KR20060124449A - A stable liquid formulation of human growth hormone - Google Patents

Abstract

A liquid formulation containing human growth hormone is provided to enhance storage stability by minimizing deamidation and agglutination of human growth hormone. The liquid formulation contains: human growth hormone; L-lysine or L-arginine; and poly(oxyethylene) poly(oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil, wherein the amount of human growth hormone is 2.5-5.5 mg/ml, the amount of L-lysine or L-arginine is 0.02-0.5 w/v% per 1mg of human growth hormone, and the amount of poly(oxyethylene) poly(oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil is 0.1-0.5 w/v%; and the liquid formulation further contains at least one component selected from buffer solution, tonicity adjustment agent, preservative and analgesia.

Description

A stable liquid formulation of human growth hormone

Figure 1 shows the residual percentage (%) of phosphorus growth hormone for the deamidation and aggregation of the liquid formulation according to the invention for 3 months at 25 ℃.

Figure 2 shows the residual percentage of phosphorus growth hormone for deamidation and aggregation for 1 year in the liquid formulation according to the present invention.

The present invention is phosphorus growth hormone; L-lysine, L-arginine, or polyethylene glycol 300; And a poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil.

Phosphorus growth hormone is a short-chain polypeptide with a molecular weight of about 22,000 consisting of 191 amino acids produced by the human pituitary gland, and is mainly used to treat pituitary dwarfism in children (Endocrinol. Rev. 4, 155, 1983).

Pharmaceutical preparations of phosphorus growth hormones tend to be unstable. For example, deamidated or sulfoxided products may be produced or degradation products in dimer or polymer form may be produced in a solution of growth hormone. These chemical degradation reactions of phosphorus growth hormone, particularly L-asp-hGH, L-iso-asp-hGH, D-asp, by direct hydrolysis or by cyclic succinimide polymer of position 149 Asn of phosphorus growth hormone -hGH, caused by deamidation to form D-iso-asp-hGH. At present, these deamidation products are not considered to be toxic or have altered biological activity or receptor binding, but exhibit the effect of reducing the structural stability of sulfoxide compared to natural phosphorus growth hormone.

Deamidation of phosphorus growth hormone is unfavorable for pharmaceuticals in that it does not change biological activity but degrades quality, and the acceptable range of phosphorus growth hormone deamide is generally described in the purity standard of fairness. In addition, the production of aggregates such as polymers can cause discomfort to patients by causing in vivo safety problems such as inducing immunogenicity and cosmetic problems such as floating matter.

Protein stability is closely related to moisture, and even in the case of phosphorus growth hormone, most commercial formulations are commercially available in the form of freeze-dried formulations that are prepared and administered during use. However, research on liquid formulations continues to be conducted to enhance the convenience of doctors and patients. For example, US Pat. No. 6,448,225 for Nutropin AQ by Genentech Inc. describes liquid phosphorus growth hormone preparations containing phosphorus growth hormone, mannitol, buffers and nonionic surfactants, in particular citrate Buffers are illustrated as preferred. In addition, Korean Patent Application No. 10-1999-0001217 discloses that dissolution of phosphorus growth hormone in a weak acid to weak acid buffer containing benzalkonium chloride, preferably maleate, provides a stable aqueous formulation. Korean Patent Application No. 10-1998-0052483 discloses that a buffer consisting of sodium acetate and sodium glutamate is preferable in providing a liquid formulation of pharmaceutically stable phosphorus growth hormone. Novo Nordisk A / S is a growth hormone and amino acid or histidine derivative selected from the group of Asp, Ile, Val, Leu or His in Korean Patent Application No. 10-1994-0702139, or at least one. A pharmaceutical preparation comprising a peptide comprising a basic amino acid residue of and at least one acidic amino acid residue of and a nonionic detergent of polysorbate or poloxamer. In addition, Norditropin SimpleXx from Novo Nordisk A / S, which is currently available, contains phosphorus growth hormone, histidine buffers, nonionic surfactants, sodium chloride and preservatives.

Despite various attempts to provide liquid formulations of growth hormone as described above, there is still a need to provide more stable liquid formulations.

An object of the present invention is to provide a liquid formulation of phosphorus growth hormone, by combining a specific amino acid or polyethylene glycol, such as L- lysine or L- arginine with a specific surfactant to minimize deamination and aggregation of the known liquid formulation Compared to providing a stable phosphorus growth hormone liquid formulations improved long-term storage and convenience.

In one embodiment, the present invention is a phosphorus growth hormone; L-lysine or L-arginine; And a poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil.

Phosphorus growth hormone to be stabilized in the liquid formulation of the present invention can be obtained from prokaryotic or eukaryotic organisms isolated from nature or transformed with DNA encoding phosphorus growth hormone using genetic recombination techniques. Methods for producing phosphorus growth hormone using E. coli or yeast as a host recombinantly, for example (Korean Patent No. 25013 or Korean Patent No. 316347 or Korean Patent Publication No. 10-1997-0006498, etc.) It is described in. The growth hormone may be in the form of a wild type or a derivative thereof.

Liquid formulations of the present invention comprise L-lysine or L-arginine as stabilizers. Such stabilizers are preferably used in the form of salts, for example L-lysine hydrochloride salts or L-arginine hydrochloride salts. These stabilizers showed an excellent effect in that agglomeration did not occur after a certain time in the liquid formulation of phosphorus growth hormone, compared to other conventional amino acid stabilizers. As can be seen from Table 8, the stabilizer did not occur at all after 14 days, and thus exhibited excellent stability compared to histidine disclosed in the publication WO 1997/39768. This result is an excellent effect not disclosed in the above-cited patents and other known documents that disclose the use of a single amino acid as a stabilizer in providing a liquid formulation of stable phosphorus growth hormone.

Poly (oxyethylene) poly (oxypropylene) copolymer (poloxamer), microcoll-15 polyoxystearate (polyethylene glycol-15 polyoxystearate) or cremophor ELP (used in the liquid formulations of the present invention) Polyethylene glycol-35 castor oil) is a nonionic surfactant. The present inventors have found that the stability of the liquid formulation can be enhanced by using a surfactant as described above among various nonionic surfactants in combination with a specific stabilizer. These surfactants showed a superior effect on deamidation and flocculation compared to conventional polysorbates such as Tween 20 or Tween 80 (Table 5). The poloxamer is preferably used poloxamer 188 (poly (oxyethylene) poly (oxypropylene) copolymer 188) or poloxamer 407 (poly (oxyethylene) poly (oxypropylene) copolymer 407), more preferably Poloxamer 188 (poly (oxyethylene) poly (oxypropylene) copolymer 188) is used.

Liquid formulations of the present invention may further comprise additives known in the art, such as buffers, tonicity agents, preservatives or analgesics, and the like.

Buffers are acetate, glutamate, lactate, malate, citrate and phosphate that do not affect the growth hormone in order to control the pH of liquid formulations. Etc. can be used. Preferably sodium citrate or sodium acetate can be used. As can be seen from the results of Example 4, the buffer does not affect the stability of the liquid formulation of the present invention. This result is because an excellent effect on the stability of the liquid formulation of the present invention is shown by the combination of a specific stabilizer and a specific surfactant. The concentration of the buffer in the liquid formulation of the present invention is 5 to 100 mM, preferably 5 to 50 mM.

Isotonic agents may be used sodium chloride, mannitol, sucrose, dextrose, sorbitol or mixtures thereof. More preferably D-mannitol is used. The concentration of the tonicity agent in the liquid formulation of the present invention is preferably 20 to 50 mg / ml.

Preservatives may be used benzyl alcohol (benzyl alcohol), phenol (phenol), methacresol (m-cresol) and the like, of which benzyl alcohol is preferred. Benzyl alcohol, in addition to preservatives, also acts as a analgesic agent. The concentration of the preservative in the liquid formulation of the present invention is preferably 1 to 9 mg / ml.

In a specific embodiment, the liquid formulation of the present invention comprises 2.5 to 7.5 mg / ml phosphorus growth hormone; L-lysine or L-arginine contains 0.01-1.0 w / v% per mg of growth hormone; Poly (oxyethylene) poly (oxypropylene) copolymers, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oils comprise 0.01 to 1.0 w / v%. In a more specific embodiment, phosphorus growth hormone is 2.5 to 5.5 mg / ml, L-lysine or L-arginine is 0.02 to 0.5 w / v% per mg of phosphorus growth hormone, poly (oxyethylene) poly (oxypropylene) copolymer, Polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil comprises 0.1 to 0.5 w / v%.

In a preferred embodiment, the phosphorus growth hormone liquid preparation of the present invention comprises 2.5 to 5.5 mg / ml phosphorus growth hormone; 0.02 to 0.5 w / v% L-lysine or L-arginine per mg of phosphorus growth hormone; 0.1 to 0.5 w / v% poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil; 5-20 mM sodium citrate or sodium acetate; 20-50 mg / ml of D-mannitol; It contains 1 to 9 mg / ml benzyl alcohol and the pH is weakly acidic to neutral, preferably pH 5.8 to 7.0, more preferably pH 6.0 to 6.2.

As another aspect, the present invention is a phosphorus growth hormone; Polyethylene glycol 300; And a poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil.

Phosphorus growth hormone and poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil included in such liquid formulations are as described above.

The liquid formulation of the present invention comprises polyethylene glycol 300 as a stabilizer. Polyethyleneglycol is a useful polymer having a property of dissolving in various organic solvents as well as water solubility and is useful for stable pharmaceutical composition because it is nontoxic and can be quickly cleared from the body (International Patent Publication WO 01/26692). . Korean Patent Application No. 10-2003-0061434 discloses a liquid phosphorus growth hormone formulation which uses polyethylene glycol instead of polysorbate, which is a nonionic surfactant, and does not add a preservative such as benzyl alcohol or phenol. have. However, the Korean patent application discloses the use of polyethylene glycol having a molecular weight of 3,000 Da or more in polyethylene glycol at a concentration of 0.001 to 20 mg / ml without the use of a surfactant, which specifies polyethylene glycol 300 having a molecular weight of 300 Da. In contrast to the present invention, which is intended to be used in combination with a surfactant to enhance the stability of the liquid formulation, the liquid formulation of the present invention can be used for a long time by lowering the occurrence of aggregation by the use of a specific polyethylene glycol and a specific surfactant as described above. A liquid formulation that can be stored could be provided.

The liquid formulations of the present invention described above may further comprise additives known in the art, for example buffers, isotonic agents, preservatives or analgesics, and the like, such buffers, isotonic agents, preservatives or analgesics same.

In a specific embodiment, the liquid formulation of the present invention comprises 2.5 to 7.5 mg / ml phosphorus growth hormone; Polyethylene glycol 300 contains 0.1 to 5.0 v / v% per 1 mg of phosphorus growth hormone; Poly (oxyethylene) poly (oxypropylene) copolymers, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oils comprise 0.01 to 1.0 w / v%. In a more specific embodiment, phosphorus growth hormone is 2.5 to 5.5 mg / ml, polyethylene glycol 300 is 0.2 to 1.0 v / v% per mg of phosphorus growth hormone, poly (oxyethylene) poly (oxypropylene) copolymer, polyethylene glycol -15 polyoxystearate or polyethyleneglycol-35 castor oil comprises 0.1 to 0.5 w / v%.

In a preferred embodiment, the phosphorus growth hormone liquid preparation of the present invention comprises 2.5 to 5.5 mg / ml phosphorus growth hormone; 0.2 to 1.0 v / v% of polyethylene glycol 300 per mg of phosphorus growth hormone; 0.1 to 0.5 w / v% poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil; 5-20 mM sodium citrate or sodium acetate; 20-50 mg / ml of D-mannitol; It contains 1 to 9 mg / ml benzyl alcohol and the pH is weakly acidic to neutral, preferably pH 5.8 to 7.0, more preferably pH 6.0 to 6.2.

The invention will be explained in more detail through the following examples. However, these examples are only presented by way of example so that the present invention can be more understood, these examples should not limit the scope of the present invention.

Example 1 Preparation and Analysis of Liquid Formulation of Phosphorus Growth Hormone

The liquid preparation of the present invention is a solution of 4.0 mg / ml of natural phosphorus growth hormone (Daewoong) in the form of a bulk solution before lyophilization, and sodium acetate (sigma) / sodium citrate (sigma) / phosphate The pH was adjusted to a range of 6.0 to 6.2 by mixing with a 10 mM aqueous solution of sodium phosphate monobasic (Sigma) and then replacing the buffer. After adjusting the concentration, excipients (D-Mannitol, Sigma), surfactants (Microcol-15 Polyoxystearate (Solutol HS 15, BASF) / Poloxamer 188 (Lutrol F 68, BASF) / Poloxamer 407 (Lutrol F 127, BASF) / Cremophor ELP (Cremophor ELP, BASF) / twin 20 (CRILLET 1 HP, croda) / twin 80 (CRILLET 4 HP, croda)), stabilizer (polyethylene glycol 300 (PEG 300, BASF) / polyethylene glycol 400 (PEG 400, BASF) / PVPK-12 (Kollidon 12 PF, BASF) / PVP-15 (Kollidon 15 PF, BASF) / L-lysine (L-Lysine, HCl, Sigma) / L-arginine (L-Arginine-HCl, Sigma)) and benzyl alcohol (Benzyl alcohol, tablet) were mixed and prepared to prepare.

After the preparation of the liquid formulation was stored for 2 weeks at 40 ℃, 75 RH% of harsh conditions to examine the stabilizing effect of phosphorus growth hormone on deamidation and polymer formation over time. Deamidation using reverse-phase HPLC (RP-HPLC) and dimer / polymer formation using size-exclusion HPLC (SEC-HPLC) according to the European Pharmacopia method. Analyzed.

Example 2 Investigation of Stability of Surfactant and Phosphorus Growth Hormone

In order to investigate the effects of pharmaceutically available surfactants on the stability of phosphorus growth hormone, a liquid formulation of phosphorus growth hormone was prepared and analyzed in the same manner as in Example 1 using the substances shown in Table 1 as components.

division chief ingredient Buffer Tonicity Surfactants Preservative solvent Control Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg - Benzyl alcohol 2.5 mg 1 mL water for injection DWF1 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Tween 20 2 mg Benzyl alcohol 2.5 mg 1 mL water for injection DWF2 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg MicroCol-15 Polyoxystearate 2 mg Benzyl alcohol 2.5 mg 1 mL water for injection DWF3 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Cremophor ELP 2 mg Benzyl alcohol 2.5 mg 1 mL water for injection DWF4 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Tween 80 2 mg Benzyl alcohol 2.5 mg 1 mL water for injection DWF5 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Poloxamer 188 2 mg Benzyl alcohol 2.5 mg 1 mL water for injection

The results for deamidation and aggregation according to the type of surfactant of the liquid formulation prepared above are shown in Table 2 below.

In Table 2, deamidation (%) is the calibrated percentage of active phosphorus growth hormone that has not been deamidated over time at 40 ° C., and aggregation is the active toughness where no dimer or polymer is formed. Corrected residual percentage of intestinal hormones.

division Control (-) DWF1 (Tween20) DWF2 (Microcol-15 Polyoxystearate) DWF3 (Cremophor ELP) DWF4 (Tween 80) DWF5 (Poloxamer 188) Deamidation (% Somatropin) 8th 65.2 ± 0.5  81.9 ± 0.3  83.1 ± 0.3  83.4 ± 0.2 82.7 ± 0.2  83.1 ± 0.3 14 days 45.5 ± 0.4  69.1 ± 0.2  68.5 ± 0.4  69.1 ± 0.2 68.9 ± 0.2  69.6 ± 0.2 Coagulation (% Somatropin) 8th 98.0 ± 0.2 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 99.9 ± 0.1 100.0 ± 0.0 14 days 80.2 ± 0.3  99.4 ± 0.1  99.1 ± 0.1  99.6 ± 0.1 99.7 ± 0.1  99.5 ± 0.1

In order to prevent deamidation and aggregation, the addition of other surfactants compared to the control group prevented deamidation and aggregation, and it was confirmed that the use of the surfactant contributed to the stabilization of the liquid formulation of phosphorus growth hormone. .

Example 3 Investigation of Stability of Stabilizer and Phosphorus Growth Hormone

In order to investigate the stability of the liquid formulation of phosphorus growth hormone according to the stabilizer, the liquid formulation of phosphorus growth hormone was prepared and analyzed in the same manner as in Example 1 using the substances shown in Table 3.

division chief ingredient Buffer Tonicity Surfactants Stabilizer Preservative solvent Control Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Tween 20 2 mg - Benzyl alcohol 2.5 mg 1 mL water for injection DWF6 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Tween 20 2 mg PEG # 300 1 v / v% Benzyl alcohol 2.5 mg 1 mL water for injection DWF7 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Tween 20 2 mg PEG # 400 1 v / v% Benzyl alcohol 2.5 mg 1 mL water for injection DWF8 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Tween 20 2 mg PVP K-12 5 mg Benzyl alcohol 2.5 mg 1 mL water for injection DWF9 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Tween 20 2 mg PVP K-17 5 mg Benzyl alcohol 2.5 mg 1 mL water for injection DWF10 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Tween 20 2 mg L-LysHCl 1 mg Benzyl alcohol 2.5 mg 1 mL water for injection DWF11 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Tween 20 2 mg L-ArgHCl 1 mg Benzyl alcohol 2.5 mg 1 mL water for injection

The results for deamidation and aggregation according to the type of stabilizer of the liquid formulation prepared above are shown in Table 4 below.

division Deamidation (% Somatropin) Coagulation (% Somatropin) 7 days 14 days 7 days 14 days Control (-) 70.8 ± 0.2 52.0 ± 0.3 100.0 ± 0.0  97.0 ± 0.2 DWF6 (PEG 300) 83.7 ± 0.1 70.6 ± 0.2 100.0 ± 0.0  99.6 ± 0.1 DWF7 (PEG 400) 79.7 ± 0.1 66.9 ± 0.1 100.0 ± 0.0  99.3 ± 0.1 DWF8 (PVP K-12) 80.8 ± 0.2 69.0 ± 0.2 100.0 ± 0.0  99.5 ± 0.1 DWF9 (PVP K-17) 80.7 ± 0.2 69.0 ± 0.2 100.0 ± 0.0  99.4 ± 0.1 DWF10 (L-LysHCl) 83.3 ± 0.1 70.4 ± 0.1 100.0 ± 0.0 100.0 ± 0.0 DWF11 (L-AgnHCl) 83.5 ± 0.1 70.2 ± 0.1 100.0 ± 0.0 100.0 ± 0.0

As shown in Table 4, it was confirmed that PEG 300 (polyethylene glycol 300) and amino acids L-Lys.HCl, L-Agn.HCl among the various stabilizers have an effect on the stability of phosphorus growth hormone, especially L The relative aggregation inhibitory effect was excellent when lysine and L-arginine were added.

Example 4 Investigation of Stability of Buffer and Phosphorus Growth Hormone

The effects of various buffers on polyethylene glycol 300 (PEG 300), arginine or lysine containing liquid formulations, which were found to contribute to the stabilization of liquid formulations of phosphorus growth hormone in accordance with Example 3, were investigated.

A liquid formulation of phosphorus growth hormone was prepared in the same manner as in Example 1 using the material shown in Table 5 as a component.

division chief ingredient Buffer Tonicity Surfactants Stabilizer Preservative solvent DWF12 Somatropin 3.3 mg Sodium acetate 10 mM D-mannitol 50 mg Tween 20 2 mg PEG 300 1 v / v% Benzyl alcohol 2.5 mg 1 mL water for injection DWF13 Somatropin 3.3 mg Sodium citrate 10 mM D-mannitol 50 mg Tween 20 2 mg PEG 300 1 v / v% Benzyl alcohol 2.5 mg 1 mL water for injection DWF14 Somatropin 3.3 mg Sodium Phosphate 10 mM D-mannitol 50 mg Tween 20 2 mg PEG 300 1 v / v% Benzyl alcohol 2.5 mg 1 mL water for injection

The stability according to the type of buffer of the prepared liquid formulation was confirmed by deamidation formation and dimer / polymer formation over time at 40 ° C., and the residual ratio of the active form is shown in Table 6.

division Deamidation (% Somatropin) Coagulation (% Somatropin) 7 days 14 days 7 days 14 days DWF12 (Sodium Acetate) 83.7 ± 0.1 70.6 ± 0.2 100.0 ± 0.0 99.27 ± 0.3 DWF13 (Sodium Citrate) 82.7 ± 0.5 70.9 ± 0.1 100.0 ± 0.0 99.62 ± 0.1 DWF14 (Sodium Phosphate) 80.8 ± 0.4 69.0 ± 0.3 100.0 ± 0.0 99.12 ± 0.4

Existing known patents (US Patent Application No. 6,448,225, Republic of Korea Patent Application No. 10-1999-0001217, Republic of Korea Patent Application No. 10-1998-0052483) describe the importance of the buffer, but according to the table The liquid formulation of the present invention showed little difference in stability depending on the type of buffer used in the existing products.

Example 5 Comparison of Stability of Liquid Formulations

In order to compare the stability of the liquid formulation of the present invention, the liquid formulations of the control groups 1 and 2 were compared with the liquid formulations of phosphorus growth hormone prepared in the same manner as in Example 1 using the substances shown in Table 7 as components. .

division chief ingredient Buffer Tonicity Surfactants Stabilizer Preservative / Painless Agent solvent Control group 1 Somatropin 2.7 mg Sodium acetate D-mannitol Tween 20 - Benzyl alcohol 1 mL water for injection Control 2 Somatropin 3.3 mg D-mannitol Poloxamer 188 Histidine phenol 1 mL water for injection DWF15 Somatropin 2.5 mg Sodium acetate 10 mM D-mannitol 35 mg MicroCol-15 Polyoxystearate 2 mg PEG # 300 1 v / v% Benzyl alcohol 2.5 mg 1 mL water for injection DWF16 Somatropin 5.0 mg Sodium acetate 10 mM D-mannitol 30 mg MicroCol-15 Polyoxystearate 2 mg PEG # 300 2 v / v% Benzyl alcohol 2.5 mg 1 mL water for injection DWF17 Somatropin 2.5 mg Sodium acetate 10 mM D-mannitol 38 mg Poloxamer 188 2 mg L-LysHCl 1 mg Benzyl alcohol 2.5 mg 1 mL water for injection DWF18 Somatropin 5.0 mg Sodium acetate 10 mM D-mannitol 38 mg Poloxamer 188 2 mg L-LysHCl 2 mg Benzyl alcohol 2.5 mg 1 mL water for injection DWF19 Somatropin 2.5 mg Sodium acetate 10 mM D-mannitol 38 mg Poloxamer 188 2 mg L-ArgHCl 1mg Benzyl alcohol 2.5 mg 1 mL water for injection DWF20 Somatropin 5.0 mg Sodium acetate 10 mM D-mannitol 38 mg Poloxamer 188 2 mg L-ArgHCl 2mg Benzyl alcohol 2.5 mg 1 mL water for injection

The liquid formulation of the phosphorus growth hormone prepared above was stored at 40 ° C. and 75 RH% for 14 days, and the residual ratio of somatropin was confirmed using SEC-HPLC. In addition, the properties were confirmed after horizontal stirring (amplitude 20 mm, 220 times / min) at 2 to 8 ° C. for 24 hours. The results are shown in Table 8.

division 40 ° C, 75 RH% conditions After stirring for 24 hours Cohesion Cohesion Constellation 14 days (Somatropin%) 14 days (Somatropin%) Clear and transparent (X), slightly produced (△), cloudy (O), very large aggregates generated (OO) Control group 1  97.00 ± 0.15 94.57 ± 0.05 OO Control 2  99.11 ± 0.05 96.26 ± 0.07 O DWF15  99.99 ± 0.01 99.99 ± 0.01 X DWF16  98.22 ± 0.06  97.01 ± 0.15 O DWF17 100.00 ± 0.00 100.00 ± 0.00 X DWF18  99.98 ± 0.01  99.97 ± 0.02 X DWF19 100.00 ± 0.00 100.00 ± 0.00 X DWF20 100.00 ± 0.00 99.98 ± 0.01 X

According to the results, the control group 1, which contains no stabilizer at all, was the most agglomerated, and the control group 2 using histidine alone as a stabilizer was the second largest aggregate. However, the liquid formulation of the present invention did not generate any aggregates except DWF16. This result indicates that a stable phosphorus growth hormone liquid formulation can be prepared by combining a specific amino acid of L-lysine or L-arginine or a specific stabilizer of polyethylene glycol 300 with a specific surfactant.

In addition, the residual ratio of somatropin with time change at 25 ° C. for 3 months and the residual rate of somatropin with time change at 2 to 8 ° C. over 1 year are shown in Table 9 and FIGS. 1 and 2.

division 25 ° C (3 months) 2 to 8 ° C (1 year) Deamidation (% Somatropin) Coagulation (% Somatropin) Deamidation (% Somatropin) Coagulation (% Somatropin) DWF15 92.34 ± 0.20  98.24 ± 0.08 94.56 ± 0.12  99.78 ± 0.02 DWF16 93.15 ± 0.15  98.45 ± 0.06 95.64 ± 0.05  99.43 ± 0.03 DWF17 95.26 ± 0.19 100.00 ± 0.00 98.06 ± 0.05 100.00 ± 0.00 DWF18 94.56 ± 0.18  99.93 ± 0.03 99.17 ± 0.09 100.00 ± 0.00

As can be seen from Table 9 and Figures 1 and 2, the liquid formulation of the present invention can be stored for a long time at a low temperature to maintain stability for at least one year.

In order to overcome the problems of the liquid preparation of phosphorus growth hormone, the present invention has a high stability against deamidation formation of phosphorus growth hormone by using a combination of a specific stabilizer and a specific surfactant. It provides a liquid formulation that is very stable against external stress during distribution and storage.

Claims (11)

Phosphorus growth hormone; L-lysine or L-arginine; And poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil. The liquid formulation of claim 1, wherein L-lysine or L-arginine is a hydrochloride salt. The liquid formulation of claim 1, wherein the poly (oxyethylene) poly (oxypropylene) copolymer is poloxamer (poly (oxyethylene) poly (oxypropylene) copolymer) 188. The method of claim 1, wherein the amount of phosphorus growth hormone is 2.5 to 5.5 mg / ml; The amount of L-lysine or L-arginine is 0.02 to 0.5 w / v% per mg of growth hormone; A liquid formulation wherein the amount of poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil is 0.1 to 0.5 w / v%. The liquid formulation of claim 1, further comprising one or more components selected from buffers, isotonic agents, preservatives, and analgesics. The method of claim 5, wherein the amount of phosphorus growth hormone is 2.5 to 5.5 mg / ml; The amount of L-lysine or L-arginine is 0.02 to 0.5 w / v% per mg of growth hormone; The amount of poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil is 0.1 to 0.5 w / v%; The amount of sodium citrate or sodium acetate is 5 to 20 mM; The amount of D-mannitol is 20-50 mg / ml; A liquid formulation wherein the amount of benzyl alcohol is 1 to 9 mg / ml. Phosphorus growth hormone; Polyethylene glycol 300; And poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil. The liquid formulation of claim 7 wherein the poly (oxyethylene) poly (oxypropylene) copolymer is poloxamer (poly (oxyethylene) poly (oxypropylene) copolymer) 188. 8. The method according to claim 7, wherein the amount of phosphorus growth hormone is 2.5 to 5.5 mg / ml; The amount of polyethylene glycol 300 is 0.2 to 1.0 v / v% per 1 mg of phosphorus growth hormone; A liquid formulation wherein the amount of poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil is 0.1 to 0.5 w / v%. 8. The liquid formulation of claim 7, further comprising at least one component selected from buffers, isotonic agents, preservatives and analgesics. The method of claim 10, wherein the amount of phosphorus growth hormone is 2.5 to 5.5 mg / ml; The amount of polyethylene glycol 300 is 0.2 to 1.0 v / v% per 1 mg of phosphorus growth hormone; The amount of poly (oxyethylene) poly (oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethyleneglycol-35 castor oil is 0.1 to 0.5 w / v%; The amount of sodium citrate or sodium acetate is 5 to 20 mM; The amount of D-mannitol is 20-50 mg / ml; A liquid formulation wherein the amount of benzyl alcohol is 1 to 9 mg / ml.

Images