Tris-HCl, glycine-NaOH buffer solution, boric acid- borax buffer solution, and Na2B7O4 buffer solution.
  18. 根据权利要求17所述的制备方法，其中， The preparation method according to claim 17, wherein
    所述缓冲盐溶液中缓冲盐的浓度为0.001M～0.6M，优选为0.01M～0.6M,更优选为0.01M～0.5M；The concentration of the buffer salt in the buffer salt solution is 0.001M to 0.6M, preferably 0.01M to 0.6M, more preferably 0.01M to 0.5M;
    优选地，所述碱性溶液的浓度为2～10M，优选为3～8M，更优选为4～6M；Preferably, the concentration of the alkaline solution is 2 to 10 M, preferably 3 to 8 M, more preferably 4 to 6 M;
    进一步优选地，所述缓冲盐溶液和所述碱性溶液的体积比为(0.1～100)：1，优选为(0.1～70)：1，更优选为(1～50)：1。Further preferably, the volume ratio of the buffered salt solution to the alkaline solution is (0.1-100):1, preferably (0.1-70):1, and more preferably (1-50):1.
  19. 根据权利要求15所述的制备方法，其中，The preparation method according to claim 15, wherein
    酸催化剂选自对甲苯磺酸、三氟乙酸、乙酸、甲磺酸、盐酸、磷酸和磺酸中的一种或两种以上。The acid catalyst is selected from one or more of p-toluenesulfonic acid, trifluoroacetic acid, acetic acid, methanesulfonic acid, hydrochloric acid, phosphoric acid and sulfonic acid.
  20. 根据权利要求15所述的制备方法，其中，The preparation method according to claim 15, wherein
    所述羧酸酯为脂肪酸酯。The carboxylic acid ester is a fatty acid ester.
  21. 根据权利要求15～20任一项所述的制备方法，其中，The preparation method according to any one of claims 15 to 20, wherein
    对所述多肽衍生物的中间体进行冻干预处理，得到冻干预处理后的多肽衍生物的中间体；Performing freeze-thaw treatment on the intermediate of the polypeptide derivative to obtain the freeze-thaw treated intermediate of the polypeptide derivative;
    对所述冻干预处理后的多肽衍生物的中间体进行脱保护，得到多肽衍生物；Deprotecting the intermediate of the polypeptide derivative after freeze-drying to obtain the polypeptide derivative;
    所述冻干预处理包括如下步骤：The freezing treatment comprises the following steps:
    预冻处理；Pre-freezing treatment;
    冷冻处理；Freezing treatment;
    一次干燥处理。One-time drying process.
  22. 根据权利要求21所述的制备方法，其中，所述冻干预处理进一步可以包括二次干燥处理。The preparation method according to claim 21, wherein the pre-freezing treatment may further include a secondary drying treatment.
  23. 根据权利要求21或22所述的制备方法，其中，所述预冻处理的预冻温度为2～8℃，预冻处理的时间为大于等于2.0小时；或者The preparation method according to claim 21 or 22, wherein the pre-freezing temperature of the pre-freezing treatment is 2 to 8° C., and the pre-freezing time is greater than or equal to 2.0 hours; or
    所述冷冻处理的冷冻温度为-30℃～-60℃，冷冻处理的时间为3小时以上；或者The freezing temperature of the freezing treatment is -30°C to -60°C, and the freezing treatment time is more than 3 hours; or
    所述一次干燥处理的温度为-10℃～0℃，一次干燥处理的真空度为0.05～2.0mbar，一次干燥处理的干燥时间为大于等于20小时；The temperature of the primary drying process is -10°C to 0°C, the vacuum degree of the primary drying process is 0.05 to 2.0 mbar, and the drying time of the primary drying process is greater than or equal to 20 hours;
    所述二次干燥处理的温度为20℃～65℃，二次干燥处理的真空度为0.05～2.0mbar，二次干燥处理的干燥时间为大于0小时且在8小时以下。The temperature of the secondary drying process is 20° C. to 65° C., the vacuum degree of the secondary drying process is 0.05 to 2.0 mbar, and the drying time of the secondary drying process is greater than 0 hours and less than 8 hours.
  24. 根据权利要求15～23任一项所述的制备方法，其中，The preparation method according to any one of claims 15 to 23, wherein
    多肽选自胰岛素、胰高血糖素、肠促胰岛素、GLP-1、甲状旁腺素(PTH)、 GLP-2、胃泌酸调节素、淀粉不溶素(Amylin)、肠高血糖素、生长激素抑制素、瘦素、YY肽、去氨加压素、骨钙蛋白、人生长激素、糖肽抗生素、非核糖体肽抗生素、促皮质素、降钙素、催产素、干扰素、纤维蛋白溶媒、抗利尿激素、白细胞介素、尿激酶、促甲状腺激素释放激素、甲硫氨酸脑啡肽、亮氨酸脑啡肽、前列腺素F2α受体调节剂、肥胖抑制素、分泌素、nesfatin、葡萄糖依赖性促胰岛素肽(GIP)及上述多肽的类似物或片段中的一种或两种以上。The polypeptide is selected from insulin, glucagon, incretin, GLP-1, parathyroid hormone (PTH), GLP-2, oxyntomodulin, amylin, enteroglucagon, somatostatin, leptin, YY peptide, desmopressin, osteocalcin, human growth hormone, glycopeptide antibiotics, non-ribosomal peptide antibiotics, corticotropin, calcitonin, oxytocin, interferon, fibrinolytic agent, antidiuretic hormone, interleukin, urokinase, thyrotropin-releasing hormone, methionine enkephalin, leucine enkephalin, prostaglandin F2α receptor modulator, obestatin, secretin, nesfatin, glucose-dependent insulinotropic peptide (GIP) and one or more of the analogs or fragments of the above polypeptides.
  25. 一种多肽衍生物，其中，所述多肽衍生物经权利要求1～24任一项所述方法制备而得。 A polypeptide derivative, wherein the polypeptide derivative is prepared by the method according to any one of claims 1 to 24.
PCT/CN2023/075022 2022-12-06 2023-02-08 Polypeptide derivative and preparation method therefor WO2024119609A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211556553.6A CN115746081B (en) 2022-12-06 2022-12-06 Polypeptide derivative and preparation method thereof
CN202211556553.6 2022-12-06

Publications (1)

Publication Number Publication Date
WO2024119609A1 true WO2024119609A1 (en) 2024-06-13

Family

ID=85344698

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/075022 WO2024119609A1 (en) 2022-12-06 2023-02-08 Polypeptide derivative and preparation method therefor

Country Status (2)

Country Link
CN (3) CN117229346A (en)
WO (1) WO2024119609A1 (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5922810A (en) * 1995-07-31 1999-07-13 Fmc Corporation Deprotection of protected functional polymers
CN107033234A (en) * 2017-01-03 2017-08-11 北京凯因科技股份有限公司 The acylated derivatives of GLP 1
CN109248323A (en) * 2017-07-14 2019-01-22 杭州先为达生物科技有限公司 Acylated GLP-1 derivative
WO2019201328A1 (en) * 2018-04-19 2019-10-24 杭州先为达生物科技有限公司 Acylated glp-1 derivative
CN110386975A (en) * 2018-04-19 2019-10-29 杭州先为达生物科技有限公司 Acylated GLP-1 derivative
CN110386974A (en) * 2018-04-19 2019-10-29 杭州先为达生物科技有限公司 GLP-1 derivative and its therapeutical uses

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110144012A1 (en) * 2005-11-21 2011-06-16 Dalian D.N. Bio-Engineering Co., Ltd. Shortened glucagon-like peptide 1(sglp-1) preparation method and application
CN114685646B (en) * 2020-12-31 2023-04-07 厦门赛诺邦格生物科技股份有限公司 Preparation method and application of polypeptide side chain analogue

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5922810A (en) * 1995-07-31 1999-07-13 Fmc Corporation Deprotection of protected functional polymers
CN107033234A (en) * 2017-01-03 2017-08-11 北京凯因科技股份有限公司 The acylated derivatives of GLP 1
CN109248323A (en) * 2017-07-14 2019-01-22 杭州先为达生物科技有限公司 Acylated GLP-1 derivative
WO2019201328A1 (en) * 2018-04-19 2019-10-24 杭州先为达生物科技有限公司 Acylated glp-1 derivative
CN110386975A (en) * 2018-04-19 2019-10-29 杭州先为达生物科技有限公司 Acylated GLP-1 derivative
CN110386974A (en) * 2018-04-19 2019-10-29 杭州先为达生物科技有限公司 GLP-1 derivative and its therapeutical uses

Also Published As

Publication number Publication date
CN117247422A (en) 2023-12-19
CN115746081B (en) 2023-11-24
CN115746081A (en) 2023-03-07
CN117229346A (en) 2023-12-15

Similar Documents

Publication Publication Date Title
AU612141B2 (en) Novel insulin derivatives
JP7075678B2 (en) Peptide ligase and its use
Palmer et al. Effects of dehydroalanine on peptide conformations
US10851146B2 (en) Method for preparing liraglutide intermediate polypeptide
Strable et al. Unnatural amino acid incorporation into virus-like particles
KR101289728B1 (en) Production of recombinant proteins by autoproteolytic cleavage of a fusion protein
KR20160093074A (en) Modified therapeutic agents, stapled peptide lipid conjugates, and compositions thereof
Huang et al. Chemical synthesis of native S‐palmitoylated membrane proteins through removable‐backbone‐modification‐assisted Ser/Thr ligation
BRPI0209685B1 (en) insulinotropic peptide derivative and method for producing an insulinotropic peptide derivative
CN100580087C (en) Conjugation of peptides
JPS6057000A (en) Novel growth hormone free peptide
Joshi et al. The degradation pathways of glucagon in acidic solutions
JP2021501172A (en) Oral delivery of GLP-1 peptide analogs
Banci et al. Molecular dynamics characterization of the active cavity of carboxypeptidase A and some of its inhibitor adducts
US20210087226A1 (en) Method for refining vasopressin
JP2009519296A (en) Polypeptide extension tag
WO2024119609A1 (en) Polypeptide derivative and preparation method therefor
US20060199812A1 (en) Method of conjugating aminothiol containing molecules to vehicles
WO2024174819A1 (en) Heteroaryl-containing unnatural amino acid and use thereof
JP2010031055A (en) Improved method for purifying tfpi and tfpi analogs
EP3411386A1 (en) A process for the preparation of guanylate cyclase 2c agonist
DK149457B (en) PROCEDURE FOR PREPARING HUMAN INSULIN OR B30 ESTERS THEREOF
AU705192B2 (en) Method for producing a correctly folded, biological active recombinant protein
JP4227193B2 (en) Acyl transfer using stabilized conversion complexes using catalysts with catalytic imidazole (eg histidine) function
CA3221611A1 (en) Long-acting dual gip/glp-1 peptide conjugates and methods of use

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23899210

Country of ref document: EP

Kind code of ref document: A1