CN114984231A - Stable preparation and preparation method thereof - Google Patents
Stable preparation and preparation method thereof Download PDFInfo
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- CN114984231A CN114984231A CN202210742400.4A CN202210742400A CN114984231A CN 114984231 A CN114984231 A CN 114984231A CN 202210742400 A CN202210742400 A CN 202210742400A CN 114984231 A CN114984231 A CN 114984231A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/26—Glucagons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
Abstract
The invention aims to provide a stable preparation containing GLP-1 or GLP-1 analogues and a preparation method thereof.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a preparation containing protein and a preparation method thereof.
Background
There are mainly 2 types of diabetes, insulin-dependent diabetes (type I diabetes) and non-insulin-dependent diabetes (type II diabetes), with more than 90% of patients with type II diabetes. Type II diabetes is a metabolic syndrome characterized primarily by disturbances in glucose metabolism, the pathogenesis of which has not been fully understood to date, and which may be related to a variety of factors, including environmental, lifestyle, and genetic factors. At present, the treatment measures of the type II diabetes, such as environment and life style intervention and adjuvant drug treatment, have insignificant effects, the insulin treatment has the defects of obesity and increase of cardiovascular and cerebrovascular disease risks, and the morbidity and mortality of people are high. These reasons have prompted the continued search for new drugs for treating type II diabetes, of which glucagon-like peptide-1 (GLP-1) receptor agonist (GLP-1RA) is one.
Peptides derived from preproglucagon (pro-glucagon) include glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2), both derived from the glucagon gene. Glucagon-like peptide-1 (GLP-1), an endogenous incretin secretagogue, plays an important role in stabilizing blood glucose, is produced by post-translational processing of preproglucan, and exists mainly in 2 bioequivalent forms: GLP-1(7-36) -NH 2 And GLP-1(7-37), GLP-1(7-36) -NH in humans 2 The occupied proportion is higher. GLP-1 also has appetite suppressant effect, and also has beneficial effect on cardiovascular disease and reducing diabetes-related nephropathy. Research finds that GLP-1 has the main action mechanism that (i) insulin secretion is stimulated in a glucose-dependent mode; ② the glucagon secretion is reduced; ③ inhibiting gastric emptying; fourthly, reducing the appetite; and promoting the growth and recovery of pancreatic beta cells.
However, while GLP-1 is a suitable candidate for the treatment of type II diabetes, it is very short-lived (half-life of about 2 minutes) in the human body due to its own GLP-1 action, which is very unstable and is rapidly degraded in vivo by dipeptidyl peptidase-4 (DPP-4). That is, GLP-1 in the human body itself is not suitable for development as a drug for clinical treatment of diabetes.
Current modification techniques for preparing long-acting GLP-1 include: fatty acid chain modifications (such as liraglutide and somaglutide), fused Fc fragment techniques (such as dolabrin), and PEG modification techniques (such as losela peptide), but modified long-acting GLP-1 formulations, especially fused Fc fragment long-acting GLP-1, still have problems with fragment peaks and inverse impurities.
CN102772787A relates to a method for preparing a stable solution of a GLP-1 compound, said method comprising heating a solution of said GLP-1 compound to a temperature above 40 ℃ for at least 5 minutes under basic pH conditions. The GLP-1 compound is liraglutide, which has 31 amino acids and is heated at higher pH and is not suitable for conventional biologics. Furthermore, the heating treatment in this patent application solves the problem of aggregation of liraglutide and does not propose a solution to the generation of fragment peaks and reverse phase impurities.
In general, peptides and proteins are sensitive to temperature, are easily physically or chemically degraded to generate fragments and other impurities, are easily aggregated, and are easily hydrolyzed under alkaline conditions. Thus, there is a need to develop stable formulations for long acting GLP-1.
Disclosure of Invention
The object of the present invention is to provide a stable formulation comprising GLP-1 or a GLP-1 analog, which shows a significant reduction in fragment peaks and impurities, and a process for the preparation thereof, comprising the step of heating the GLP-1 containing formulation to a temperature of 55 ℃ to 65 ℃ under weakly acidic conditions.
In a first aspect, the present invention provides a stable formulation comprising GLP-1 or a GLP-1 analog, and the stable formulation has been subjected to a treatment step at a temperature of 55 ℃ to 65 ℃.
In some embodiments, the stability formulation has a pH of 6.5 to 7.0, and is subjected to a step of treating at a temperature of 55 ℃ to 65 ℃.
Preferably, the stability formulation has a pH of 6.5 to 7.0 and is subjected to a treatment step at a temperature of 55 ℃ to 65 ℃ for 30 to 90 minutes.
Preferably, the stability formulation has a pH of 6.5 to 7.0 and is subjected to a treatment step at a temperature of 55 ℃ to 65 ℃ for 50 to 70 minutes.
Preferably, the stability formulation has a pH of 6.6 to 6.8 and is subjected to a treatment step at a temperature of 55 ℃ to 65 ℃ for 30 to 90 minutes.
Preferably, the stability formulation has a pH of 6.6 to 6.8 and is subjected to a treatment step at a temperature of 55 ℃ to 65 ℃ for 50 to 70 minutes.
In some embodiments, the stability formulation has a pH of 6.7 and is subjected to a step of treatment at a temperature of 55 ℃ to 65 ℃ for 30 to 90 minutes.
In some embodiments, the stable formulation is subjected to a step of treatment at a temperature of 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃ or 65 ℃ for 50-70 minutes.
In some embodiments, the stability formulation has a pH of 6.7 and is subjected to a step of treatment at a temperature of 60 ℃ for 60 minutes.
In some embodiments, the GLP-1 analog is selected from the group consisting of an ortholog of human GLP-1, a mutant of an ortholog of human GLP-1, a modified human GLP-1, a mutant of an ortholog of modified human GLP-1, and the GLP-1 analog retains the native biological activity of GLP-1.
In some embodiments, the modified human GLP-1, ortholog of modified human GLP-1, mutant of modified human GLP-1, or mutant of ortholog of modified human GLP-1 comprises a modification selected from the group consisting of: pegylation; glycosylation, polysialylation, or hydroxyethylation; a fusion maltose binding protein; fusion of albumin; albumin fusion by acylation; fusing Fc; and fusion PEG mimetics.
In some embodiments, the GLP-1 analog is selected from the group consisting of exenatide (exenatide), liraglutide (liraglutide), lixisenatide (lixisenatide), albiglutide (albiclutide), dolabrus peptide (dulaglutide), somaglutide (semaglutide), benaglutide (beinaglutide), polyethylene glycol loxapide (PEG-loxatenide), and sapaglutide (supaglutide).
In some embodiments, the stable formulation further comprises a pharmaceutical excipient selected from one or more of a buffer, a preservative, an emulsifier, a suspending agent, a diluent, a stabilizer, a pH adjuster, and a surfactant.
It is known to those skilled in the art that if a pharmaceutical excipient is unstable or degrades upon processing at temperatures between 55 ℃ and 65 ℃, the pharmaceutical excipient may be processed at temperatures between 55 ℃ and 65 ℃ and cooled prior to addition to a stable formulation.
In some embodiments, the buffering agent is selected from one or more of citrate, histidine, succinate.
In some embodiments, the surfactant is selected from one or more of poloxamer 188, polysorbate 20, and polysorbate 80.
In some embodiments, the stabilizing agent is selected from one or more of lactose, sucrose, trehalose, mannitol, and cellobiose.
In some embodiments, the stable formulation comprises a GLP-1 analog, a buffer, a stabilizer, and a surfactant, and the stable formulation has a pH of 6.5 to 7.0, the GLP-1 analog is a human GLP-1-Fc fusion protein comprising an Fc domain, an ortholog Fc fusion protein of human GLP-1, a mutant Fc fusion protein of human GLP-1, or a mutant Fc fusion protein of an ortholog of human GLP-1, treated at a temperature of 55 ℃ to 65 ℃ for 30 to 90 minutes,
the buffer is selected from one or more of citrate, histidine and succinate;
the surfactant is selected from one or more of poloxamer 188, polysorbate 20 and polysorbate 80; and is provided with
The stabilizer is selected from one or more of lactose, sucrose, trehalose, mannitol and cellobiose.
In some embodiments, the stable formulation comprises dolabrin, citrate, polysorbate 80 and mannitol,
and the pH of the stable formulation is 6.5-7.0, and the step of treating at a temperature of 55-65 ℃ for 30-90 minutes is performed.
In some embodiments, the stable formulation comprises dolabrin, citrate, polysorbate 80 and mannitol,
and the stability formulation has a pH of 6.7, and is subjected to a treatment step at a temperature of 60 ℃ for 50 to 70 minutes.
In some embodiments, the stable formulation comprises dolabrin, citrate, polysorbate 80 and mannitol,
and the stability formulation has a pH of 6.7, and is subjected to a treatment step at a temperature of 60 ℃ for 60 minutes.
In the case where polysorbate 80 is included in the stability formulation, after heat treatment and cooling, polysorbate 80 is further added.
In a second aspect, the present invention provides a method of preparing a stable formulation, said method comprising the step of treating a formulation comprising GLP-1 or a GLP-1 analog at a temperature of 55 ℃ to 65 ℃.
In some embodiments, the method comprises the step of treating a formulation comprising GLP-1 or a GLP-1 analog at a pH of 6.5 to 7.0 at a temperature of 55 ℃ to 65 ℃.
Preferably, the method comprises the step of treating a formulation comprising GLP-1 or a GLP-1 analog at pH6.5-7.0 at a temperature of 55 ℃ to 65 ℃ for 30-90 minutes.
Preferably, the method comprises the step of treating a formulation comprising GLP-1 or a GLP-1 analog at pH6.5-7.0 at a temperature of 55 ℃ to 65 ℃ for 50-70 minutes.
Preferably, the method comprises the step of treating a formulation comprising GLP-1 or a GLP-1 analog at pH6.6-6.8 at a temperature of 55 ℃ to 65 ℃ for 30-90 minutes.
Preferably, the method comprises the step of treating a formulation comprising GLP-1 or a GLP-1 analog at pH6.6-6.8 at a temperature of 55 ℃ to 65 ℃ for 50-70 minutes.
In some embodiments, the method comprises the step of treating a formulation comprising GLP-1 or a GLP-1 analog at pH6.7 at a temperature of 55 deg.C to 65 deg.C for 30 to 90 minutes.
In some embodiments, the method comprises the step of treating a formulation comprising GLP-1 or a GLP-1 analog at a temperature of 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃ or 65 ℃ for 50-70 minutes.
In some embodiments, the method comprises the step of treating a formulation comprising GLP-1 or a GLP-1 analog at pH6.7 at a temperature of 60 ℃ for 60 minutes.
In some embodiments, the GLP-1 analog is selected from the group consisting of an ortholog of human GLP-1, a mutant of an ortholog of human GLP-1, a modified human GLP-1, a mutant of an ortholog of modified human GLP-1, and the GLP-1 analog retains the native biological activity of GLP-1.
In some embodiments, the modified human GLP-1, ortholog of modified human GLP-1, mutant of modified human GLP-1, or mutant of ortholog of modified human GLP-1 comprises a modification selected from the group consisting of: pegylation; glycosylation, polysialylation, or hydroxyethylation; a fusion maltose binding protein; fusion of albumin; albumin fusion by acylation; fusing Fc; and fusion PEG mimetics.
In some embodiments, the GLP-1 analog is selected from the group consisting of exenatide (exenatide), liraglutide (liraglutide), lixisenatide (lixisenatide), albiglutide (albiclutide), dolabrus peptide (dulaglutide), somaglutide (semaglutide), benaglutide (beinaglutide), polyethylene glycol loxapide (PEG-loxatenide), and sapaglutide (supaglutide).
In some embodiments, the formulation comprising GLP-1 or a GLP-1 analogue further comprises a pharmaceutical excipient selected from one or more of a buffer, a preservative, an emulsifier, a suspending agent, a diluent, a stabilizer, a pH adjusting agent and a surfactant.
It is known to those skilled in the art that if a pharmaceutical excipient is unstable or degrades when processed at temperatures between 55 ℃ and 65 ℃, the pharmaceutical excipient may be added to the stable formulation after processing at temperatures between 55 ℃ and 65 ℃ and cooling.
In some embodiments, the buffering agent is selected from one or more of citrate, histidine and succinate.
In some embodiments, the surfactant is selected from one or more of poloxamer 188, polysorbate 20, and polysorbate 80.
In some embodiments, the stabilizing agent is selected from one or more of lactose, sucrose, trehalose, mannitol, and cellobiose.
In some embodiments, the method comprises the step of treating a formulation having a pH of 6.5 to 7.0 comprising a GLP-1 analog, a buffer, a stabilizer, and a surfactant, said GLP-1 analog being a human GLP-1-Fc fusion protein comprising an Fc domain, an ortholog Fc fusion protein of human GLP-1, a mutant Fc fusion protein of human GLP-1, or a mutant Fc fusion protein of an ortholog of human GLP-1, at a temperature of 55 ℃ to 65 ℃ for 30 to 90 minutes,
the buffer is selected from one or more of citrate, histidine and succinate;
the surfactant is selected from one or more of poloxamer 188, polysorbate 20 and polysorbate 80; and is
The stabilizer is selected from one or more of lactose, sucrose, trehalose, mannitol and cellobiose.
In some embodiments, the method includes the step of treating a formulation comprising dulaglutide, citrate, polysorbate 80 and mannitol at a ph of 6.5-7.0 at a temperature of 55 ℃ -65 ℃ for 30-90 minutes.
In some embodiments, the method includes the step of treating a formulation comprising 1-10mg/mL dolabrin, 10-20mM citrate, 0.01-0.1% polysorbate 80 and 4-5% mannitol at ph6.5-7.0 at a temperature of 55 ℃ -65 ℃ for 30-90 minutes.
In some embodiments, the method comprises the step of treating a formulation comprising dulaglutide, citrate, polysorbate 80 and mannitol at ph6.7 at a temperature of 60 ℃ for 50-70 minutes.
In some embodiments, the method comprises the step of treating a formulation comprising 1-5mg/mL dolabrin, 13-18mM citrate, 0.01-0.05% polysorbate 80 and 4.5-5% mannitol at ph6.7 at a temperature of 60 ℃ for 50-70 minutes.
In some embodiments, the method comprises the step of treating a formulation comprising dulaglutide, citrate, polysorbate 80 and mannitol at ph6.7 at a temperature of 60 ℃ for 60 minutes.
In some embodiments, the method comprises the step of treating a formulation comprising 3mg/mL dolabrin, 15mM citrate, 0.02% polysorbate 80, and 4.6% mannitol at ph6.7 at a temperature of 60 ℃ for 60 minutes.
In the case where polysorbate 80 is included in the stability formulation, after heat treatment and cooling, polysorbate 80 is further added.
Detailed Description
The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that various changes and modifications based on the inventive concept herein by those skilled in the art are intended to be included within the scope of the invention. The reagents used are not indicated by the manufacturer, and are all conventional products commercially available.
In the present application "GLP-1" is native human GLP-1. In the present application, "GLP-1 analogs" include orthologs of human GLP-1, mutants of orthologs of human GLP-1, modified human GLP-1, mutants of modified human GLP-1, or mutants of orthologs of modified human GLP-1, said GLP-1 analogs maintaining the native biological activity of GLP-1. The term "modified" refers to altering one or more properties of human GLP-1, an ortholog of human GLP-1, a mutant of human GLP-1, or a mutant of an ortholog of human GLP-1, which alteration does not alter the primary amino acid sequence of native human GLP-1, an ortholog of human GLP-1, a mutant of human GLP-1, or a mutant of an ortholog of human GLP-1, including but not limited to solubility, circulating half-life, stability, clearance rate, immunogenicity, allergenicity, and/or manufacturability. For example, the "modification" includes covalent chemical modifications that do not alter the primary amino acid sequence of native human GLP-1, an orthologue of human GLP-1, a mutant of human GLP-1, or a mutant of an orthologue of human GLP-1. "modifications" to native human GLP-1, an orthologue of human GLP-1, a mutant of human GLP-1, or a mutant of an orthologue of human GLP-1 may include, but are not limited to, one or more of the following: pegylation (covalent attachment of one or more polyethylene glycol (PEG) molecules or derivatives thereof); glycosylation (e.g., N-glycosylation), polysialylation, or hesylation; a maltose binding protein fusion protein; albumin fusion proteins (e.g., HSA fusion proteins); albumin binding by, for example, conjugation to a fatty acid chain (acylation); an Fc-fusion protein; and fusion with PEG mimetics, and the like.
In some embodiments, the GLP-1 analog comprises a deletion, substitution, or insertion of 1-10 amino acids in the human GLP-1 sequence or ortholog thereof, thereby having an extended half-life in vivo, and retained native biological activity.
In some embodiments, the GLP-1 analog comprises C at 1-5 amino acid residues of a human GLP-1 sequence or ortholog thereof 12-24 Modification of long chain fatty acids to have an extended half-life in vivo, and retained natural biological activity.
In some embodiments, the GLP-1 analog is a fusion protein that includes an Fc domain.
In this application, an "Fc domain" consists of the hinge, CH2, and CH3 constant region structures of an antibody. The Fc domain has a longer plasma half-life. The Fc domain may provide a longer half-life when fused to GLP-1 or GLP-1 analogs, or be involved in functions such as Fc receptor binding, protein A binding, complement binding, and the like. As used herein, "Fc domain" refers to a wild-type Fc from a native antibody, e.g., the Fc of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof comprising deletion, substitution, and/or addition of one or more amino acids. In some embodiments, the Fc variant possesses the activity of a wild-type Fc, such as binding to an Fc receptor, and increases the in vivo half-life of an Fc fusion protein, such as a mutated Fc domain known in the art.
In some embodiments, the fusion protein is such that the C-terminus of the GLP-1 or GLP-1 analog is linked to the N-terminus of the Fc domain, either directly or via a peptide linker, or vice versa. The peptide linker is a peptide linker well known in the art.
In some embodiments, GLP-1 analogs herein include, but are not limited to, exenatide (exenatide), liraglutide (liraglutide), lissenitide (lixisenatide), albiglutide (albicidin), dolaglutide (dulagllutide), somaglutide (semaglutide), benaglutide (benaglutide), pegolopside (PEG-loxatenide), and sapaglutide (supaglutide).
Exenatide is a GLP-1 analogue isolated from lizard saliva and composed of 39 amino acids, and has 53% homology with human GLP-1. The 2 nd site of the N end of the polypeptide is glycine (GLP-1 is alanine), the polypeptide is not easily degraded by DPP-4 enzyme, and the C end in the amino acid sequence has 9 more amino acid residues (PSSGAPPPS) compared with GLP-1 and is not easily degraded by endopeptidase, so the polypeptide has longer half life and stronger biological activity. Exenatide can reduce the level of glycated hemoglobin (HbA1c) and postprandial blood glucose in type II diabetics and reduce weight.
The liraglutide is prepared by substituting arginine for lysine at position 34 of a human GLP-1(7-37) chain, and connecting a 16-carbon palmitic acid side chain connected with glutamic acid to the lysine at position 26, has 97 percent of sequence homology with the human GLP-1, and can bind and activate GLP-1R. GLP-1 can be combined with albumin after being modified by fatty chain, has higher enzyme stability to DPP-4 and Neutral Endopeptidase (NEP), and thus has longer plasma half-life. The elimination half-life of liraglutide is 13h, and the injection is only needed to be injected 1 time per day. In addition to lowering blood glucose, liraglutide achieves weight reduction by reducing energy intake through appetite suppression, but the cost and the need for daily injections may limit its use in obesity.
The linatide is a short-acting GLP-1RA medicament taken 1 time a day, and structurally, the linatide is prepared by removing proline at the 38 th position on the basis of the structure of exenatide and connecting 6 lysines behind serine at the 39 th position, wherein the 6 lysine residues increase the rigidity of the molecular structure, so that the medicinal property of the linatide is improved. Compared with exenatide injected 2-3 times per day, the risperidone is a more stable structure, reduces protein degradation in systemic circulation, prolongs circulation time, and ensures that 1 injection is performed every day.
The albiglutide is long-acting GLP-1RA injected subcutaneously for 1 time per week, and structurally, the albiglutide substitutes glycine for alanine at position 8 of GLP-1(7-36) chain, and then 2 modified GLP-1 peptide chains are fused on Human Serum Albumin (HSA), so that the half-life period is greatly prolonged.
The dolaglutide is a long-acting GLP-1RA medicine which only needs to be administrated 1 time per week, and structurally, the dolaglutide is prepared by replacing 8 th position of GLP-1(7-37) chain with glycine for alanine, 22 th position with glutamic acid for glycine, 36 th position with glycine for arginine, and fusing the dolaglutide to a constant region (Fc) of modified human immunoglobulin G4(IgG4) through a "- (Gly-Gly-Gly-Ser) 3-Ala-" coupling bridge to form a dimer structure, and the average biological half-life is as long as 90 hours. The dolabrus peptide is the first macromolecule GLP-1RA with the curative effect equivalent to that of the liraglutide. Moreover, the administration frequency of 1 administration of dolabrus peptide per week can greatly improve the compliance of patients.
Compared with exenatide, liraglutide and linatide are single-chain polypeptides with the length of only more than 30 amino acids respectively, and dolafeptide is a single-chain dimer peptide with the length of 275 amino acids, and the difference between the liraglutide and the linatide is about 240 amino acids. Taking liraglutide as an example, the amino acid length of dolabrlutide is 8.9 times that of liraglutide, and the difference between the amino acid lengths is so large that the physical and chemical properties of the two are far from each other. For example, GLP-1-Fc fusion proteins with an Fc region involve the problem of oxidation of the Fc region and the loss of the corresponding intact main peak. The prior art stabilizing formulations or stabilization methods for exenatide, liraglutide and linatide are not necessarily applicable to dolarrtide which differs from them by about 240 amino acids in length and which is a double-chain dimer structure.
The present invention unexpectedly found that a formulation comprising GLP-1 or GLP-1 analog, which is capable of retaining the fragment peak and antiphase impurities at a low level for up to 3 weeks, shows significantly reduced fragment peak and antiphase impurities compared to an untreated formulation comprising GLP-1 or GLP-1 analog, is heat treated under neutral mildly acidic conditions at a temperature of 55 ℃ to 65 ℃ for a period of 30min to 90min, especially for a GLP-1-Fc fusion protein comprising an Fc domain or a GLP-1 analog Fc fusion protein. Thus, the formulations of the present invention show significantly improved stability.
The other parts except the main peak of RP in the reversed phase chromatographic result are collectively called reversed phase impurities.
In particular, the preparation of the invention, after said treatment, shows a decrease in the rate of decrease of the monomer peak by up to 40% and a decrease in the rate of decrease of the fusion protein in fragment form by nearly 40% at 60 ℃ in comparison with the treatment at 50 ℃ and 70 ℃ over a period of 3 weeks. Furthermore, the longer the heating time, the lower the monomer peak drop rate, the lower the LMW growth rate, and the lower the GLP-1-Fc fusion protein main peak drop rate, from 0 to 60 minutes. When the heating temperature reaches 60 ℃, the formation of fragment peaks and reverse phase impurities can be obviously reduced, and the stability of the preparation is improved.
Examples
The GLP-1-Fc fusion protein used in the embodiment of the invention is dolaglutide;
sample preparation: CHO-K1 cells are used for expressing GLP-1-Fc fusion protein, and the fusion protein is used for subsequent experiments after purification;
the abbreviation is:
PS 80: polysorbate 80
PFS: prefilled syringe
LMW: low molecular weight
HMW: high molecular weight
SEC: size exclusion
Reverse Phase (RP) chromatography was used to monitor the formation of GLP-1-Fc fusion protein in fragment form, oxidation of the Fc region and corresponding loss of the major intact peak. Size Exclusion (SEC) chromatography was used to monitor polymer formation and corresponding monomer loss.
Protein solutions containing 15mM citrate, 4.6% mannitol and pH6.7 are respectively placed in water baths with different temperatures for different time periods to be heated, after the treatment is finished, the solutions are cooled to room temperature, 0.02% PS80 is added and mixed evenly, finally the preparation is filtered through a 0.22um filter membrane and filled into PFS, and the PFS is placed in a stabilizing box at 40 ℃ for stability investigation.
Example 1
And (3) observing heating temperature:
the experimental design is shown in the following table:
the stability results were as follows:
F4/F5/F6 generates a large amount of turbid precipitates after being heated, so that the subsequent liquid preparation process is cancelled. It is shown that the heating temperature higher than 70 deg.C is not good for the stability of the preparation.
From the above table results, it can be seen that the monomer peak drop rate of F3 is significantly lower than that of F1 and F2, mainly because the LMW formation rate is reduced, indicating fewer fragment peaks. Meanwhile, the RP main peak descending rate of F3 is obviously lower than that of F1 and F2. Therefore, the above results show that when the heating temperature reaches 60 ℃, the formation of fragment peaks and reverse phase impurities can be significantly reduced, and the stability of the preparation can be improved.
Example 2
Examination of heating time and heating temperature:
the experimental design is shown in the following table:
the stability results were as follows:
as can be seen from the above table, the longer the heating time, the lower the SEC monomer peak drop rate, the lower the LMW growth rate, and the lower the RP peak drop rate. When the heating time reaches 60min, the SEC monomer peak is basically unchanged, and the stability of the preparation is obviously improved compared with the non-heating group.
Claims (10)
1. A stable formulation comprising a GLP-1 analogue and which has been subjected to a treatment step at a temperature of 55 ℃ to 65 ℃.
2. The stable formulation of claim 1, having a pH of 6.5 to 7.0, having been subjected to a treatment step at a temperature of 55 ℃ to 65 ℃,
preferably, the pH of the stable formulation is 6.5 to 7.0, and the stable formulation is subjected to a treatment step at a temperature of 55 ℃ to 65 ℃ for 30 to 90 minutes,
preferably, the pH of the stable formulation is 6.5 to 7.0, and the stable formulation is subjected to a treatment step at a temperature of 55 ℃ to 65 ℃ for 50 to 70 minutes,
preferably, the stability formulation has a pH of 6.7 and is subjected to a treatment step at a temperature of 60 ℃ for 60 minutes.
3. The stable formulation of claim 1, the GLP-1 analog is selected from the group consisting of an ortholog of human GLP-1, a mutant of an ortholog of human GLP-1, a modified human GLP-1, a mutant of an ortholog of modified human GLP-1, and the GLP-1 analog retains the native biological activity of GLP-1,
preferably, the modified human GLP-1, orthologues of modified human GLP-1, mutants of modified human GLP-1, or mutants of orthologues of modified human GLP-1 comprise modifications selected from the group consisting of: pegylation; glycosylation, polysialylation, or hydroxyethylation; a fusion maltose binding protein; fusion of albumin; albumin fusion by acylation; fusing Fc; and a fusion PEG mimetic;
preferably, the GLP-1 analog is selected from a human GLP-1-Fc fusion protein comprising an Fc domain, an orthologue Fc fusion protein of human GLP-1, a mutant Fc fusion protein of human GLP-1, or a mutant Fc fusion protein of an orthologue of human GLP-1;
preferably, the GLP-1 analogue is selected from exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, benaglutide, peglofenade and sapaglutide.
4. The stable formulation of claim 1, further comprising a pharmaceutical excipient selected from one or more of a buffer, a preservative, an emulsifier, a suspending agent, a diluent, a stabilizer, a pH adjuster, and a surfactant,
preferably, the buffering agent is selected from one or more of citrate, histidine and succinate;
preferably, the surfactant is selected from one or more of poloxamer 188, polysorbate 20 and polysorbate 80;
preferably, the stabilizer is selected from one or more of lactose, sucrose, trehalose, mannitol and cellobiose.
5. The stable formulation of claim 1, comprising a GLP-1 analog, a buffer, a stabilizer, and a surfactant, and the stable formulation has a pH of 6.5 to 7.0, the GLP-1 analog selected from the group consisting of a human GLP-1-Fc fusion protein comprising an Fc domain, an ortholog Fc fusion protein of human GLP-1, a mutant Fc fusion protein of human GLP-1, or a mutant Fc fusion protein of an ortholog of human GLP-1, treated for a step of 30-90 minutes at a temperature of 55 ℃ to 65 ℃,
the buffer is selected from one or more of citrate, histidine and succinate;
the surfactant is selected from one or more of poloxamer 188, polysorbate 20 and polysorbate 80;
the stabilizer is selected from one or more of lactose, sucrose, trehalose, mannitol and cellobiose;
preferably, the stable formulation comprises dolabrin, citrate, polysorbate 80 and mannitol,
and the pH of the stable formulation is 6.5-7.0, subjected to a step of treatment at a temperature of 55-65 ℃ for 30-90 minutes, preferably, for 50-70 minutes;
preferably, the stable formulation comprises dolabrin, citrate, polysorbate 80 and mannitol,
and the stability formulation has a pH of 6.7, and is subjected to a step of treatment at a temperature of 60 ℃ for 60 minutes.
6. A method of making a stable formulation, the method comprising the step of treating a formulation comprising a GLP-1 analogue at a temperature of 55 ℃ to 65 ℃.
7. The method of claim 6, comprising the step of treating a formulation of GLP-1 analog at pH6.5-7.0 at a temperature of 55 ℃ -65 ℃,
preferably, the method comprises the step of treating a formulation comprising a GLP-1 analog at pH6.5-7.0 for 30-90 minutes at a temperature of 55 ℃ -65 ℃,
preferably, the method comprises the step of treating a formulation comprising a GLP-1 analog at pH6.5-7.0 for 50-70 minutes at a temperature of 55 ℃ -65 ℃,
preferably, the method comprises the step of treating the formulation comprising the GLP-1 analog at pH6.7 at a temperature of 60 ℃ for 60 minutes.
8. The method of claim 6, wherein the GLP-1 analog is selected from the group consisting of an ortholog of human GLP-1, a mutant of an ortholog of human GLP-1, a modified human GLP-1, a mutant of an ortholog of modified human GLP-1, and wherein the GLP-1 analog retains the native biological activity of GLP-1,
preferably, the modified human GLP-1, orthologues of modified human GLP-1, mutants of modified human GLP-1, or mutants of orthologues of modified human GLP-1 comprise modifications selected from the group consisting of: pegylation; glycosylation, polysialylation, or hydroxyethylation; a fusion maltose binding protein; fusion of albumin; albumin fusion by acylation; fusing Fc; and a fusion PEG mimetic;
preferably, the GLP-1 analog is selected from a human GLP-1-Fc fusion protein comprising an Fc domain, an orthologue Fc fusion protein of human GLP-1, a mutant Fc fusion protein of human GLP-1, or a mutant Fc fusion protein of an orthologue of human GLP-1;
preferably, the GLP-1 analogue is selected from exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, benaglutide, peglofenade and sapaglutide.
9. The method of claim 6, wherein the GLP-1 analog-containing formulation further comprises a pharmaceutical excipient selected from one or more of a buffer, a preservative, an emulsifier, a suspending agent, a diluent, a stabilizer, a pH adjuster, and a surfactant,
preferably, the buffering agent is selected from one or more of citrate, histidine and succinate;
preferably, the surfactant is selected from one or more of poloxamer 188, polysorbate 20 and polysorbate 80;
preferably, the stabilizer is selected from one or more of lactose, sucrose, trehalose, mannitol and cellobiose.
10. The method of claim 6, wherein the stable formulation comprises a GLP-1 analog, a buffer, a stabilizer, and a surfactant, the stable formulation has a pH of 6.5-7.0, and the method comprises the step of treating at a temperature of 55 ℃ -65 ℃ for 30-90 minutes,
the GLP-1 analog is selected from a human GLP-1-Fc fusion protein comprising an Fc domain, an orthologue Fc fusion protein of human GLP-1, a mutant Fc fusion protein of human GLP-1, or a mutant Fc fusion protein of an orthologue of human GLP-1,
the buffer is one or more of citrate, histidine and succinate,
the surfactant is selected from one or more of poloxamer 188, polysorbate 20 and polysorbate 80,
the stabilizer is selected from one or more of lactose, sucrose, trehalose, mannitol and cellobiose;
preferably, the stable formulation comprises dolaglutide, citrate, polysorbate 80 and mannitol,
and the method comprises the step of treating at a temperature of 55 ℃ to 65 ℃ for 30 to 90 minutes, preferably, for 50 to 70 minutes;
preferably, the stable formulation comprises dolaglutide, citrate, polysorbate 80 and mannitol,
and the method comprises the step of treating at a temperature of 60 ℃ for 60 minutes.
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| US20180021409A1 (en) * | 2014-06-25 | 2018-01-25 | Glaxosmithkline Llc | Pharmaceutical Composition |
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| CN111808201A (en) * | 2020-09-04 | 2020-10-23 | 迈威(上海)生物科技股份有限公司 | Preparation method of recombinant human glucagon-like peptide-1 analogue fusion protein |
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| CN101056650A (en) * | 2004-11-12 | 2007-10-17 | 诺和诺德公司 | Stable formulations of insulinoptropic peptides |
| US20180021409A1 (en) * | 2014-06-25 | 2018-01-25 | Glaxosmithkline Llc | Pharmaceutical Composition |
| CN107661288A (en) * | 2016-07-29 | 2018-02-06 | 江苏泰康生物医药有限公司 | Stable liquid preparation and its preparation containing the analog fusions of GLP 1 |
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