Liquid formulation of long acting insulinotropic peptide conjugate
09801950 · 2017-10-31
Assignee
Inventors
- Hyun Uk Kim (Busan, KR)
- Hyung Kyu LIM (Hwaseong-si, KR)
- Sung Hee Hong (Suwon-si, KR)
- Dae Jin KIM (Hwaseong-si, KR)
- Sung Min Bae (Seongnam-si, KR)
- Se Chang Kwon (Seoul, KR)
Cpc classification
A61P5/50
HUMAN NECESSITIES
A61K47/6811
HUMAN NECESSITIES
A61K47/68
HUMAN NECESSITIES
International classification
C07K16/00
CHEMISTRY; METALLURGY
A61K47/26
HUMAN NECESSITIES
Abstract
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, insulinotropic peptide, and 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, and a method for preparing the formulation. For the purpose of preventing microbial contamination, a preservative may be added. The liquid formulation of the present invention is free of human serum albumin and other potentially hazardous factors to body, having no risk of viral contamination, and thus can provide excellent storage stability for insulinotropic peptide conjugates at high concentration.
Claims
1. A liquid formulation of a long-acting insulinotropic peptide conjugate, the liquid formulation consisting essentially of: a pharmaceutically effective amount of the long-acting insulinotropic peptide conjugate wherein an insulinotropic peptide, which is a physiologically active peptide, is linked 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, wherein the insulinotropic peptide is glucagon-like peptide-1, glucagon-like peptide-2, exendin-3, exendin-4 or imidazo-acetyl exendin-4; wherein the buffer has a pH of 5.2 to 7.0; and wherein the non-ionic surfactant has a concentration of 0.001% (w/v) to 0.05% (w/v).
2. The liquid formulation according to claim 1, wherein the immunoglobulin Fc region is a Fc region derived from IgG, IgA, IgD, IgE, or IgM.
3. The liquid formulation according to claim 2, wherein the immunoglobulin Fc region is a hybrid of domains of different origins derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.
4. The liquid formulation according to claim 2, wherein the immunoglobulin Fc region is a dimer or multimer consisting of single-chain immunoglobulins composed of domains of same origin.
5. The liquid formulation of according to claim 2, wherein the immunoglobulin Fc region is an IgG4 Fc region.
6. The liquid formulation according to claim 5, wherein the immunoglobulin Fc region is a human aglycosylated IgG4 Fc region.
7. The liquid formulation according to claim 1, wherein the insulinotropic peptide is linked to the immunoglobulin Fc region via a non-peptidyl polymer or a fusion protein.
8. The liquid formulation according to claim 7, wherein the non-peptidyl polymer is a polyethylene glycol.
9. The liquid formulation according to claim 7, wherein the non-peptidyl polymer is selected from the group consisting of a biodegradable polymer; a lipid polymer; chitins; hyaluronic acid; and a combination thereof, wherein said biodegrdable polymer is selected from the group consisting of sepolypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid, and polylactic-glycolic acid.
10. The liquid formulation according to claim 1, wherein the pharmaceutically effective amount of the long-acting insulinotropic peptide conjugate has a concentration of 0.5 mg/ml to 150 mg/ml.
11. The liquid formulation according to claim 1, wherein the sugar alcohol is one or more selected from the group consisting of mannitol, sorbitol, and sucrose.
12. The liquid formulation according to claim 11, wherein a concentration of the sugar alcohol is 3% (w/v) to 15% (w/v) based on a total volume of the liquid formulation.
13. The liquid formulation according to claim 1, wherein the buffer is a citrate buffer, an acetate buffer, or a histidine buffer.
14. The liquid formulation according to claim 1, wherein the pH range of the buffer is 5.2 to 6.0.
15. The liquid formulation according to claim 1, wherein the isotonic agent is sodium chloride having a concentration of 0 mM to 200 mM.
16. The liquid formulation according to claim 1, wherein the non-ionic surfactant is polysorbate 80 or polysorbate 20.
17. The liquid formulation according to claim 1, wherein the stabilizer further comprises methionine.
18. The liquid formulation according to claim 17, wherein a concentration of the methionine is 0.005% (w/v) to 0.1% (w/v) based on a total volume of the liquid formulation.
19. The liquid formulation according to claim 1, wherein the stabilizer further comprises one or more substances selected from the group consisting of a sugar, a polyalcohol, and an amino acid.
20. A liquid formulation of a long-acting insulinotropic peptide conjugate, the liquid formulation consisting essentially of: the long-acting insulinotropic peptide conjugate wherein an insulinotropic peptide and an immunoglobulin Fc region are linked by polyethylene glycol; and an albumin-free stabilizer, wherein the stabilizer comprises citrate buffer, mannitol, polysorbate 20, and sodium chloride, wherein the insulinotropic peptide is giucagon-like peptide-1, glucagon-like pepetide-2, exendin-3, exendin-4 or imidazo-acetyl exendin-4; wherein the buffer has a pH of 5.2 to 7.0; and wherein the polysorbate 20 has a concentration of 0.001% to 0.05% (w/v).
21. The liquid formulation according to claim 1, further comprising one or more preservatives selected from the group consisting of m-cresol, phenol, and benzyl alcohol.
22. The liquid formulation according to claim 21, wherein a concentration of the one or more preservatives is 0.001% to 1% (w/v) based on a total volume of the liquid formulation.
23. The liquid formulation according to claim 21, wherein the one or more preservatives is m-cresol.
24. A multiple-use liquid formulation of a long-acting insulinotropic peptide conjugate, the multiple-use liquid formulation consisting essentially of: a pharmaceutically effective amount of the long-acting insulinotropic peptide conjugate wherein an insulinotropic peptide, which is a physiologically active peptide, is linked to an immunoglobulin Fc region; an albumin-free stabilizer, wherein the stabilizer comprises a buffer, a sugar alcohol, a non-ionic surfactant, and an isotonic agent; and one or more preservatives selected from the group consisting of m-cresol, phenol, and benzyl alcohol, wherein the insulinotropic peptide is glucagon-like peptide-1, glucagon-like peptide-2, exendin-3 ,exendin-4 or imidazo-acetyl exendin-4; wherein the buffer has a pH of 5.2 to 7.0; and wherein the polysorbate 20 has a concentration of 0.001% (w/v) to 0.05% (w/v).
25. A method for preparing the liquid formulation of claim 1, the method comprising: mixing a long-acting insulinotropic peptide conjugate wherein an insulinotropic peptide, which is a physiologically active peptide, is linked to an immunoglobulin Fc region with a stabilizer comprising a buffer, a sugar alcohol, a non-ionic surfactant and sodium chloride as an isotonic agent; wherein the insuhnotropic peptide is glucagon-like peptide-1, glucagon-like peptide-2, exendin-3 exendin-4 or imidazo-acetyl exendin-4; wherein the buffer has of 5.2 to 7.0; and wherein the non-ionic surfactant has a concentration of 0.001% (w/v) to 0.05% (w/v).
26. A method for preparing the liquid formulation of claim 21, comprising: mixing a long-acting insulinotropic peptide conjugate wherein an insulinotropic peptide, which is a physiologically active peptide, is linked to an immunoglobulin Fc region with a stabilizer comprising a buffer, a sugar alcohol, a non-ionic surfactant and sodium chloride as an isotonic agent; methionine; and a preservative; wherein the insulinotropic peptide is glucagon-like peptide-1, glucagon-like peptide-2, exendin-3, exendin-4or imidazo-acetyl exendin-4: wherein the buffer has a pH of 5.2 to 7.0; and wherein the non-ionic surfactant has a concentration of 0.001% (w/v) to 0.05% (w/v).
Description
DESCRIPTION OF DRAWINGS
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BEST MODE
(5) As 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 wherein an insulinotropic peptide is linked 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.
(6) In addition, the present invention provides a liquid formulation of long-acting insulinotropic peptide conjugate for multiple administrations, further comprising a preservative in addition to the insulinotropic peptide conjugate and albumin-free stabilizer.
(7) As used herein, “long-acting insulinotropic peptide conjugate” refers to a conjugate wherein a physiologically active insulinotropic peptide comprising a derivative, variant, precursor, and fragment and an immunoglobulin Fc region are linked, and it may further refer to a conjugate having increased in vivo duration of physiological activity compared to a wild-type insulinotropic peptide.
(8) As used herein, the term “long-acting” refers to an enhancement of duration of physiological activity compared to that of a wild-type. The term “conjugate” refers to the form wherein an insulinotropic peptide and immunoglobulin Fc region are combined.
(9) The insulinotropic peptide used in the present invention has a function of secreting insulin and it stimulates the synthesis and expression of insulin in pancreatic β-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 (CA) exendin-4, and more preferably, imidazoacetyl (CA) exendin-4. Any insulinotropic peptide, either native or recombinant, may be used and preferably it is a recombinant insulinotropic peptide generated by using E. coli as a host cell. As long as its biological activity is not significantly affected, any derivatives thereof, which are generated by substitution, deletion, or insertion of amino acids, may be used in the present invention.
(10) The sequence of the insulinotropic peptide may be obtained from known database such as GenBank of NCBI, and it can have 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more, and most preferably 98% or more sequence homology with a wild-type protein, as long as it demonstrates the activity of an insulinotropic peptide.
(11) Furthermore, the immunoglobulin Fc useful of the present invention may be a human immunoglobulin Fc or its closely related analog or immunoglobulin Fc derived from animals such as cow, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs. In addition, the immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. Preferably, the immunoglobulin Fc 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 improve half-life of ligand-binding protein. Also, the immunoglobulin Fc region may be a dimer or multimer of single-chain immunoglobulins having domains of same origin Immunoglobulin Fc may be generated by treating a native IgG with a certain protease, or by transformed cells using a genetic recombination technique. Preferably, the immunoglobulin Fc is a recombinant human immunoglobulin Fc produced in E. coli.
(12) Meanwhile, IgG may be divided into the IgG1, IgG2, IgG3 and IgG4 subclasses, and in the present invention a combination or hybrid thereof may be used. Preferred are the IgG2 and IgG4 subclasses, and most preferred is the Fc region of IgG4 which rarely has the effector function such as complement dependent cytotoxicity (CDC). That is, the most preferred immunoglobulin Fc region as a drug carrier of the present invention is a human IgG4-derived aglycosylated Fc region. The human-derived Fc region is more preferable than a non-human derived Fc region, which may act as an antigen in the human body and cause undesirable immune responses such as producing a new antibody.
(13) The long-acting insulinotropic peptide conjugate used in the present invention is prepared by combining the synthesized insulinotropic peptide and an immunoglobulin Fc region. The method for combining the two may be cross-linking an insulinotropic peptide and an immunoglobulin Fc region via a non-peptidyl polymer or the production of a fusion protein in which insulinotropic peptide and an immunoglobulin Fc region are linked by genetic recombination.
(14) The non-peptidyl polymer used for the cross-linking may be selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers such as PLA (poly (lactic acid) and PLGA (poly (lactic-glycolic acid), lipid polymers, chitins, hyaluronic acid or a combination thereof. Preferably, polyethylene glycol may be used but is not limited thereto. Their derivatives well known in the art and derivatives which can be readily prepared using a method known in the art are also within the scope of the present invention.
(15) For preparing a long-acting insulinotropic peptide conjugate used in the present invention, one may refer to Korean Patent Registration No. 10-0725315, Korean Patent Publication No. 10-2009-0008151, and Korean Patent Registration No. 10-1058290. Those skilled in the art can produce the long-acting insulinotropic peptide conjugate of the present invention by referring to these references.
(16) The liquid formulation of long-acting insulinotropic peptide conjugate of the present invention comprises a long-acting insulinotropic peptide conjugate in a therapeutically effective amount. In general, the therapeutically effective amount of insulinotropic peptide, especially exendin-4 (Byetta), refers to 250 mcg in a pen-injector. 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 may preferably be a long-acting CA exendin-4 conjugate. The liquid formulation of long-acting insulinotropic peptide conjugate of the present invention can stably store the conjugate without precipitation, not only when the insulinotropic peptide conjugate is present at low concentration, but also when it is present at high concentration. Therefore, the present formulation can stably provide the insulinotropic peptide at high concentration into the body.
(17) As used herein, the term “stabilizer” refers to a substance that allows stable storing of the long-acting insulinotropic peptide conjugate. The term “stabilization” refers to the state wherein loss of an active ingredient is less than a certain amount, typically less than 10% during a certain period and under specific storage conditions. A formulation is regarded as a stable formulation when the residual purity of long-acting insulinotropic peptide conjugate therein is 90% or more, and more preferably 92 to 95% after being stored at 5±3° C. for 2 years, at 25±2° C. for 6 months, or at 40±2° C. for 1 to 2 weeks. As for the proteins like long-acting insulinotropic peptide conjugates, the storage stability thereof is important for providing an accurate dosage as well as for suppressing the potential formation of antigenic substances against the long-acting insulinotropic peptide conjugate. During storage, 10% loss of long-acting insulinotropic peptide conjugate is acceptable for a substantial administration unless it causes the formation of aggregates or fragments in the composition leading to the formation of antigenic compounds.
(18) The stabilizer of the present invention preferably comprises a buffer, a sugar alcohol, an isotonic agent such as sodium chloride, and a non-ionic surfactant, and more preferably comprises methionine in addition, for stabilizing the long-acting insulinotropic peptide conjugate.
(19) The buffer works to maintain the pH of solution to prevent a sharp pH change in the liquid formulation for stabilizing long-acting insulinotropic peptide conjugate. The buffer may include an alkaline salt (sodium or potassium phosphate or hydrogen or dihydrogen salts thereof), sodium citrate/citric acid, sodium acetate/acetic acid, histidine/histidine hydrochloride, any other pharmaceutically acceptable pH buffer known in the art, and a combination thereof. The preferred example of such buffer includes a citrate buffer, an acetate buffer, and a histidine buffer. The concentration of buffer is preferably 5 mM to 100 mM, more preferably 10 mM to 50 mM. The pH of buffer is preferably 4.0 to 7.0, more preferably 5.0 to 7.0, even more preferably 5.2 to 7.0, and even far more preferably 5.2 to 6.0.
(20) 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 a total volume of solution, more preferably 3 to 10% (w/v) based on a total volume of solution. A sugar alcohol may be one or more selected from the group consisting of mannitol, sorbitol, and sucrose, but is not limited thereto.
(21) An isotonic agent acts to maintain an appropriate osmotic pressure when the long-acting insulinotropic peptide conjugate in solution is administered into the body, and also acts to stabilize the long-acting insulinotropic peptide conjugate in solution. The osmotic pressure of formulation is adjusted to be isotonic with blood. These isotonic liquid formulations have osmotic pressure of about 300 mOsm/kg in general. A representative example of isotonic agent includes a sugar alcohol, water-soluble inorganic salt, and amino acid, and preferred example is a water-soluble inorganic salt, i.e. sodium chloride. The concentration of sodium chloride as isotonic agent is preferably 0 to 150 mM, and it can be adjusted depending on the type and amount of components included in formulation such that the liquid formulation including all the mixture becomes isotonic.
(22) The non-ionic surfactant reduces the surface tension of the protein solution to prevent the absorption or aggregation of proteins onto a hydrophobic surface. Examples of the non-ionic surfactant useful in the present invention include polysorbates, poloxamers and combinations thereof, with preference for polysorbates. Among the non-ionic surfactants of polysorbates are polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. The most preferred non-ionic surfactant is polysorbate 20.
(23) It is inappropriate to use a non-ionic surfactant at high concentration in liquid formulation, and this is due to the fact that non-ionic surfactant at high concentration induces interference effects when measuring protein concentration and determining protein stability through analytic methods such as UV-spectroscopy or isoelectric focusing, thereby causing difficulty in examining the protein stability accurately. Therefore, the liquid formulation of the present invention comprises the non-ionic surfactant preferably at a low concentration no more than 0.2%(w/v), more preferably at 0.001% to 0.05%(w/v).
(24) According to one example of the present invention, it was demonstrated that when sodium chloride was added as isotonic agent in the presence of buffer, sugar alcohol, and non-ionic surfactant, the storage stability of long-acting insulinotropic peptide conjugate at low concentration was significantly increased. This indicates that use of sodium chloride as isotonic agent simultaneously with buffer, sugar alcohol, and non-ionic surfactant induces synergic effects, thereby allowing the long-acting insulinotropic peptide conjugate to have a high stability. However, as for a long-acting insulinotropic peptide conjugate at high concentration, when sodium chloride was excluded, the occurrence of precipitation was prevented and the solubility of protein was improved. These results suggest that when sodium chloride is used as an isotonic agent, the content thereof may be adjusted according to the concentration of long-acting insulinotropic peptide conjugate.
(25) In addition, it was confirmed that a long-acting insulinotropic peptide conjugate at low concentration is most stable in a buffer at a pH of 5.2, whereas a long-acting insulinotropic peptide conjugate at high concentration is most stable in a buffer at a pH of 5.4 or 5.6. Thus, it was determined that the pH of buffer can be appropriately adjusted depending on the concentration of conjugate.
(26) Methionine comprised in the stabilizer of the present invention suppresses the formation of impurities which may occur by oxidation of protein in solution, thereby stabilizing a target protein even further. The concentration of methionine is 0.005 to 0.1% (w/v) based on a total volume of solution, preferably 0.01 to 0.1% (w/v).
(27) It is preferred that the stabilizer of the present invention does not contain albumin. Since the human serum albumin available as a stabilizer of protein is produced from human serum, there is always the possibility that it may be contaminated with pathogenic viruses of human origin. Gelatin or bovine serum albumin may cause diseases or may be apt to induce an allergic response 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 has no possibility of causing viral contamination.
(28) In addition, the stabilizer of the present invention may further comprise sugars, polyalcohol, or amino acids. Preferable examples of sugars, which may be further added to increase the storage stability of the 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.
(29) The liquid formulation of the present invention may further comprise a preservative in addition to the above-described conjugate, buffer, isotonic agent, sugar alcohol, and non-ionic surfactant, or additionally methionine, for the purpose of preventing microbial contamination in multiple-use formulation.
(30) As used herein, “preservative” refers to a compound that is added to a pharmaceutical formulation to act as an antimicrobial. Example of preservative includes benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, benzalconium chloride, phenylmercuric nitrate, thimerosal, and benzoic acid, but is not limited thereto. A single type of preservative may be used individually, or a random combination of two or more types of preservative may be used. Preferably, the liquid formulation of the present invention may comprise one or more of m-cresol, phenol, and benzyl alcohol as a preservative.
(31) The liquid formulation of the present invention may comprise 0.001% to 1% (w/v) preservative, and preferably 0.001% to 0.5% (w/v) preservative, and more preferably 0.001 to 0.25% (w/v) preservative.
(32) In one example of the present invention, 0.22% (w/v) m-cresol was added as a preservative in the liquid formulation of the present invention, and the effect of cresol on the stability of insulinotropic peptide conjugate was evaluated. As a result, it was confirmed that the conjugate remained stable in the formulation added with preservative, without precipitation. Therefore, the liquid formulation of insulinotropic peptide conjugate of the present invention, which comprises preservative in addition to the stabilizer, may be used for multiple administrations.
(33) The liquid formulation of the present invention may further comprise other substances and materials known in the art selectively in addition to the above-described buffer, isotonic agent, sugar alcohol, and non-ionic surfactant, or additionally methionine and preservative, as long as the effect of the present invention is not affected.
(34) The albumin-free liquid formulation of long-acting insulinotropic peptide conjugate according to the present invention providing stability to the long-acting insulinotropic peptide conjugate does not have a risk of viral contamination, while providing an excellent storage stability with a simple formulation, and thus the present formulation can be provided more cost-effectively compared to other stabilizer or free-dried formulation.
(35) Also, since the liquid formulation of the present invention comprises the long-acting insulinotropic peptide conjugate which has an enhanced duration of physiological activity compared to a wild-type, it can be used as an effective drug formulation by retaining the protein activity in the body for a longer period compared to the conventional insulinotropic peptide formulation. Also, the present liquid formulation provides an excellent stability for storing a long-acting insulinotropic peptide conjugate at high concentration as well as at low concentration.
(36) As another aspect, the present invention provides a method for preparing the liquid formulation of the present invention.
(37) A stable liquid formulation of long-acting insulinotropic peptide conjugate can be prepared through generating long-acting insulinotropic peptide conjugate, and mixing the generated long-acting insulinotropic peptide conjugate with a stabilizer comprising a buffer, sugar alcohol, non-ionic surfactant, and isotonic agent. Also, for multiple uses, a stable liquid formulation of long-acting insulinotropic peptide conjugate may be generated by further mixing a preservative in addition to the stabilizers.
(38) [Mode for Invention]
(39) Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.
EXAMPLE 1
Evaluation of the Stability of Long-Acting Insulinotropic Peptide Conjugate in the Presence or Absence of Isotonic Agent Such as Salt
(40) The stability of long-acting insulinotropic peptide conjugate (15.41 μg/mL CA exendin-4, Nominal Conc.) was evaluated in the presence or absence of sodium chloride as an isotonic agent in the formulation comprising a buffer, a sugar alcohol, and a non-ionic surfactant as a stabilizer; and in the formulation comprising a buffer, a sugar alcohol, a non-ionic surfactant, and methionine as a stabilizer. For this purpose, the long-acting insulinotropic peptide conjugate was stored at 25° C. and 40° C. for 0 to 4 weeks in the following compositions of Table 1, and then the stability of the conjugate was analyzed by reverse phase-high performance liquid chromatography (RP-HPLC) and size exclusion-high performance liquid chromatography (SE-HPLC). Citrate buffer was used as a buffer, mannitol was used as a sugar alcohol, and polysorbate 20 was used as a non-ionic surfactant. In Tables 2 and 3, RP-HPLC (%) and SE-HPLC (%) represent the value of “area %/start area %” showing the residual purity of the long-acting insulinotropic peptide conjugate compared to the initial purity. Table 2 shows the residual purity of long-acting insulinotropic peptide conjugate after being stored at 25° C., and Table 3 shows the residual purity of long-acting insulinotropic peptide conjugate after being stored at 40° C.
(41) TABLE-US-00001 TABLE 1 Concen- Sugar No. tration Buffer Salt alcohol surfactant 1 0.2 mg/ 2.0 mM Na- 150 mM 5% 0.005% mL Citrate (pH NaCl Mannitol Polysorbate 5.2) 20 2 0.2 mg/ 2.0 mM Na- — 5% 0.005% mL Citrate (pH Mannitol Polysorbate 5.2) 20 3 0.2 mg/ 2.0 mM Na- — 5% 0.005% mL Citrate (pH Mannitol/ Polysorbate 5.2) 0.1 mg/mL 20 Methionine 4 0.2 mg/ 2.0 mM Na- 150 mM 5% 0.005% mL Citrate (pH NaCl Mannitol/ Polysorbate 5.2) 0.1 mg/mL 20 Methionine
(42) TABLE-US-00002 TABLE 2 RP-HPLC (%) SE-HPLC (%) No. 0 week 1 week 2 weeks 4 weeks 0 week 1 week 2 weeks 4 weeks 1 100 99.3 99.2 97.1 100 99.8 100.1 100.2 2 100 99.1 99.1 96.9 100 99.8 100.1 100.1 3 100 99.8 99.5 97.9 100 99.8 100.0 100.1 4 100 99.5 99.8 98.4 100 99.9 100.2 100.2
(43) TABLE-US-00003 TABLE 3 RP-HPLC (%) SE-HPLC (%) No. 0 week 1 week 2 weeks 4 weeks 0 week 1 week 2 weeks 4 weeks 1 100 96.6 93.7 88.0 100 100.0 98.8 97.2 2 100 precipitation precipitation Precipitation 100 precipitation precipitation precipitation 3 100 98.1 precipitation Precipitation 100 99.9 precipitation precipitation 4 100 96.9 95.2 90.6 100 100.0 99.0 97.2
(44) Based on the comparison between Test groups #1 and #2, and between #3 and #4 in Tables 2 and 3, it is evident that when the liquid formulation of long-acting insulinotropic peptide conjugate was stored at 25° C. and 40° C., especially at 40° C. for 4 weeks, and in the presence of NaCl as isotonic agent, particularly 150 mM NaCl, the stability of the long-acting insulinotropic peptide conjugate was maintained remarkably high (Tables 2 and 3).
EXAMPLE 2
Evaluation of the Stability of Long-Acting Insulinotropic Peptide Conjugate at Various pH of Buffer
(45) While the pH range of the general liquefied protein drug is in 5 to 7, the pH of liquid formulation of exendin-4 (Byetta), an insulinotropic peptide drug, is 4.5, which is lower than the general pH range. Therefore, in this Example, the effect of pH of buffer on stability of conjugate was examined for a long-acting insulinotropic peptide conjugate comprising insulinotropic peptide and immunoglobulin Fc protein, preferably long-acting imidazoacetyl (CA) exendin-4 conjugate.
(46) Citrate buffer was used as a buffer, 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 the compositions of long-acting insulinotropic peptide conjugate were stored at 25±2° C. for 4 weeks and the stability thereof was analyzed by size exclusion chromatography (SE-HPLC) and reverse phase chromatography (RP-HPLC). RP-HPLC (%) and SE-HPLC (%) in Table 5 represent “area %/start area %” demonstrating the residual purity of the long-acting insulinotropic peptide conjugate in comparison with the initial purity.
(47) TABLE-US-00004 TABLE 4 Formu- Concen- lation tration Sugar Isotonic No. (mcg/mL) Buffer Surfactant alcohol agent 1 197.6 20 mM Na- 0.005% 5% 150 nM Citrate Polysorbate Manntiol NaCl (pH 5.2) 80 2 197.6 20 mM Na- 0.005% 5% 150 nM Citrate Polysorbate Manntiol NaCl (pH 5.5) 80 3 197.6 20 mM Na- 0.005% 5% 150 nM Citrate Polysorbate Manntiol NaCl (pH 6.0) 80
(48) TABLE-US-00005 TABLE 5 Formu- RP-HPLC SE-HPLC lation (Area %/Start Area %) % (Area %/Start Area %) % No. pH 0 W 1 W 2 W 4 W 0 W 1 W 2 W 4 W 1 5.2 100.0 99.1 98.6 97.5 100.0 100.0 100.5 100.5 2 5.5 100.0 99.8 97.7 95.0 100.0 99.6 100.8 100.7 3 6.0 100.0 98.3 98.1 94.8 100.0 99.6 100.7 100.7
(49) As shown above, when the pH was 5.2 in the above liquid formulation, the long-acting insulinotropic peptide conjugate was most stable (Table 5).
EXAMPLE 3
Evaluation of the Stability of Long-Acting Insulinotropic Peptide Conjugate Depending on the Type and Concentration of Non-Ionic Surfactant
(50) The stability of long-acting insulinotropic peptide conjugate was examined using different types and concentrations of polysorbate which is a non-ionic surfactant in the stabilizer of the present invention.
(51) The non-ionic surfactants, i.e., polysorbate 80 and polysorbate 20, were examined at both concentrations of 0.005% and 0.01%. The composition of stabilizer comprises a buffer, a sugar alcohol, and an isotonic agent as well as surfactant, as used in the above example for providing stability to the long-acting insulinotropic peptide conjugate. Citrate buffer at a pH of 5.2, which showed high stability in Example 2, was used as a buffer, mannitol was used as a sugar alcohol, and sodium chloride was used as an isotonic agent.
(52) The following compositions shown in Table 6 were used as a stabilizer for long-acting insulinotropic peptide conjugate, preferably for long-acting CA exendin-4 conjugate. Then the compositions were stored at 25±2° C. for 8 weeks and the stability thereof 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 as compared to the initial purity.
(53) TABLE-US-00006 TABLE 6 Formu- Con- lation centration Sugar Isotonic No. (mcg/mL) Buffer Surfactant alcohol agent 1 197.6 20 mM Na- 0.005% 5% 150 nM Citrate Polysorbate Manntiol NaCl (pH 5.2) 80 2 197.6 20 mM Na- 0.01% 5% 150 nM Citrate Polysorbate Manntiol NaCl (pH 5.2) 80 3 197.6 20 mM Na- 0.005% 5% 150 nM Citrate Polysorbate Manntiol NaCl (pH 5.2) 20 4 197.6 20 mM Na- 0.01% 5% 150 nM Citrate Polysorbate Manntiol NaCl (pH 5.2) 20
(54) TABLE-US-00007 TABLE 7 RP-HPLC (Area %/Start SE-HPLC Formulation Area %) % (Area %/Start Area %) % No. Surfactant 0 W 2 W 4 W 8 W 0 W 2 W 4 W 8 W 1 0.005% 100.0 97.5 94.1 90.9 100.0 100.0 100.0 99.9 Polysorbate 80 2 0.01% 100.0 98.3 95.2 92.6 100.0 99.9 99.9 99.8 Polysorbate 80 3 0.005% 100.0 98.9 97.5 93.8 100.0 100.0 99.9 99.9 Polysorbate 20 4 0.01% 100.0 98.69 97.0 92.2 100.0 100.0 100.0 99.1 Polysorbate 20
(55) As shown above, based on the SE-HPLC analysis results, the stability of the long-acting insulinotropic peptide conjugate was almost the same even when different types and concentrations of polysorbates were used. However, based on the RP-HPLC analysis results, it was observed that when polysorbate 20 was used, the stability of peptide conjugate was similar to or higher than when the same concentration of polysorbate 80 was used. Also, the stability of long-acting insulinotropic peptide conjugate was higher in the liquid formulation comprising 0.005% polysorbate 20, compared to the one comprising 0.01% polysorbate 20 (Table 7).
EXAMPLE 4
Comparison of Stability Between the Finally Selected Liquid Formulation of Long-Acting Insulinotropic Peptide Conjugate and the Commercially Available Liquid Formulation of Peptide or Protein Drug Comprising the Same
(56) In the present example, the stability of the formulation that was selected through stability tests in Examples 1 to 3 was evaluated. The finally selected formulation of long-acting insulinotropic peptide conjugate comprises citrate buffer at a pH of 5.2, sodium chloride, mannitol, and polysorbate 20. For this purpose, the stability of drug formulations was compared between the liquid formulations which are generated by applying the long-acting insulinotropic peptide conjugate to a liquid formulation of commercially available insulinotropic peptide drug, exendin-4 (Byetta); and to a liquid formulation of immunoglobulin fusion protein drug, Etanercept (TNFR-Fc fusion protein, ENBREL).
(57) Using the following compositions shown in Table 8, the following formulations were prepared: a liquid formulation of long-acting insulinotropic peptide conjugate, more preferably long-acting CA exendin-4 conjugate (Liquid Formulation #1); a liquid formulation prepared by applying the long-acting insulinotropic peptide conjugate to the stabilizer composition of the liquid formulation of insulinotropic peptide drug, exendin-4 (Byetta) (Liquid Formulation #2); and a liquid formulation prepared by applying the long-acting insulinotropic peptide conjugate to the stabilizer composition of the liquid formulation of immunoglobulin fusion protein drug, Etanercept (TNFR-Fc fusion protein, ENBREL) (Liquid Formulation #3). As a control group, a liquid formulation was prepared by applying the long-acting insulinotropic peptide conjugate to a stabilizer composition comprising PBS only (Liquid Formulation #4). Subsequently, the formulations were stored at 25±2° C. for 8 weeks, and the stability thereof 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 as compared to the initial purity.
(58) TABLE-US-00008 TABLE 8 Sugar Formu- Concen- alcohol lation tration and Isotonic No. (mcg/mL) Buffer Surfactant other agent 1 197.6 20 mM Na- 0.005% 5% 150 mM Citrate Polysorbate Manntiol NaCl (pH 5.2) 20 2 197.6 20 mM Na- — 5% — Acetate Manntiol (pH 4.5) 3 197.6 20 mM Na- — 1% 100 mM Phosphate Sucrose NaCl (pH 6.3) 25 mM L- Arginine 4 197.6 PBS — — —
(59) TABLE-US-00009 TABLE 9 RP-HPLC SE-HPLC (Area %/Start Area %) % (Area %/Start Area %) % No. 0 W 2 W 4 W 8 W 0 W 2 W 4 W 8 W 1 100.0 98.9 97.5 93.8 100.0 100.0 100.0 99.9 2 100.0 98.4 96.6 90.9 100.0 100.1 99.9 99.2 3 100.0 95.4 89.1 N/A 100.0 100.0 100.0 99.7 4 100.0 92.7 84.1 69.2 100.0 100.0 99.9 99.6
(60) As a result of stability test, it was observed that the liquid formulation of long-acting insulinotropic peptide conjugate of the present invention showed higher stability than the liquid formulations prepared by applying the long-acting insulinotropic peptide conjugate to the liquid formulations of a commercially available insulinotropic peptide drug, exendin-4 (Byetta), and an immunoglobulin fusion protein drug, Etanercept (TNFR-Fc usion protein, ENBREL), as shown in
EXAMPLE 5
Evaluation of the Stability of Long-Acting Insulinotropic Peptide Conjugate Depending on the Addition of Methionine
(61) In order to determine the effect of methionine on the stability of the conjugate, the liquid formulation was prepared by adding methionine for preventing oxidation, to the composition comprising citrate buffer at a pH of 5.2, sodium chloride, mannitol, and polysorbate 20, which were selected in the above Examples. The formulations were stored at 25±2° C. for 4 weeks and at 40±2° C. for 4 weeks, and then the stability thereof were analyzed.
(62) The liquid formulation of long-acting insulinotropic peptide conjugate, more preferably the long-acting CA exendin-4 conjugate was prepared in the following compositions shown in Table 10 and the stability thereof was analyzed. RP-HPLC (%) and SE-HPLC (%) in Tables 11 to 14 represent the proportions of long-acting insulinotropic peptide conjugate and impurities at each time point. Table 11 shows the results of accelerated stability test by RP-HPLC (25±2° C.) and Table 12 shows the results of accelerated stability test by SE-HPLC (25±2° C.). Table 13 shows the results of instability severity test by RP-HPLC (40±2° C.) and Table 14 shows the results of instability severity test by SE-HPLC (40±2° C.) Impurity #3 represents the oxidized form of long-acting insulinotropic peptide conjugate. However, since SE-HPLC separates the sample by molecular weight and the difference in molecular weight between oxidized form and non-oxidized form is minor, it was hard to isolate the oxidized form of long-acting insulinotropic peptide conjugate through SE-HPLC.
(63) TABLE-US-00010 TABLE 10 Concen- Sugar tration alcohol& Isotonic No. (mcg/mL) Buffer Surfactant methionine agent 1 200 20 mM Na- 0.005% 5% 150 mM Citrate Polysor- Mannitol NaCl (pH 5.2) bate 20 2 200 20 mM Na- 0.005% 5% 150 mM Citrate Polysor- Mannitol NaCl (pH 5.2) bate 20 0.01% Methionine
(64) TABLE-US-00011 TABLE 11 Formulation Storage Proportion of conjugate and impurity (Area %) No. duration #1 #2 #3 Conjugate #4 #5 #6 Others 1 0 week 0.1 0.1 0.8 93.5 3.2 1.7 0.4 0.1 1 week 0.1 0.2 1.0 92.8 3.8 1.8 0.3 <0.1 2 weeks 0.2 0.2 1.4 92.7 3.2 2.0 0.3 <0.1 4 weeks 0.1 0.3 1.8 90.8 4.6 1.7 0.3 0.6 2 0 week 0.1 0.2 0.7 93.7 3.5 1.4 0.4 <0.1 1 week 0.1 0.2 0.7 93.2 3.8 1.6 0.3 <0.1 2 weeks 0.1 0.2 0.8 93.5 3.2 1.8 0.3 <0.1 4 weeks 0.1 0.3 0.6 92.2 4.3 2.0 0.4 0.2
(65) TABLE-US-00012 TABLE 12 Formulation Storage Proportion of conjugate and impurity (Area %) No. Duration #1 #2 #3 Conjugate #4 #5 Others 1 0 week 0.2 0.3 0.0 99.5 0.0 0.0 0.0 1 week 0.2 0.5 0.0 99.3 0.0 0.0 0.0 2 weeks 0.2 0.2 0.0 99.6 0.0 0.0 0.0 4 weeks 0.1 0.2 0.0 99.7 0.0 0.0 0.0 2 0 week 0.3 0.2 0.0 99.5 0.0 0.0 0.0 1 week 0.3 0.3 0.0 99.4 0.0 0.0 0.0 2 weeks 0.2 0.1 0.0 99.7 0.0 0.0 0.0 4 weeks 0.2 0.1 0.0 99.7 0.0 0.0 0.0
(66) TABLE-US-00013 TABLE 13 Formu- lation Storage Proportion of conjugate and impurity (Area %) No. Duration #1 #2 #3 Conjugate #4 #5 #6 Others 1 0 week 0.1 0.1 0.8 93.5 3.2 1.7 0.4 0.1 1 week 0.2 0.3 1.5 90.3 5.0 2.4 0.3 <0.1 2 weeks 0.1 0.5 2.1 87.6 6.2 3.2 0.3 <0.1 4 weeks 0.1 1.1 3.7 82.3 8.6 3.8 0.3 0.2 2 0 week 0.1 0.2 0.7 93.7 3.5 1.4 0.4 <0.1 1 week 0.1 0.4 0.7 90.8 4.9 2.8 0.3 0.1 2 weeks 0.1 0.5 0.7 89.2 5.9 3.2 0.3 0.0 4 weeks 0.1 1.0 0.8 84.9 8.5 3.9 0.3 0.5
(67) TABLE-US-00014 TABLE 14 Formulation Storage Proportion of conjugate and impurity (Area %) No. Duration #1 #2 #3 Conjugate #4 #5 Others 1 0 week 0.2 0.3 0.0 99.5 0.0 0.0 0.0 1 week 0.2 0.3 0.0 99.5 0.0 0.0 0.0 2 weeks 0.2 0.0 0.0 98.3 1.3 0.3 0.0 4 weeks 0.1 0.0 0.0 96.7 2.7 0.4 0.0 2 0 week 0.3 0.2 0.0 99.5 0.0 0.0 0.0 1 week 0.2 0.3 0.0 99.5 0.0 0.0 0.0 2 weeks 0.1 0.0 0.0 98.5 1.1 0.3 0.0 4 weeks 0.1 0.0 0.0 96.7 2.8 0.5 0.0
(68) As results of the accelerated stability test and instability severity test and as shown in
EXAMPLE 6
Evaluation of the Long-Term Storage Stability of the Finally Selected Liquid Formulation of Long-Acting Insulinotropic Peptide Conjugate
(69) In the present example, the liquid formulation that was finally selected by the above examples was evaluated for the long-term storage stability and accelerated stability. The finally selected liquid formulation 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±3° C. for 6 months and at 25±2° C. for 6 months and the stability thereof were analyzed. The results are shown in Tables 15 and 16, and RP-HPLC (%), SE-HPLC (%), protein content (%), and specific activity test (%) represent the residual purity of the conjugate compared to the initial purity. Table 15 shows the results of testing long-term storage stability of formulation after storing the same at 5±3° C., and Table 16 shows the results of accelerated stability test after storing the same at 25±2° C.
(70) TABLE-US-00015 TABLE 15 Evaluation of long-term storage stability (stored at 5 ± 3° C.) Confirmation Purity test test RP- SE- Protein Specific Storage RP- Western SDS- HPLC HPLC Content activity Duration Color pH HPLC blot PAGE (%) (%) Endotoxin (%) (%) Start No 5.2 Match Acceptable acceptable 100.0 100.0 acceptable 100.0 100.0 color/ Transparent 1 month No 5.2 Match acceptable acceptable 100.1 99.7 acceptable 105.8 114.3 color/ Transparent 3 months No 5.2 Match acceptable acceptable 100.1 99.6 acceptable 100.0 115.7 color/ Transparent 6 months No 5.2 Match acceptable acceptable 100.0 99.5 acceptable 100.0 97.0 colour/ Transparent
(71) TABLE-US-00016 TABLE 16 Accelerated Stability Test (stored at 25 ± 2° C.) Conformation Purity Test Test RP- SE- Protein Specific Storage RP- Western SDS- HPLC HPLC Content activity Duration Color pH HPLC blot PAGE (%) (%) Endotoxin (%) (%) Start No 5.2 match acceptable acceptable 100.0 100.0 acceptable 100.0 100.0 color/ Transparent 1 month No 5.2 match acceptable acceptable 99.6 99.4 acceptable 105.8 116.4 color/ Transparent 3 months No 5.2 match acceptable acceptable 98.0 98.6 acceptable 103.8 95.8 color/ Transparent 6 months No 5.2 match acceptable acceptable 95.4 97.7 acceptable 103.8 90.5 color/ Transparent
(72) As a result of long-term storage stability test, the long-acting insulinotropic peptide conjugate was stable for more than 6 months in the liquid formulation of the present invention. Also, even when stored in the accelerated condition for 6 months, RP-HPLC analysis results showed that 95.4% or more of the peptide conjugate was remained intact in the formulation, thereby confirming that the present liquid formulation provides excellent storage stability to the long-acting insulinotropic peptide conjugate.
EXAMPLE 7
Evaluation of the Stability of Long-Acting Insulinotropic Peptide Conjugate Depending on the Concentration of Protein
(73) The effect of high conjugate concentration was examined for the finally selected liquid formulation, comprising citrate buffer at a pH of 5.2, sodium chloride, mannitol, polysorbate 20, and methionine for preventing oxidation. For this purpose, the precipitation in the formulation was monitored with naked eyes at 40° C. and at various conjugate concentrations shown in Table 17. After 72 hours of monitoring, precipitation occurred in all of the present formulations at high concentration (4 mg/ml or more). Also, as the concentration increased, the occurrence of precipitation was increased as well.
(74) TABLE-US-00017 TABLE 17 Sugar Concen- alcohol Surfac- No. tration Buffer Salt and others tant 1 0.52 mg/mL 20 mM Na- 150 mM 5% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine 2 4.0 mg/mL 20 mM Na- 150 mM 5% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine 3 5.0 mg/mL 20 mM Na- 150 mM 5% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine 4 8.0 mg/mL 20 mM Na- 150 mM 5% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine 5 10.0 mg/mL 20 mM Na- 150 mM 5% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine 6 13.0 mg/mL 20 mM Na- 150 mM 5% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine
EXAMPLE 8
Evaluation of Stability of Long-Acting Insulinotropic Peptide Conjugate at High Concentration Depending on the Concentration of a Salt and a Sugar Alcohol, and the Presence of Methionine
(75) The effect of the concentration of NaCl and mannitol as a sugar alcohol on preventing the precipitation was examined for the finally selected liquid formulation of long-acting insulinotropic peptide conjugate at high concentration. The formulations were prepared in the following compositions shown in Table 18 and monitored for occurrence of precipitation with naked eyes at 40° C. for 48 hours. The duration of absence of precipitation shown in
(76) TABLE-US-00018 TABLE 18 Sugar Concen- alcohol Surfac- No. tration Buffer Salt and others tant 1 5.0 mg/mL 20 mM Na- 150 mM 5% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine 2 5.0 mg/mL 20 mM Na- 150 mM 10% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine 3 5.0 mg/mL 20 mM Na- 200 mM 5% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine 4 5.0 mg/mL 20 mM Na- 150 mM 5% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.2) bate 20
(77) As shown in the above results, it was confirmed that the concentration of NaCl did not significantly affect the occurrence of precipitation and stability of the insulinotropic peptide conjugate at high concentration, based on the observation by naked eyes. However, when the concentration of mannitol as a sugar alcohol was increased from 5% to 10%, the precipitation could be suppressed significantly (
EXAMPLE 9
Evaluation of Stability of Long-Acting Insulinotropic Peptide Conjugate at High Concentration Depending on the Presence of a Salt and at Various pH
(78) Having 10% mannitol as selected by Example 8, the effect of pH was examined on the suppression of precipitation and the promotion of stability of long-acting insulinotropic conjugate at high concentration. Citrate buffer was used as a buffer, and polysorbate 20 was used as a non-ionic surfactant. According to Example 8, precipitation could be suppressed by exclusion of methionine from formulation. However methionine was still added to the formulation for the purpose of preventing oxidation of the protein. Furthermore, in order to confirm the synergic effect of NaCl and pH, 150 mM NaCl was added or excluded in the formulation. The long-acting insulinotropic peptide conjugate at high concentration was prepared in the following compositions shown in Table 19 and monitored for the occurrence of precipitation at 40° C. for 7 days. After 7 days of storing, the samples were analyzed by RP-HPLC and SE-HPLC.
(79) The duration of the absence of precipitation shown in
(80) TABLE-US-00019 TABLE 19 Sugar Concen- alcohol Surfac- No. tration Buffer Salt and others tant 1 5.0 mg/mL 20 mM Na- — 10% 0.005% Citrate Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine 2 5.0 mg/mL 20 mM Na- 150 mM 10% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine 3 5.0 mg/mL 20 mM Na- — 10% 0.005% Citrate Mannitol/ Polysor- (pH 5.4) 0.1 mg/mL bate 20 Methionine 4 5.0 mg/mL 20 mM Na- 150 mM 10% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.4) 0.1 mg/mL bate 20 Methionine 5 5.0 mg/mL 20 mM Na- — 10% 0.005% Citrate Mannitol/ Polysor- (pH 5.6) 0.1 mg/mL bate 20 Methionine 6 5.0 mg/mL 20 mM Na- 150 mM 10% 0.005% Citrate NaCl Mannitol/ Polysor- (pH 5.6) 0.1 mg/mL bate 20 Methionine
(81) TABLE-US-00020 TABLE 20 RP-HPLC (Area %) No. 0 D 1 D 2 D 3 D 4 D 7 D 1 98.5 Precipitation Precipitation Precipitation Precipitation Precipitation 2 98.4 98.0 Precipitation Precipitation Precipitation Precipitation 3 98.4 97.9 97.7 97.6 97.3 96.8 4 98.3 98.0 97.7 97.6 97.2 96.3 5 98.2 97.8 97.8 97.5 97.4 96.5 6 98.3 98.1 97.9 97.5 97.2 96.5
(82) TABLE-US-00021 TABLE 21 SE-HPLC (Area %) No. 0 D 1 D 2 D 3 D 4 D 7 D 1 98.3 Precipitation Precipitation Precipitation Precipitation Precipitation 2 98.3 95.6 Precipitation Precipitation Precipitation Precipitation 3 98.3 98.0 97.8 97.5 97.4 97.4 4 98.4 98.1 97.9 97.4 97.3 97.6 5 98.5 98.0 98.0 97.9 97.8 97.6 6 98.5 98.1 98.1 98.0 97.9 97.8
(83) As shown above, the precipitation was suppressed better at the high pH of 5.4 and 5.6 than at the pH of 5.2. After 7 days of storing, precipitation was observed in all formulations. However, in the composition comprising 10% mannitol and 150 mM NaCl at a pH of 5.6 (Composition No. 6), the amount of impurity generated was smallest. At the pH of 5.4 and 5.6, the presence of NaCl did not have a significant effect on the stability of long-acting insulinotropic peptide conjugate at high concentration, except for the precipitation (Tables 20 and 21, and
EXAMPLE 10
Evaluation of Stability of Long-Acting Insulinotropic Peptide Conjugate at High Concentration Depending on the Concentration of Sugar Alcohol and at Various pH
(84) Based on the above Examples, the effect of concentration of sugar alcohol and pH on the stability of long-acting insulinotropic peptide conjugate at high concentration was examined. Citrate buffer was used as a buffer, and polysorbate 20 was used as a non-ionic surfactant. Also, methionine was added to the formulation for the purpose of preventing oxidation. In addition, based on the results observed in Example 9, NaCl was excluded in the formulation of long-acting insulinotropic peptide conjugate at high concentration. The long-acting insulinotropic peptide conjugate at high concentration was formulated in the following compositions as shown in Table 22 and stored at 40° C. for 5 days and moved to the temperature of 25° C. and stored for 4 more weeks. Every week, the stability of protein was analyzed by SE-HPLC, IE-HPLC, and RP-HPLC. SE-HPLC (%) of Table 23, IE-HPLC (%) of Table 24, and RP-HPLC (%) of Table 25 represent the residual purity of the long-acting insulinotropic peptide conjugate.
(85) TABLE-US-00022 TABLE 22 Sugar Concen- alcohol Surfac- No. tration Buffer Salt and other tant 1 10.0 mg/mL 20 mM Na- — 10% 0.005% Citrate Mannitol/ Polysor- (pH 5.6) 0.1 mg/mL bate 20 Methionine 2 10.0 mg/mL 20 mM Na- — 10% 0.005% Citrate Mannitol/ Polysor- (pH 5.2) 0.1 mg/mL bate 20 Methionine 3 10.0 mg/mL 20 mM Na- — 10% 0.005% Citrate Mannitol/ Polysor- (pH 6.0) 0.1 mg/mL bate 20 Methionine 4 10.0 mg/mL 20 mM Na- — 2% 0.005% Citrate Mannitol/ Polysor- (pH 6.0) 0.1 mg/mL bate 20 Methionine 5 10.0 mg/mL 20 mM Na- — 2% 0.005% Citrate Mannitol/ Polysor- (pH 6.4) 0.1 mg/mL bate 20 Methionine 6 10.0 mg/mL 20 mM Na- — 5% 0.005% Citrate Mannitol/ Polysor- (pH 6.0) 0.1 mg/mL bate 20 Methionine 7 10.0 mg/mL 20 mM Na- — 5% 0.005% Citrate Mannitol/ Polysor- (pH 6.4) 0.1 mg/mL bate 20 Methionine
(86) TABLE-US-00023 TABLE 23 SE-HPLC (Area %) No. 0 W 1 W 2 W 3 W 4 W 1 99.4 98.3 98.3 98.2 98.0 2 99.3 98.0 98.0 97.8 97.6 3 99.3 98.1 98.1 98.0 97.9 4 99.3 97.9 97.8 97.8 97.7 5 99.0 97.7 97.6 97.6 97.5 6 99.3 98.1 98.0 98.0 97.7 7 99.0 97.7 97.7 97.6 97.5
(87) TABLE-US-00024 TABLE 24 IE-HPLC (Area %) No. 0 W 1 W 2 W 3 W 4 W 1 95.6 81.7 78.2 75.7 65.3 2 95.7 73.8 69.0 64.6 53.3 3 95.7 81.7 79.9 77.1 69.1 4 95.6 80.3 78.0 75.3 66.4 5 95.6 72.7 70.8 68.3 60.2 6 95.7 80.6 77.1 72.3 61.6 7 95.7 74.1 72.0 69.8 62.7
(88) TABLE-US-00025 TABLE 25 RP-HPLC (Area %) No. 0 W 1 W 2 W 3 W 4 W 1 97.5 87.2 84.1 81.0 75.7 2 97.6 80.0 77.6 67.5 60.3 3 97.5 87.5 84.9 81.0 76.0 4 97.5 86.6 83.4 79.2 72.9 5 96.5 84.5 79.5 76.2 72.9 6 97.5 86.1 82.8 77.1 69.7 7 96.6 82.9 80.1 77.2 71.9
(89) As shown above, when the pH was low, the stability was also reduced, compared to when the pH was high. The stability of the conjugate was highest at 10% mannitol, while 2% and 5% mannitol did not affect the stability of long-acting insulinotropic peptide conjugate at high concentration.
EXAMPLE 11
Evaluation of the Stability of Long-Acting Insulinotropic Peptide Conjugate at High Concentration Depending on the Type and Concentration of Sugar Alcohol
(90) For developing an isotonic liquid formulation, the effect of the type and concentration of a sugar alcohol, which affects the osmotic pressure of the formulation most significantly, on the stability of insulinotropic peptide conjugate was examined under the same condition as in the above Examples. The type of sugar alcohol was changed to sucrose. Based on Formulation No. 1 of Example 10, 10% mannitol was replaced by 5% and 7% sucrose (Table 26). The formulations were stored at 25° C. for 4 weeks and the stability thereof was analyzed every week by SE-HPLC, IE-HPLC, and RP-HPLC. SE-HPLC (%) of Table 27, IE-HPLC (%) of Table 28, and RP-HPLC (%) of Table 29 represent the residual purity of the long-acting insulinotropic peptide conjugate.
(91) TABLE-US-00026 TABLE 26 Sugar Concen- alcohol Surfac- No. tration Buffer Salt and others tant 1 10.0 mg/mL 20 mM Na- — 10% 0.005% Citrate Mannitol/ Polysor- (pH 5.6) 0.1 mg/mL bate 20 Methionine 2 10.0 mg/mL 20 mM Na- — 5% 0.005% Citrate Sucrose/ Polysor- (pH 5.6) 0.1 mg/mL bate 20 Methionine 3 10.0 mg/mL 20 mM Na- — 7% 0.005% Citrate Sucrose/ Polysor- (pH 5.6) 0.1 mg/mL bate 20 Methionine
(92) TABLE-US-00027 TABLE 27 SE-HPLC (Area %) No. 0 W 1 W 2 W 3 W 4 W 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
(93) TABLE-US-00028 TABLE 28 IE-HPLC (Area %) No. 0 W 1 W 2 W 3 W 4 W 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
(94) TABLE-US-00029 TABLE 29 RP-HPLC (Area %) No. 0 W 1 W 2 W 3 W 4 W 1 97.5 87.2 84.1 81.0 75.7 2 97.5 88.5 85.0 80.9 75.3 3 97.5 90.1 85.8 82.5 76.0
(95) As shown above, when sucrose was used instead of mannitol, the stability of the conjugate was maintained, and the stability of conjugate was increased slightly in 7% sucrose rather than in 5% sucrose, but there was no significant difference.
EXAMPLE 12
Evaluation of Stability of Long-Acting Insulinotropic Peptide Conjugate at High Concentration Depending on the Type of Buffer, Adjustment of Osmotic Pressure, and Addition of Preservative
(96) In order to develop an isotonic liquid formulation, the concentration of sugar alcohol, which has the greatest effect on osmotic pressure, was adjusted and different types of buffers were tested for providing conjugate stability under the conditions of the above Examples. Also, under the same condition, 0.22% m-cresol was added as a preservative, and the effect thereof on the conjugate stability was tested as well. The long-acting insulinotropic peptide conjugate was formulated in the following compositions shown in Table 30 and stored at 25° C. for 2 weeks. Then every week, the stability of the samples were analyzed by SE-HPLC, IE-HPLC, and RP-HPLC. SE-HPLC (%) of Table 31, IE-HPLC (%) of Table 32, and RP-HPLC (%) of Table 33 represent the residual purity of the long-acting insulinotropic peptide conjugate.
(97) TABLE-US-00030 TABLE 30 Suger alcohal and No. Concentration Buffer Salt others Surfactant Preservative 1 10.0 mg/mL 20 mM Na- — 10% 0.005% — Citrate Mannitol/ Polysorbate (pH 5.6) 0.1 mg/mL 20 Methionine 2 10.0 mg/mL 20 mM Na- — 5% 0.005% 0.22% m- Citrate Sucrose/ Polysorbate cresol (pH 5.6) 0.1 mg/mL 20 Methionine 3 10.0 mg/mL 20 mM — 7% 0.005% — Histidine- Sucrose/ Polysorbate Cl (pH 0.1 mg/mL 20 5.6) Methionine 4 10.0 mg/mL 20 mM — 10% 0.005% 0.22% m- Histidine- Mannitol/ Polysorbate cresol Cl (pH 0.1 mg/mL 20 5.6) Methionine 5 10.0 mg/mL 20 mM Na- — 5% 0.005% — Acetate Sucrose/ Polysorbate (pH 5.6) 0.1 mg/mL 20 Methionine 6 10.0 mg/mL 20 mM Na- — 7% 0.005% 0.22% m- Acetate Sucrose/ Polysorbate cresol (pH 5.6) 0.1 mg/mL 20 Methionine
(98) TABLE-US-00031 TABLE 31 SE-HPLC (Area %) No. 0 W 1 W 2 W 1 99.4 98.9 97.0 2 99.3 99.2 98.6 3 99.2 98.9 98.4 4 99.1 98.8 98.0 5 99.3 99.1 98.7 6 99.2 99.1 98.4
(99) TABLE-US-00032 TABLE 32 IE-HPLC (Area %) No. 0 W 1 W 2 W 1 90.4 88.9 84.7 2 90.5 90.2 88.2 3 89.0 83.5 78.0 4 89.2 85.0 80.1 5 89.6 84.4 79.8 6 90.1 86.9 83.2
(100) TABLE-US-00033 TABLE 33 RP-HPLC (Area %) No. 0 W 1 W 2 W 1 93.2 91.3 90.1 2 93.2 91.9 90.1 3 91.8 88.2 80.3 4 92.4 88.6 84.4 5 91.5 87.6 83.8 6 92.2 89.4 86.0
(101) As shown above, when different types of buffers were used, the peptide conjugate of each formulation was stable. Also, addition of m-cresol did not affect the peptide stability.
(102) These results support that the composition of the liquid formulation of the present invention could maintain a high stability of the insulinotropic peptide conjugate at high concentration.
(103) Based on the above description, it will be apparent to those skilled in the art that various modifications and changes may be made without departing from the scope and spirit of the invention. Therefore, it should be understood that the above embodiment is not limitative, but illustrative in all aspects. The scope of the invention is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within metes and bounds of the claims, or equivalents of such metes and bounds are therefore intended to be embraced by the claims.