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    • 专利摘要:
    "liquid formulation of long-acting insulinotropic peptide conjugate". the present invention relates to a liquid formulation of long-acting insulinotropic peptide conjugate, comprising a pharmaceutically effective amount of long-acting insulinotropic peptide conjugate consisting of a physiologically active peptide, which is an insulinotropic peptide, and an immunoglobulin fc region; and an albumin-free stabilizer, wherein the stabilizer comprises a buffer, a sugar alcohol, a nonionic surfactant, and an isotonic agent, and refers to a method of preparing the formulation. a preservative can be added to prevent microbial contamination. the liquid formulation of the present invention is free from human serum albumin and other factors potentially hazardous to the organism, with no risk of viral contamination, and can therefore provide excellent storage stability to the concentrated insulinotropic peptide conjugate.
    公开号:BR112015001735A2
    申请号:R112015001735-5
    申请日:2013-07-25
    公开日:2021-04-13
    发明作者:Hyun Uk Kim；Hyung Kyu Lim；Sung Hee Hong；Dae Jin Kim；Sung Min Bae；Se Chang Kwon
    申请人:Hanmipharm. Co., Ltd.；
    IPC主号:
    专利说明:

    [001] [001] The present invention relates to a liquid formulation of long-acting insulinotropic peptide conjugate, which comprises a pharmaceutically effective amount of long-acting insulinotropic peptide conjugate, in which a physiologically active peptide, which is an insulinotropic peptide, is linked to an immunoglobulin Fc region, and an albumin-free stabilizer, in which the stabilizer comprises a buffer, a sugar alcohol, a non-ionic surfactant, and an isotonic agent, and refers to a method for the preparation of the formulation. STATE OF THE PREVIOUS TECHNIQUE
    [002] [002] Diabetes is a disease derived from multiple pathogenic factors and there are usually two types of diabetes. Patients with type I diabetes or insulin-dependent diabetes mellitus (DMID) barely produce or do not produce insulin, which is a hormone that regulates the use of carbohydrates. Patients with type II diabetes or non-insulin-dependent diabetes mellitus (NIDDM) have a plasma insulin level equal to or greater than that of individuals without diabetes. However, patients with type II diabetes develop resistance to insulin-stimulated glucose metabolism and lipid metabolism in insulin-sensitive main tissues, that is, muscle, liver, and adipose tissue. Although the level of insulin in the plasma may increase, it is not enough to overcome the considerable insulin resistance, thus causing hyperglycemia. Continued or unregulated hyperglycemia is associated with an increase in the rate of morbidity and early mortality. Often, an abnormal increase in the level of sugar is directly and indirectly related to metabolic and hemodynamic changes in diseases associated with the metabolism of lipids, lipoproteins, apolipoproteins, and others. For example, type II diabetes mellitus patients in particular have a high risk of developing coronary heart disease, stroke, peripheral vascular disease, hypertension, nephropathy, and neuropathy, as well as hemangiomas and microvascular complications.
    [003] [003] Therapies currently used to treat type II diabetes include administration of exogenous insulin, oral administration of drugs, a therapeutic diet and exercise. In 2005, exenatide (Exendin-4: Byetta®) was approved by the FDA as a complementary therapy for patients with type II diabetes who do not achieve adequate glucose regulation, even with metformin and / or sulfonylurea treatment.
    [004] [004] Exenatide (exendin-4) is a potent agonist of the GLP-1 receptor and is produced by the lizard salivary gland. Exendin-4 has an affinity for insulin, suppresses food intake and gastric emptying, and has an affinity for β cells in rodents (Parks et. Al., Metabolism. 50: 583-589, 2001; Aziz and Anderson, J Nutr. 132: 990-995, 2002; and Egan et al., J. Clin. Endocrinol. Metab. 87: 1282-1290, 2002). In addition, since glycine is present at position 2 of the N-terminus of exidine-4, it is not a substrate for DPP IV, unlike GLP-1. The disadvantage of exenatide is its short half-life (t1 / 2), which is only 2 to 4 hours, and therefore has to be injected twice a day (Kolterman et al., J. Clin. Endocrinol. Metab. 88: 3082-3089, 2003 and Fineman et al., Diabetes Care. 26: 2370-2377, 2003).
    [005] [005] Peptides such as the exenatide described above are easily denatured or degraded by proteases in the body due to their low stability and loss of activity. In addition, the size of exenatides is relatively small and is therefore easily removed by the kidney. Thus, drugs containing peptides as pharmaceutically active principles must be administered frequently to patients in order to maintain the desired serum level and titer. In most cases, peptide drugs are administered by injection and at a high frequency to maintain the serum level of the physiologically active peptide, but this is very painful for patients.
    [006] [006] Many efforts have been made to solve these problems, one of which was to administer a peptide drug by oral or nasal inhalation, increasing the permeability of the biomembrane of the peptide drug. However, this method has a very low efficiency of delivering the peptide to the body compared to the injection route. Therefore, there are still many difficulties in maintaining the activity of the peptide drug in vivo at the required level.
    [007] [007] On the other hand, continuous efforts have been made to maximize the therapeutic effects of the drug, improving the stability of the peptide drug in the blood and maintaining a high serum drug level over a long period. These long-acting peptide drug formulations should promote an increase in the stability of the peptide drug and also maintain a sufficiently high titre of the drug itself, without inducing immune responses in patients.
    [008] [008] As a method of stabilizing peptides and preventing their degradation by protease, there have been many attempts to modify a specific sequence of amino acids sensitive to protease. For example, GLP-1 (amide 7-37 or 7-36), which is effective for the treatment of type II diabetes by reducing the blood glucose level, has a very short half-life of less than four minutes ( Kreymann et al., 1987). The short half-life results from the loss of GLP-1 title due to peptide cleavage between amino acids No. 8 (Ala) and No. 9 (Asp) of GLP-1 by dipeptidyl-pepdidase IV (DPP IV). Thus, many studies have been conducted on the development of GLP-1 derivatives resistant to DPP IV, and in these studies Ala8 was replaced by Gly (Deacon et al., 1998; Burcelin et al., 1999), or by Leu or D- Ala (Xiao et al., 2001) to increase resistance to DPP IV, while maintaining the activity of the peptide. In addition, the N-terminal amino acid of
    [009] [009] In addition to these variants, exenatide (exendin-4, US 5,424,686), which is a derivative of GLP-1 purified from the salivary gland of the gila monster, has resistance to DPP IV and bioactivity higher than that of GLP-1, thus having a half-life in the body of 2 to 4 hours, which is much longer than that of GLP-1. However, a sufficient duration of bioactivity in vivo cannot be achieved solely by increasing the resistance of the peptide to DPP IV. For example, exendin-4 (exenatide) currently available must be administered twice a day by injection, which is an excessive burden for patients.
    [010] [010] A limitation of these insulinotropic peptides is that the size of the peptide is too small to be captured in the kidney and therefore is easily lost outside the body. Therefore, in order to prevent the loss of the peptide in the kidney, a highly soluble macromolecule, such as polyethylene glycol (PEG), has been attached to the surface of the peptide.
    [011] [011] PEG binds to a specific site, or not specifically to several sites of a target peptide and increases its molecular weight, which then prevents the loss of the peptide in the kidney and hydrolysis of it, without the occurrence of side effects . For example, WO2006 / 076471 reports that by fixing PEG to a type B natriuretic peptide (BNP), which activates the production of cGMP through binding to NPR-A and reduces intra-arterial pressure, being effective as a therapeutic agent for congestive heart failure, BNP bioactivity can be maintained. Similarly, US 6 924 264 describes a method for increasing the in vivo durability of exidin-4 by binding PEG to the lysine residue of an exidine-4. However, although these methods can prolong the in vivo durability of a peptide drug by increasing the molecular weight of PEG, the peptide drug titer decreases markedly when the molecular weight of PEG increases. In addition, the reactivity of PEG with the peptide decreases, and consequently the yield as well.
    [012] [012] As an alternative to increase the in vivo stability of the physiologically active peptide, a method of producing a fusion protein has been developed in which the genes for the peptide and for the physiologically active protein are joined by genetic recombination and cells transformed with the recombinant gene are cultured. As an example, an exendin-4-producing fusion protein, fused with transferrin (Tf) via a polypeptide linker, has been disclosed (Korean Patent Application No. 10-2009-7003679). Likewise, as a method of using immunoglobulin, a fusion protein of a GLP-1 derivative, with the GLP-1 derivative fused to IgG4 Fc, has also been described before (Korean Patent Application No. 10- 2007-7014068).
    [013] [013] Recently, as a pharmaceutical peptide and protein formulation, of long duration action, capable of promoting a minimum reduction in activity and an increase in stability, a conjugate generated by the combination of the immunoglobulin Fc region, a non-peptide polymer and a physiologically active polypeptide, has been disclosed in Korean Patent No. 10- 0567902 (Physiologically active polypeptide conjugate with improved in vivo durability) and in Korean Patent No. 10-0725315 (Protein complex using an immunoglobulin fragment and method for the preparation).
    [014] [014] In the previous method, an insulinotropic peptide is used as a physiologically active polypeptide in the preparation of a long-acting insulinotropic peptide conjugate (Korean Patent No. 10- 2008-0001479). In order to produce the long-acting insulinotropic peptide conjugate drug, it is essential to avoid physical-
    [015] [015] Proteins and peptides generally have a short half-life and may undergo denaturation in the form of monomer aggregation, precipitation by aggregation, and adsorption to the surface of the container when exposed to inadequate temperatures, the air-water interface, high pressure, or physical or mechanical stress, organic solvents, and microbial contamination. Denatured proteins and peptides lose their inherent physicochemical properties and physiological activity. Since protein denaturation is irreversible in most cases, denatured proteins and peptides cannot recover their inherent properties. In addition, proteins are likely to be unstable and easily affected by external factors, such as temperature, humidity, oxygen, ultraviolet rays, and therefore undergo physical or chemical changes, including aggregation, polymerization, or oxidation, thereby losing activity .
    [016] [016] In addition, adsorbed proteins and peptides can aggregate when denaturing and when aggregated proteins and peptides are introduced into the body they can cause the formation of antibodies. Thus, sufficiently stable proteins and peptides should be administered. In this regard, several methods have been developed to avoid denaturing proteins and peptides in solution (John Geigert, J. Parenteral Sci. Tech., 43, No5, 220-224, 1989, David Wong, Pharm. Tech. October , 34-48, 1997, Wei Wang., Int. J. Pharm., 185, 129-188, 1999, Willem Norde, Adv. Colloid Interface Sci., 25, 267- 340, 1986, Michelle etal., Int. J. Pharm. 120, 179-188, 1995).
    [017] [017] In the production of some protein and peptide drugs, a lyophilization process has been used to solve the stability problems. This process, however, is inconvenient in that the lyophilized products have to be dissolved in solvents for injection before use and because it requires a large-scale investment, since a large number of freezer-dryers is necessary to carry out the process. freeze-drying of the manufacturing process. Alternatively, a spray method has been used which employs spray drying. However, this method is not very profitable due to the low yield of the product and can have a negative effect on the stability of the product since the proteins are exposed to high temperature.
    [018] [018] In an alternative approach to solving these limitations, the addition of stabilizers to the protein and peptide solution was investigated to prevent physico-chemical changes in the protein drug, while maintaining its in vivo efficacy during long-term storage. One type of protein, human serum albumin, has been widely used as a stabilizer in several protein drugs and its effectiveness has been approved (Edward Tarelli et al., Biologicals (1998) 26, 331-346).
    [019] [019] Purification of human serum albumin requires the inactivation of biological contaminants, such as mycoplasma, prions, bacteria and viruses, or the screening or inspection of one or more biological contaminants or pathogens, but even through these processes, these contaminants they may not be completely removed or inactivated. In this way, patients may be exposed to biological contaminants or pathogens when administered with human serum albumin. For example, although the screening process involves inspecting certain viruses in the donor's blood sample, the inspection process is not always reliable and cannot detect certain viruses that are present in small numbers.
    [020] [020] Due to their chemical differences, different proteins can be inactivated gradually at different rates, under different conditions, during storage. That is, the prolongation of the storage period achieved by a stabilizer is not the same for different proteins. For this reason, the proportion, concentration and type of stabilizers that are appropriate for improving protein storage stability vary according to the physicochemical properties of the target protein. In addition, when different stabilizers are used together, they can induce adverse effects different from the desired effects, due to the interaction and competing side effects. In addition, during storage, the properties of proteins or their concentration may change, causing different effects.
    [021] [021] Therefore, much effort and care are needed to stabilize proteins in solution. In particular, a long-acting insulinotropic peptide conjugate with improved in vivo durability and stability is in the form of an insulinotropic peptide combined with the immunoglobulin Fc region, and therefore has a significantly different molecular weight and volume than the insulinotropic peptide. base. As such, a special composition is needed to stabilize the protein. In addition, an insulinotropic peptide and an Fc region of the immunoglobulin are physiochemically different peptides or proteins and thus have to be stabilized simultaneously. However, as previously described, due to their physical-chemical differences, different peptides or proteins can be gradually inactivated at different rates, under different conditions, in storage. In addition, when the appropriate stabilizers for the peptide or protein are used together, they can induce adverse effects different from the desired effects due to the interaction and competing side effects. Therefore, it is very difficult to find a stabilizing composition for a long-acting insulinotropic peptide conjugate, which can simultaneously stabilize an insulinotropic peptide and an immunoglobulin Fc region.
    [022] [022] Recently, a protein and peptide formulation has been developed that can be used repeatedly for the convenience of patients.
    [023] [023] However, the use of preservative can affect protein stability. The most well-known problem with the use of preservatives is that of precipitation. Precipitation of the protein can reduce the therapeutic effects of the drug and, when administered to the body, can induce an unexpected immune response. Therefore, it is crucial to choose a type and concentration of preservative that maintain the ability to prevent microbial contamination without simultaneously affecting the stability of the protein. DISCLOSURE OF THE INVENTION TECHNICAL PROBLEM
    [024] [024] In an effort to provide a stable liquid formulation of a long-acting insulinotropic peptide conjugate, in which it can be stored without the risk of viral contamination for a long period, the present invention has found that a formulation that improves the stability of the long-acting insulinotropic peptide conjugate can be obtained by means of a stabilizer consisting of a buffer, a sugar alcohol, a non-ionic surfactant, and an isotonic agent, or additionally methionine, and that the formulation can be used several times when additionally a preservative is included, thus completing a cost effective and stable liquid formulation. SOLUTION TO THE PROBLEM
    [025] [025] An object of the present invention is to provide a liquid formulation of a long-acting insulinotropic peptide conjugate comprising a pharmaceutically effective amount thereof, in which a physiologically active peptide, that is, an insulinotropic peptide, is bound an Fc region of the immunoglobulin; and an albumin-free stabilizer, wherein the stabilizer comprises a buffer, a sugar alcohol, a non-ionic surfactant, and an isotonic agent.
    [026] [026] Another object of the present invention is to provide a liquid formulation of a long-acting insulinotropic peptide conjugate for multiple administrations, which additionally comprises a preservative in addition to the long-acting insulinotropic peptide conjugate and the exempt stabilizer. of albumin.
    [027] [027] Another objective of the present invention is to provide a method for the preparation of the liquid formulation of a long-acting insulinotropic peptide conjugate. ADVANTAGE EFFECTS OF THE INVENTION
    [028] [028] Since the liquid long-acting insulinotropic peptide conjugate formulation of the present invention comprises a buffer, an isotonic agent, a sugar alcohol, and a nonionic surfactant, or additionally, methionine, but is free of human serum albumin and other factors potentially dangerous to the body, there is no risk of viral contamination. In addition, it can provide excellent storage stability to the long-acting insulinotropic peptide conjugate comprised of an insulinotropic peptide and an immunoglobulin Fc region, thereby having a higher molecular weight and an improved in vivo physiological activity duration. relation to the wild-type protein. Such a liquid formulation of the present invention can provide excellent storage stability with a simple formulation, obtaining a more economical peptide drug than other stabilizer and lyophilizer. If a preservative is added to the formulation, the formulation can be used several times. In addition, the present formulation can retain protein activity in the body for a longer period than a conventional insulinotropic peptide formulation, and therefore can be used as an effective pharmaceutical formulation. BRIEF DESCRIPTION OF THE DRAWINGS
    [029] [029] Figure 1 is a graph of RP-HPLC analysis of peptide stability in the liquid formulation of choice at a pH of 5.2 (Liquid Formulation # 1), in the liquid formulation prepared by the addition of an insulinotropic peptide conjugate. long-acting effects on a stabilizing composition of a commercially available liquid insulinotropic peptide drug formulation, exenatide, ie exendin-4 (Byetta) (Liquid Formulation # 2), in the liquid formulation prepared by the addition of a conjugate of long-acting insulinotropic peptide to a stabilizing composition of liquid immunoglobulin fusion protein drug formulation, etanercept (TNFR-Fc fusion protein, ENBREL) (Liquid Formulation # 3), and in a control group (Formulation Liquid # 4), having all been stored at 25 ± 2ºC for 8 weeks.
    [030] [030] Figure 2 is a graph of the RP-HPLC analysis of the proportion of oxidized long-acting insulinotropic peptide conjugate in the liquid formulation at a pH of 5.2, without methionine (Liquid Formulation # 1) and in liquid formulation at pH 5.2, with methionine (Liquid Formulation # 2), stored at 25 ± 2ºC and 40 ± 2ºC for 4 weeks.
    [031] [031] Figure 3 shows the results of monitoring the occurrence of precipitation in long-acting insulinotropic peptide conjugate compositions according to Table 18, with the naked eye and at 40ºC, for 48 hours. The duration of the absence of precipitation indicates the period of time after storage of the peptide in which no protein precipitation occurred.
    [032] [032] Figure 4 shows the results of monitoring the occurrence of precipitation in long-acting insulinotropic peptide conjugate compositions according to Table 19, with the naked eye and at 40ºC, for 7 days. The duration of the absence of precipitation indicates the period of time after storage of the peptide in which no protein precipitation occurred. BEST MODE FOR CARRYING OUT THE INVENTION
    [033] [033] In one aspect, the present invention provides a liquid formulation of long-acting insulinotropic peptide conjugate, comprising a pharmaceutically effective amount of long-acting insulinotropic peptide conjugate, in which an insulinotropic peptide is bound to an immunoglobulin Fc region; and an albumin-free stabilizer, wherein the stabilizer comprises a buffer, a sugar alcohol, a non-ionic surfactant, and an isotonic agent.
    [034] [034] In addition, the present invention provides a liquid formulation of long-acting insulinotropic peptide conjugate for multiple administrations, which further comprises a preservative in addition to the insulinotropic peptide conjugate and the albumin-free stabilizer.
    [035] [035] As used herein, "long-acting insulinotropic peptide conjugate" refers to a conjugate in which a physiologically active insulinotropic peptide, comprising a derivative, variant, precursor, and fragment and an Fc region of the immunoglobulin are linked, and may also refer to a conjugate with an increased in vivo duration of physiological activity in relation to a wild-type insulinotropic peptide.
    [036] [036] As used herein, the term "long duration of action" refers to an increase in the duration of physiological activity compared to that of a wild type. The term "conjugate" refers to the way an insulinotropic peptide and an Fc region of the immunoglobulin are combined.
    [037] [037] The insulinotropic peptide used in the present invention has the function of secreting insulin and stimulates the synthesis and expression of insulin in β pancreatic cells. The type of insulinotropic peptide includes precursor, agonist, derivatives, fragments and variants. Preferably, the insulinotropic peptide may be a glucagon-like peptide-1 (GLP-1), a glucagon-like peptide-2 (GLP-2), exendin-3, exendin-4, and imidazoacetyl exendin-4 (CA) , and most preferably imidazoacetyl exendin-4 (CA). Any insulinotropic peptide, native or recombinant can be used and, preferably, is a recombinant insulinotropic peptide generated from E. coli as a host cell. As long as their biological activity is not significantly affected, any derivatives generated by substitution, deletion or insertion of amino acids can be used in the present invention.
    [038] [038] The insulinotropic peptide sequence can be obtained from known databases, such as NCBI's GenBank, and can be 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more of sequence homology with a wild-type protein, provided it demonstrates the activity of an insulinotropic peptide.
    [039] [039] In addition, the immunoglobulin Fc useful in the present invention can be a human immunoglobulin Fc or its closely related analog, or animal derived immunoglobulin Fc, such as cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs. In addition, the immunoglobulin Fc region can be derived from IgG, IgA, IgD, IgE and IgM, or from a combination or hybrid thereof. Preferably, it is derived from IgG or IgM, which are most abundant in human blood, and most preferably it is derived from IgG, which is known to increase the half-life of the ligand-binding protein. In addition, the immunoglobulin Fc region can be a dimer or multimer of single chain immunoglobulins with domains of the same origin. The immunoglobulin Fc region can be generated by treating a native IgG with a particular protease, or by cells transformed by a genetic recombination technique. Preferably, the immunoglobulin Fc is a recombinant human immunoglobulin Fc produced in E. coli.
    [040] [040] On the other hand, IgG can be divided into subclasses IgG1, IgG2, IgG3 and IgG4, and in the present invention, a combination or hybrid of them can be used. The IgG2 and IgG4 subclasses are preferred, and the most preferred is the IgG4 Fc region, which rarely has the effector function, such as complement-dependent cytotoxicity (CDC). That is, as the pharmaceutical carrier of the present invention, the most preferred Fc portion of the immunoglobulin is a non-glycosylated Fc portion derived from human IgG4. The Fc portion of human origin is preferred over an Fc portion of non-human origin, which can act as an antigen in the human body and elicit undesirable immune responses, such as the production of a new antibody.
    [041] [041] The non-peptide polymer used in the crosslinking can be selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol and propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinylethyl ether (such as biodegradable polymers (such as biodegradable polymers) )) and PLGA (poly (lactic-co-glycolic acid)), polymers of lipids, chitins, hyaluronic acid or a combination thereof. Polyethylene glycol is preferably used but not limited to it. Its derivatives well known in the art and derivatives that can be easily prepared by a method known in the art are included within the scope of the present invention
    [042] [042] For the preparation of the long-acting insulinotropic peptide conjugate used in the present invention, reference can be made to Korean Patent No. 10-0725315, Korean Patent Publication No. 10-2009- 0008151, and Korean Patent No. 10-1058290. Those skilled in the art will be able to produce a long-acting insulinotropic peptide conjugate of the present invention by consulting these references.
    [043] [043] The liquid long-acting insulinotropic peptide conjugate formulation of the present invention comprises a long-acting insulinotropic peptide conjugate in a therapeutically effective amount. In general, the therapeutically effective amount of the insulinotropic peptide, especially exendin-4 (Byetta), refers to 250 mcg in an injection pen. The concentration of long-acting insulinotropic peptide conjugate used in the present invention ranges from 0.1 mg / ml to 200 mg / ml, and preferably from 0.5 mg / ml to 150 mg / ml. The insulinotropic peptide can preferably be a long-acting exendin-4 CA conjugate. The liquid formulation of the long-acting insulinotropic peptide conjugate of the present invention can store the conjugate stably, without precipitation, not only when the insulinotropic peptide conjugate is present in low concentration, but also in high concentration. Therefore, the present formulation provides stable insulinotropic peptide, in high concentration, in the body.
    [044] [044] As used herein, the term "stabilizer" refers to a substance that allows stable storage of the long-acting insulinotropic peptide conjugate. The term "stabilization" refers to the state in which the loss of an active ingredient is less than a certain value, typically less than 10%, during a given period and under specific storage conditions. A formulation is considered to be a stable formulation when the residual purity of the long-acting insulinotropic peptide conjugate contained therein is 90% or more, and more preferably 92 to 95% after storage at 5 ± 3 ° C for 2 years. 25 ± 2ºC for 6 months, or 40 ± 2ºC for 1 to 2 weeks. As for proteins similar to the long-acting insulinotropic peptide conjugate, their stability in storage is important to obtain an exact dose, as well as to suppress the potential formation of antigenic substances against the long-acting insulinotropic peptide conjugate. . During storage, a 10% loss of long-acting insulinotropic peptide conjugate is acceptable for substantial administration, unless it causes the formation of aggregates or fragments in the composition that lead to the formation of antigenic compounds.
    [045] [045] The stabilizer of the present invention preferably comprises a buffer, a sugar alcohol, an isotonic agent such as sodium chloride, and a nonionic surfactant, and more preferably additionally comprises methionine to stabilize the long-acting insulinotropic peptide conjugate. duration.
    [046] [046] The buffer serves to maintain the pH of the solution and prevent a sudden change in pH in the liquid formulation in order to stabilize the long-acting insulinotropic peptide conjugate. The buffer may include an alkaline salt (sodium or potassium phosphate or its hydrogen or dihydrogen salts), sodium citrate / citric acid, sodium acetate / acetic acid, histidine / histidine hydrochloride, any other pharmaceutically pH buffer acceptable amount known in the art, and a combination thereof. The preferred example of buffer includes a citrate buffer, an acetate buffer and a histidine buffer. The buffer concentration is preferably from 5 mM to 100 mM, more preferably from 10 mM to 50 mM. The pH of the buffer is preferably from 4.0 to 7.0, more preferably from 5.0 to 7.0, even more preferably from 5.2 to 7.0, and even more preferably from 5.2 to 6, 0.
    [047] [047] Sugar alcohol acts to increase the stability of the long-acting insulinotropic peptide conjugate. The concentration of the sugar alcohol used in the present invention is preferably 1 to 20% (w / v) based on the total volume of solution, more preferably 3 to 10% (w / v) based on the total volume of solution. Sugar alcohol may be one or more of the group consisting of mannitol, sorbitol, and sucrose, but is not limited to them.
    [048] [048] An isotonic agent acts to maintain an adequate osmotic pressure when the long-acting insulinotropic peptide conjugate in solution is administered to the body, and also acts to stabilize the long-acting insulinotropic peptide conjugate. in solution. The osmotic pressure of the formulation is adjusted to be isotonic with the blood. These liquid isotonic formulations generally have an osmotic pressure of about 300 mOsm / kg. Representative examples of an isotonic agent include a sugar alcohol, a water-soluble inorganic salt, an amino acid, and the preferred example is a water-soluble inorganic salt, i.e., sodium chloride. The concentration of sodium chloride as an isotonic agent is preferably from 0 to 150 mM, and can be adjusted depending on the type and quantity of components included in the formulation, such that the liquid formulation, including the entire mixture, is isotonic.
    [049] [049] The non-ionic surfactant decreases the surface tension of the protein solution to prevent absorption or aggregation of proteins on a hydrophobic surface. Examples of nonionic surfactants useful in the present invention include polysorbates, poloxamers and their combinations, with preference for polysorbates. Polysorbate nonionic surfactants include polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. The most preferred nonionic surfactant is polysorbate 20.
    [050] [050] A nonionic surfactant at a high concentration should not be used in the liquid formulation, as the nonionic surfactant at a high concentration induces interference effects in the measurement of protein concentration and in the determination of protein stability by analytical methods, such as such as UV spectroscopy or isoelectric focusing, thus causing difficulties in accurately assessing protein stability. Therefore, the liquid formulation of the present invention comprises the non-ionic surfactant, preferably at a low concentration, not exceeding 0.2% (w / v), more preferably from 0.001% to 0.05% (w / v) .
    [051] [051] According to an example of the present invention, it has been shown that when sodium chloride is added as an isotonic agent in the presence of buffer, sugar alcohol, and non-ionic surfactant, long-term storage stability of the peptide conjugate long-acting insulinotropic agent in low concentration increases significantly. This suggests that the use of sodium chloride as an isotonic agent simultaneously with buffer, sugar alcohol, and non-ionic surfactant induces synergistic effects, thus allowing the long-acting insulinotropic peptide conjugate to have a high stability. However, with regard to the long-acting insulinotropic peptide conjugate in high concentration, when sodium chloride is excluded, precipitation did not occur and the solubility of the protein improved. These results suggest that when sodium chloride is used as an isotonic agent, its content can be adjusted according to the concentration of the long-acting insulinotropic peptide conjugate.
    [052] [052] In addition, it was confirmed that a long-acting insulinotropic peptide conjugate at low concentration is more stable in a buffer at a pH of 5.2, while a long-acting insulinotropic peptide conjugate at a high concentration is more stable in a buffer at a pH of 5.4 or 5.6. It was thus determined that the pH of the buffer can be adjusted accordingly depending on the concentration of conjugate.
    [053] [053] The methionine comprised in the stabilizer of the present invention suppresses the formation of impurities that can occur by oxidation of proteins in solution, thus further stabilizing the target protein. The concentration of methionine is from 0.005 to 0.1% (w / v) in the total volume of solution, preferably from 0.01 to 0.1% (w / v).
    [054] [054] Preferably, the stabilizer of the present invention does not contain albumin. Since the human serum albumin available as a protein stabilizer is produced from human serum, it is always possible that it is contaminated with pathogenic viruses of human origin. Bovine serum gelatin or albumin can cause disease or induce an allergic reaction in some patients. Free of heterologous proteins, such as serum albumins of human or animal origin or purified gelatin, the stabilizer of the present invention cannot cause viral contamination.
    [055] [055] In addition, the stabilizer of the present invention may further comprise sugars, polyalcohol or amino acids. Preferred examples of sugars that can be added to further increase stability during long-term storage of long-acting insulinotropic peptide conjugate include monosaccharides such as mannose, glucose, fucose and xylose, and polysaccharides such as lactose, maltose, sucrose , raffinose and dextran. Preferred examples of polyalcohol include propylene glycol, low molecular weight polyethylene glycol, glycerol, low molecular weight polypropylene glycol and a combination thereof.
    [056] [056] The liquid formulation of the present invention can also comprise a preservative, in addition to the aforementioned conjugate, buffer, isotonic agent, sugar alcohol, and non-ionic surfactant, or additionally methionine, in order to prevent microbial contamination of the formulation multiple use.
    [057] [057] As used herein, "preservative" refers to a compound that is added to a pharmaceutical formulation to act as an antimicrobial agent. Examples of preservatives include benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, benzalkonium chloride, phenylmercury nitrate, thimerosal, benzoic acid, but not being limited to them. A single type of preservative can be used individually, or a random combination of two or more types of preservative can be used. Preferably, the liquid formulation of the present invention can comprise one or more of m-cresol, phenol and benzyl alcohol as a preservative.
    [058] [058] The liquid formulation of the present invention can comprise
    [059] [059] In an example of the present invention, 0.22% (w / v) of m-cresol was added as a preservative to the liquid formulation of the present invention and the effect of cresol on the stability of the insulinotropic peptide conjugate was evaluated. As a result, it was confirmed that the conjugate remained stable, without precipitating, in the formulation with the added preservative. Therefore, the liquid insulinotropic peptide conjugate formulation of the present invention, which comprises a preservative in addition to a stabilizer, can be used for multiple administrations.
    [060] [060] The liquid formulation of the present invention may further selectively comprise other substances and materials known in the art, in addition to those described above, buffer, isotonic agent, sugar alcohol, and non-ionic surfactant, or additionally, methionine and preservative, provided that the effect of the present invention is not affected.
    [061] [061] The long-acting insulinotropic peptide conjugate albumin-free liquid formulation according to the present invention, which provides stability to the long-acting insulinotropic peptide conjugate, poses no risk of viral contamination and simultaneously provides a excellent storage stability, with a simple formulation, thus being able to be supplied more economically than other stabilizing or lyophilized formulations.
    [062] [062] Furthermore, since the liquid formulation of the present invention comprises the long-acting insulinotropic peptide conjugate with an increased physiological activity duration over the wild type, it can be used as an effective pharmaceutical formulation, retaining the activity of the protein in the body for a longer period than the conventional insulinotropic peptide formulation. In addition, the present liquid formulation provides excellent stability, suitable for the storage of a long-acting insulinotropic peptide conjugate in high concentration, as well as in low concentration.
    [063] [063] In another aspect, the present invention provides a method for preparing the liquid formulation of the present invention.
    [064] [064] A stable liquid formulation of long-acting insulinotropic peptide conjugate can be prepared by generating the long-acting insulinotropic peptide conjugate, followed by mixing the generated long-acting insulinotropic peptide conjugate with a stabilizer. consisting of a buffer, sugar alcohol, non-ionic surfactant and isotonic agent. In addition, for multiple uses, a stable liquid formulation of long-acting insulinotropic peptide conjugate can be produced by mixing an additional preservative in addition to the stabilizers. Mode for the Invention
    [065] [065] The present invention will now be described in more detail with reference to the Examples. However, these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Example 1: Evaluation of the stability of the long-acting insulinotropic peptide conjugate in the presence or absence of an isotonic agent such as salt
    [066] [066] The stability of the long-acting insulinotropic peptide conjugate (15.41 µg / mL exendin-4 CA, nominal concentration) was evaluated in the presence or absence of sodium chloride as an isotonic agent in the formulation composed of a buffer , a sugar alcohol, and a nonionic surfactant as a stabilizer; and in the formulation composed of a buffer, a sugar alcohol, a nonionic surfactant, and methionine as a stabilizer. For this purpose, the long-acting insulinotropic peptide conjugate was stored at 25ºC and 40ºC between 0 and 4 weeks in the compositions shown in Table 1, and the stability of the conjugate was analyzed by reverse-phase high-performance liquid chromatography ( RP-HPLC) and high performance liquid chromatography-molecular exclusion (SE-HPLC). As a buffer, citrate buffer was used, mannitol was used as the sugar alcohol, and polysorbate 20 was used as the non-ionic surfactant.
    [067] [067] When comparing test groups # 1 and # 2, and groups # 3 and # 4 in Tables 2 and 3, it is evident that when the long-acting liquid insulinotropic peptide conjugate formulation was stored at 25ºC and 40ºC, especially at 40ºC for 4 weeks and in the presence of NaCl as an isotonic agent, particularly 150 mM NaCl, the stability of the long-acting insulinotropic peptide conjugate remained extremely high (Tables 2 and 3). Example 2: Evaluation of the stability of the long-acting insulinotropic peptide conjugate at various pH values of the buffer
    [068] [068] While the pH range of a liquefied protein drug is generally 5 to 7, the pH of the liquid formulation of exendin-4 (Byetta), an insulinotropic peptide drug, is 4.5, which is lower the usual pH range. Therefore, in this example, the effect of buffer pH on conjugate stability for a long-acting insulinotropic peptide conjugate comprising an insulinotropic peptide and immunoglobulin Fc protein, preferably an imidazoacetyl exendin-4 (CA) conjugate, was evaluated. ) of long duration action.
    [069] [069] As a buffer, citrate buffer was used, mannitol was used as a sugar alcohol, sodium chloride was used as an isotonic agent, and polysorbate 80 was used as a non-ionic surfactant. The following compositions shown in Table 4 were used as a stabilizer for the long-acting insulinotropic peptide conjugate. Then, long-acting insulinotropic peptide conjugate compositions were stored at 25 ± 2ºC for 4 weeks and their stability was assessed by molecular exclusion chromatography (SE-HPLC) and reverse phase chromatography (RP-HPLC) . RP-HPLC (%) and SE-HPLC (%) in Table 5 represent «area% / starting area%», which reflects the residual purity of the long-acting insulinotropic peptide conjugate compared to the initial purity. Table 4 Concentration formulation. Surfactant Buffer Agent Alcohol No. (mcg / ml) sugar and isotonic other Citrate-Na 0.005% 5% NaCl 1 197.6 20 mM (pH Polysorbate 80 Mannitol 150 mM 5.2) Citrate-Na 0.005% 5% NaCl 2 197.6 20 mM (pH Polysorbate 80 Mannitol 150 mM 5.5) Citrate-Na 0.005% 5% NaCl 3 197.6 20 mM (pH Polysorbate 80 Mannitol 100 mM 6.0) Table 5 Formul. RP-HPLC (%) SE-HPLC (%) pH No. (Area% / Initial Area%) (Area% / Initial Area%) 0S 1S 2S 4S 0S 1S 2S 4S 1 5.2 100 99.1 98.6 97.5 100 100 100.5 100.5 2 5.5 100 99.8 97.7 95.0 100 99.6 100.8 100.7 3 6.0 100 98.3 98.1 94.8 100 99.6 100.7 100.7
    [070] [070] As can be seen above, the greatest stability of the long-acting insulinotropic peptide conjugate was recorded at a pH of 5.2 of the liquid formulation (Table 5). Example 3: Evaluation of the stability of the long-acting insulinotropic peptide conjugate as a function of the type and concentration of the non-ionic surfactant
    [071] [071] The stability of the long-acting insulinotropic peptide conjugate has been examined for different types and concentrations of polysorbate, which is a nonionic surfactant present in the stabilizer of the present invention.
    [072] [072] Non-ionic surfactants, that is, polysorbate 80 and polysorbate 20, were tested in both concentrations, 0.005% and 0.01%. The stabilizer composition comprises a buffer, a sugar alcohol, an isotonic agent and a surfactant, as in the previous example, to provide stability to the long-acting insulinotropic peptide conjugate. Citrate buffer at a pH of 5.2, which resulted in high stability in Example 2, was used as a buffer, mannitol was used as sugar alcohol, and sodium chloride was used as an isotonic agent.
    [073] [073] The following compositions shown in Table 6 were used as a stabilizer for the long-acting insulinotropic peptide conjugate, preferably for the long-acting exendin-4 CA conjugate. Then, the compositions were stored at 25 ± 2 ° C for 8 weeks and their stability was analyzed by RP-HPLC and SE-HPLC. RP-HPLC (%) and SE-HPLC (%) in Table 7 represent the residual purity of the long-acting insulinotropic peptide conjugate compared to the initial purity. Table 6 Concent. Surfactant Buffer Agent Alcohol No. (mcg / ml) sugar and isotonic others Citrate-Na 0.005% 5% NaCl 1 197.6 20 mM (pH Polysorbate 80 Mannitol 150 Mm 5.2) Citrate-Na 0.001% 5% NaCl 2 197.6 20 mM (pH Polysorbate 80 Mannitol 150 mM 5.2) Citrate-Na 0.005% 5% NaCl 3 197.6 20 mM (pH Polysorbate 20 Mannitol 150 mM 5.2) Citrate-Na 0.01% 5% 197.6 20 mM NaCl (pH Polysorbate 20 Mannitol 150 mM
    [074] [074] As can be seen above based on the results of the SE-HPLC analysis, the stability of the long-acting insulinotropic peptide conjugate remained practically constant, even in the presence of different types and concentrations of polysorbates. However, based on the results of the analysis by RP-HPLC, it was observed that in the presence of polysorbate 20, the stability of the peptide conjugate was similar or higher than in the presence of polysorbate 80 at the same concentration. In addition, higher long-acting insulinotropic peptide conjugate stability was recorded in the liquid formulation of 0.005% polysorbate 20 than that of 0.01% polysorbate 20 (Table 7). Example 4: Comparison of stability between the liquid formulation of choice of long-acting insulinotropic peptide conjugate and the commercially available liquid formulation of peptide drug or protein containing the same
    [075] [075] In the present example, the stability of the selected formulation was assessed using the stability tests of Examples 1 to 3. The long-acting insulinotropic peptide conjugate formulation of choice comprises citrate buffer at a pH of 5 , 2, sodium chloride,
    [076] [076] Using the compositions shown below in Table 8, the following formulations were prepared: a liquid formulation of long-acting insulinotropic peptide conjugate, more preferably long-acting exendin-4 CA conjugate (Liquid Formulation # 1 ); a liquid formulation prepared by adding the long-acting insulinotropic peptide conjugate to the stabilizing composition of the liquid insulinotropic peptide drug formulation, exendin-4 (Byetta) (Liquid Formulation # 2); and a liquid formulation prepared by adding the long-acting insulinotropic peptide conjugate to the stabilizing composition of the liquid immunoglobulin fusion protein drug formulation, Etanercept (TNFR-Fc fusion protein, ENBREL) (Liquid Formulation # 3). As a control group, a liquid formulation was prepared by adding the long-acting insulinotropic peptide conjugate to a stabilizing composition composed only of PBS (Liquid Formulation # 4). Subsequently, the formulations were stored at 25 ± 2 ° C for 8 weeks and their stability was analyzed by RP-HPLC and SE-HPLC. RP-HPLC (%) and SE-HPLC (%) in Table 9 show the residual purity of the long-acting insulinotropic peptide conjugate compared to the initial purity. Table 8 Concent. Surfactant Buffer Agent Alcohol No. (mcg / ml) sugar and isotonic others
    [077] [077] As a result of the stability test, the long-acting insulinotropic peptide conjugate liquid formulation of the present invention was found to have greater stability than the liquid formulations prepared by adding the long-acting insulinotropic peptide conjugate. duration to the commercially available liquid insulinotropic peptide drug formulations, exendin-4 (Byetta), and immunoglobulin fusion protein drug, Etanercept (TNFR-Fc fusion protein, ENBREL), as shown in Figure 1 and in Table 9. Example 5: Evaluation of the stability of the long-acting insulinotropic peptide conjugate as a function of the addition of methionine
    [078] [078] To determine the effect of methionine on the stability of the conjugate, the liquid formulation was prepared by adding methionine, which is intended to prevent oxidation, to the composition composed of citrate buffer at a pH of
    [079] [079] The liquid formulation of long-acting insulinotropic peptide conjugate, more preferably the long-acting exendin-4 CA conjugate, was prepared with the compositions shown below in Table 10 and their stability was analyzed. RP-HPLC (%) and SE-HPLC (%) in Tables 11 to 14 represent the proportions of the long-acting insulinotropic peptide conjugate and the impurities at each point in time. Table 11 shows the results of the RP-HPLC accelerated stability test (25 ± 2ºC) and Table 12 shows the results of the SE-HPLC accelerated stability test (25 ± 2ºC). Table 13 shows the results of the test of the intensity of instability by RP-HPLC (40 ± 2ºC) and Table 14 shows the results of the test of intensity of instability by SE-HPLC (40 ± 2ºC). Impurity # 3 represents the oxidized form of the long-acting insulinotropic peptide conjugate. However, since SE-HPLC separates the sample by molecular weight and the difference in molecular weight between the oxidized form and the non-oxidized form is insignificant, it was difficult to isolate the oxidized form of the long-acting insulinotropic peptide conjugate by SE-HPLC. Table 10 Concent. Surfactant Buffer Agent Alcohol No. (mcg / ml) sugar and isotonic methionine Citrate-Na 0.005% 5% Mannitol NaCl 1 200 20 mM (pH Polysorbate 20 150 mM 5.2) Citrate-Na 0.005% 5% Mannitol NaCl 2 200 20 mM (pH Polysorbate 20 0.01% Methionine 150 mM 5.2)
    [080] [080] As the results of the accelerated stability test and the instability intensity test show, and as also illustrated in Figure 2, it was observed that the proportion of oxidized long-acting insulinotropic peptide conjugate (Impurity # 3 in RP-HPLC analysis) increased in the liquid formulation without methionine, but did not increase in the liquid formulation with 0.01% methionine (Figure 2). Therefore, it has been confirmed that the liquid formulation containing methionine can provide stability to the long-acting insulinotropic peptide conjugate more effectively. Example 6: Evaluation of long-term storage stability of the liquid formulation of choice for the long-acting insulinotropic peptide conjugate
    [081] [081] In the present example, the liquid formulation that was ultimately chosen through the previous examples was evaluated for its stability in long-term storage and its accelerated stability. The liquid formulation of choice comprises citrate buffer at a pH of 5.2, sodium chloride, mannitol, polysorbate 20 and methionine. For this purpose, the formulations were stored at 5 ± 3ºC for 6 months, and at 25 ± 2ºC for 6 months, and their stability was analyzed. The results are shown in Tables 15 and 16, in which RP-HPLC (%), SE-HPLC (%), protein content (%) and specific activity test (%) represent the residual purity of the conjugate compared to initial purity. Table 15 shows the results of the long term storage stability tests of the formulation after its storage at 5 ± 3ºC and Table 16 shows the results of the accelerated stability test after its storage at 25 ± 2ºC. Table 15 Evaluation of long-term storage stability (stored at 5 ± 3ºC) Duration Color p Test Purity test Content Activity Storage H confirmation Specific protein n RP- Wester SDS- RP- RP- Endotoxin aa HPLC n blot PAGE HPL HPL a ( %) (%)
    [082] [082] The results of the long-term storage stability test show that the long-acting insulinotropic peptide conjugate remained stable for more than 6 months in the liquid formulation of the present invention. In addition, even when stored under accelerated conditions for 6 months, the results of the RP-HPLC analysis show that 95.4% or more of the peptide conjugate remained intact in the formulation, thus confirming that the present liquid formulation provides the peptide conjugate long-acting insulinotropic with excellent storage stability. Example 7: Evaluation of the stability of the long-acting insulinotropic peptide conjugate as a function of protein concentration
    [083] [083] The effect of a high concentration of conjugate was analyzed for the liquid formulation of choice, composed of citrate buffer at a pH of 5.2, sodium chloride, mannitol, polysorbate 20, and methionine to prevent oxidation. For this purpose, precipitation in the formulation was monitored with the naked eye and at 40ºC for various concentrations of conjugate, as shown in Table 17. After 72 hours of monitoring, precipitation was observed in all high concentration formulations (4 mg / ml or more). In addition, with increasing concentration, the occurrence of precipitation has also increased. Table 17 Concent. No. Buffer Salt Sugar alcohol and others Surfactant Citrate-Na NaCl 5% Mannitol / 0.005% 1 0.52 mg / ml 20 mM (pH 5.2) 150 mM 0.1 mg / ml Methionine Polysorbate 20 Citrate-Na NaCl 5% Mannitol / 0.005% 2 4.0 mg / ml 20 mM (pH 5.2) 150 mM 0.1 mg / ml Methionine Polysorbate 20 Citrate-Na NaCl 5% Mannitol / 0.005% 3 5.0 mg / ml 20 mM ( pH 5.2) 150 mM 0.1 mg / ml Methionine Polysorbate 20 Citrate-Na NaCl 5% Mannitol / 0.005% 4 8.0 mg / ml 20 mM (pH 5.2) 150 mM 0.1 mg / ml Methionine Polysorbate 20
    [084] [084] The effect of the concentration of NaCl and mannitol as sugar alcohol in preventing precipitation was examined for the liquid formulation of choice for long-acting insulinotropic peptide conjugate at high concentration. The formulations were prepared with the compositions shown below in Table 18 and monitored with the naked eye and at 40ºC for the occurrence of precipitation, for 48 hours. The duration of the absence of precipitation shown in Figure 3 shows the time after storage in which protein precipitation did not occur. Table 18 Concent. No. Buffer Salt Sugar alcohol and others Surfactant Citrate-Na NaCl 5% Mannitol / 0.005% 1 5.0 mg / ml 20 mM (pH 5.2) 150 mM 0.1 mg / ml Methionine Polysorbate 20 Citrate-Na NaCl 10% Mannitol / 0.005% 2 5.0 mg / ml 20 mM (pH 5.2) 150 mM 0.1 mg / ml Methionine Polysorbate 20 Citrate-Na NaCl 5% Mannitol / 0.005% 3 5.0 mg / ml 20 mM ( pH 5.2) 150 mM 0.1 mg / ml Methionine Polysorbate 20 Citrate-Na NaCl 5% Mannitol / 0.005% 4 5.0 mg / ml 20 mM (pH 5.2) 150 mM Polysorbate 20
    [085] [085] As can be seen from these results, it is confirmed that the concentration of NaCl does not significantly affect neither the occurrence of precipitation nor the stability of the insulinotropic peptide conjugate at high concentration, based on observation with the naked eye. However, when the concentration of mannitol as sugar alcohol is increased from 5% to
    [086] [086] The duration of the absence of precipitation shown in Figure 4 indicates the time elapsed after storage in which there was no protein precipitation. RP-HPLC (%) from Table 20 and SE-HPLC (%) from Table 21 indicate the residual purity of the long-acting insulinotropic peptide conjugate compared to the initial purity. Table 19 No. Concent. Buffer Salt Sugar alcohol and others Surfactant Na-Citrate 5% Mannitol / 0.005% 1 5.0 mg / ml 20 mM (pH 5.2) 0.1 mg / ml Methionine Polysorbate 20
    [087] [087] As can be seen from these results, precipitation was better suppressed at a high pH of 5.4 and 5.6 than at a pH of 5.2. After 7 days of storage, precipitation was observed in all formulations. However, in the composition composed of 10% mannitol and 150 mM NaCl at a pH of 5.6 (Composition No. 6), the amount of impurities generated was less. At pH 5.4 and 5.6, the presence of NaCl did not have a significant effect on the stability of the long-acting insulinotropic peptide conjugate at high concentration, except for precipitation (Tables 20 and 21, and Figure 4) . Example 10: Evaluation of the stability of the long-acting insulinotropic peptide conjugate at high concentration as a function of sugar alcohol concentration and at various pH
    [088] [088] Based on the previous Examples, the effect of the concentration of sugar alcohol and pH on the stability of the long-acting insulinotropic peptide conjugate at high concentration was examined. As a buffer, citrate and polysorbate 20 buffer was used as a non-ionic surfactant. Methionine was added to the formulation to prevent oxidation. In addition, based on the results observed in Example 9, NaCl was excluded from the long-acting insulinotropic peptide conjugate formulation at high concentration. The high-acting, long-acting insulinotropic peptide conjugate was formulated with the compositions shown below in Table 22, which were stored at 40 ° C for 5 days, changed to a temperature of 25 ° C, and stored for an additional 4 weeks. Every week, protein stability was analyzed by SE-HPLC, IE-HPLC and RP-HPLC. SE- HPLC (%) from Table 23, IE-HPLC (%) from Table 24, and RP-HPLC (%) from Table 25 represent the residual purity of the long-acting insulinotropic peptide conjugate. Table 22 No. Concent. Buffer Salt Sugar alcohol and others Surfactant Citrate-Na 10% Mannitol / 0.005% 1 10.0 mg / ml 20 mM (pH 0.1 mg / ml Methionine Polysorbate 20 5.6)
    [089] [089] As the previous results indicate, at low pH values, stability is less than at high pH values. The highest stability of the conjugate was observed for 10% mannitol, while 2% and 5% mannitol did not affect the stability of the long-acting insulinotropic peptide conjugate at high concentration. Example 11: Evaluation of the stability of the long-acting insulinotropic peptide conjugate at high concentration depending on the type and concentration of the sugar alcohol
    [090] [090] In order to develop an isotonic liquid formulation, the effect that the type and concentration of a sugar alcohol, which significantly affects the osmotic pressure of the formulation, has been evaluated on the stability of the insulinotropic peptide conjugate of action long-term under the same conditions as the previous Examples. The type of sugar alcohol was changed to sucrose. Based on Formulation No. 1 of Example 10, 10% mannitol was replaced with 5% and 7% sucrose (Table 26). The formulations were stored at 25 ° C for 4 weeks and their stability was analyzed every week by SE-HPLC, IE-HPLC and RP-HPLC.
    SE-HPLC (%) from Table 27, IE-HPLC (%) from Table 28, and RP-HPLC (%) from Table 29 represent the residual purity of the long-acting insulinotropic peptide conjugate.
    Table 26
    At the.
    Concent.
    Buffer Salt Sugar alcohol and others Surfactant Citrate-Na 10% Mannitol / 0.005% 1 10.0 mg / ml 20 mM (pH 5.6) 0.1 mg / ml Methionine Polysorbate 20 Citrate-Na 5% Sucrose / 0.005% 2 10.0 mg / ml 20 mM (pH 5.6) 0.1 mg / ml Methionine Polysorbate 20 Citrate-Na 7% Sucrose / 0.005% 3 10.0 mg / ml 20 mM (pH 5.6) 0, 1 mg / ml Methionine Polysorbate 20 Table 27 No.
    SE-HPLC (Area%) 0S 1S 2S 3S 4S 1 99.4 98.3 98.3 98.2 98.0 2 99.5 98.4 98.3 98.2 98.0 3 99.5 98, 5 98.4 98.3 98.1 Table 28 No.
    IE-HPLC (Area%) 0S 1S 2S 3S 4S 1 95.6 81.7 78.2 75.7 65.3 2 95.6 83.1 79.9 76.5 68.8 3 95.7 83, 8 81.3 78.1 69.6 Table 29 No.
    SE-HPLC (Area%) 0S 1S 2S 3S 4S 1 97.5 87.2 84.1 81.0 75.7 2 97.5 88.5 85.0 80.9 75.3
    权利要求:
    Claims (30)
    [1]
    1. Liquid formulation of long-acting insulinotropic peptide conjugate, characterized by comprising a pharmaceutically effective amount of long-acting insulinotropic peptide conjugate, in which an insulinotropic peptide, which is a physiologically active peptide, is linked to an immunoglobulin Fe region; and an albumin-free stabilizer, wherein the stabilizer comprises a buffer, a sugar alcohol, a non-ionic surfactant, and an isotonic agent.
    [2]
    2. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 1, characterized in that the insulinotropic peptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), exendin-3, exendin-4, the precursors, agonists, derivatives, fragments, and variants thereof, and a combination thereof.
    [3]
    Liquid long-acting insulinotropic peptide conjugate formulation according to claim 2, characterized in that the insulinotropic peptide variant is imidazo-acetyl exendin-4.
    [4]
    4. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 1, characterized in that the Fe region of the immunoglobulin is a Fe region derived from lgG, lgA, lgD, lgE, or lgM.
    [5]
    5. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 4, characterized in that the Fe region of the immunoglobulin is a hybrid of domains of different origins derived from immunoglobulins selected from the group consisting of lgG, lgA, lgD, lgE, and lgM.
    [6]
    6. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 4, characterized in that the Fe region of the immunoglobulin is a dimer or multimer consisting of single-chain immunoglobulins, composed of domains of the same origin.
    , .., - · 2/5
    [7]
    7. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 4, characterized in that the immunoglobulin Fe region is an IgG4 Fe region.
    [8]
    8. Long-acting liquid insulinotropic peptide conjugate formulation according to claim 7, characterized in that the immunoglobulin Fe region is a non-glycosylated human IgG4 Fe region.
    [9]
    Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 1, characterized in that the conjugate is generated using a non-peptide polymer or recombinant technique.
    [10]
    1 O. Long-acting liquid insulinotropic peptide conjugate formulation according to claim 9, characterized in that the non-peptide polymer is a polyethylene glycol.
    [11]
    11. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 10, characterized in that the non-peptide polymer is selected from the group consisting of a biodegradable polymer such as a polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, a polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinylethyl ether, polylactic acid (PLA) and polylactic-glycolic acid (PLGA); a lipid polymer; chitins; a hyaluronic acid; and a combination of these.
    [12]
    12. Long-acting insulinotropic peptide conjugate liquid formulation according to claim 1, characterized in that the pharmaceutically effective amount of the long-acting insulinotropic peptide conjugate corresponds to a concentration of 0.5 mg / ml at 150 mg / ml.
    [13]
    13. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 1, characterized in that the sugar alcohol is one or more selected from the group consisting of mannitol,: ... •
    sorbitol, and sucrose.
    [14]
    14. Long-acting liquid insulinotropic peptide conjugate formulation according to claim 13, characterized in that the sugar alcohol concentration is from 3% (w / v) to 15% (w / v) based on the volume total solution. ~
    [15]
    Liquid long-acting insulinotropic peptide conjugate formulation according to claim 1, characterized in that the buffer is a citrate buffer, an acetate buffer, or a histidine buffer.
    [16]
    16. Long-acting liquid insulinotropic peptide conjugate formulation according to claim 1, characterized in that the pH range of the buffer is 4 to 7.
    [17]
    17. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 16, characterized in that the pH range of the buffer is 5 to 7.
    [18]
    18. Liquid formulation of long-acting insulinotropic peptide conjugate of .. I agree with claim 1, characterized in that the isotonic agent is sodium chloride with a concentration of O mM to 200 mM.
    [19]
    19. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 1, characterized in that the nonionic surfactant is polysorbate 80 or polysorbate 20.
    [20]
    20. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 19, characterized in that the nonionic surfactant is in a concentration of 0.001% (w / v) to 0.05% (w / v).
    [21]
    21. Long-acting liquid insulinotropic peptide conjugate formulation according to claim 1, characterized in that the stabilizer additionally comprises methionine.
    [22]
    22. Liquid long-acting insulinotropic peptide conjugate formulation according to claim 21, characterized in that the methionine concentration is from 0.005% (w / v) to 0.1% (w / v) based in the total volume of solution.
    [23]
    23. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 1, characterized in that the stabilizer additionally comprises one or more substances selected from the group consisting of sugars, polyalcohols and amino acids.
    [24]
    24. Liquid formulation of long-acting insulinotropic peptide conjugate comprising a long-acting insulinotropic peptide conjugate, characterized in that the insulinotropic peptide and an immunoglobulin Fe region are linked by polyethylene glycol; and an albumin-free stabilizer, wherein the stabilizer comprises citrate buffer, mannitol, polysorbate 20 and sodium chloride.
    [25]
    25. Liquid formulation of long-acting insulinotropic peptide conjugate according to claim 1, characterized in that it additionally comprises one or more preservatives selected from the group consisting of m-cresol, phenol and benzyl alcohol.
    [26]
    26. Long-acting liquid insulinotropic peptide conjugate formulation according to claim 25, characterized in that the concentration of the preservative is from 0.001% to 1% (w / v) based on the total volume of solution.
    [27]
    27. Long-acting liquid insulinotropic peptide conjugate formulation according to claim 25, characterized in that the preservative is m-cresol.
    [28]
    28. Long-acting liquid insulinotropic peptide conjugate formulation according to claim 25, characterized in that it is for multiple uses.
    [29]
    29. Method of preparing the long-acting liquid insulinotropic peptide conjugate formulation as described in any one of claims 1 to 24, characterized in that it comprises
    .
    5/5 '"(a) preparing a long-acting insulinotropic peptide conjugate; and (b) mixing the long-acting insulinotropic peptide conjugate prepared in step (a) with a stabilizer consisting of buffer, alcohol sugar, non-ionic surfactant, sodium chloride as isotonic agent, and methionine.
    [30]
    30. Method of preparing the liquid long-acting insulinotropic peptide conjugate formulation as described in any of claims 27 to 30, characterized in that it comprises (a) preparing a long-acting insulinotropic peptide conjugate; and (b) mixing the long-acting insulinotropic peptide conjugate prepared in step (a) with a stabilizer consisting of buffer, sugar alcohol, non-ionic surfactant, sodium chloride as isotonic agent, methionine and a preservative.
    H·:
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    同族专利:
    公开号 | 公开日
    WO2014017845A3|2014-03-20|
    PT2877158T|2019-12-17|
    CN104487053A|2015-04-01|
    CA2880026C|2020-09-01|
    EP2877158B1|2019-09-11|
    TW201408337A|2014-03-01|
    KR20140018798A|2014-02-13|
    HK1204565A1|2015-11-27|
    ES2759987T3|2020-05-12|
    US20150238629A1|2015-08-27|
    KR102161177B1|2020-09-29|
    US9801950B2|2017-10-31|
    JP2018119006A|2018-08-02|
    WO2014017845A2|2014-01-30|
    JP6385345B2|2018-09-05|
    CN108125909A|2018-06-08|
    RU2015104494A|2016-09-20|
    AU2013293716A1|2015-02-19|
    JP2015522654A|2015-08-06|
    PL2877158T3|2020-04-30|
    EP2877158A2|2015-06-03|
    CA2880026A1|2014-01-30|
    RU2671576C2|2018-11-02|
    TWI643637B|2018-12-11|
    CN104487053B|2018-01-30|
    MX2015001088A|2015-07-06|
    JP6685343B2|2020-04-22|
    AR094821A1|2015-09-02|
    EP2877158A4|2016-03-23|
    MX369479B|2019-11-08|
    AU2013293716B2|2018-04-26|
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    法律状态:
    2021-05-04| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2021-05-04| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |
    2021-05-25| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|
    2021-08-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    优先权:
    申请号 | 申请日 | 专利标题
    KR20120081476|2012-07-25|
    KR10-2012-0081476|2012-07-25|
    PCT/KR2013/006670|WO2014017845A2|2012-07-25|2013-07-25|A liquid formulation of long acting insulinotropic peptide conjugate|
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