BOTULINUM TOXIN PRE-FILLED INJECTABLE FORMULATION THAT FACILITATES DISCHARGE RATE CONTROL AND IS STABLE

Abstract

Provided is a syringe formulation that is pre-filled with botulinum toxin, which allows easy control of a discharge rate and may be stable.

Claims

1. A syringe formulation pre-filled with botulinum toxin, comprising: a syringe comprising a syringe barrel, a plunger rod, and a plunger stopper; and a botulinum toxin liquid preparation filled in the syringe, wherein an amount of increase of a force acting on an end of the plunger rod decreases as a rate of discharge of the botulinum toxin liquid preparation increases.

2. The syringe formulation of claim 1, wherein a ratio of a length to an inner diameter of the syringe barrel is 10 to 22.

3. The syringe formulation of claim 2, wherein the inner diameter of the syringe barrel is 3.5 mm to 6.5 mm, and the length of the syringe barrel is 60 mm to 100 mm.

4. The syringe formulation of claim 1, wherein a material of the syringe barrel is glass, COC, or COP.

5. The syringe formulation of claim 1, wherein a material of the plunger stopper is isoprene rubber (IS), butadiene rubber (BR), butyl rubber, halogenated butyl rubber, styrene-butadiene rubber, or a mixture thereof.

6. The syringe formulation of claim 1, wherein the botulinum toxin liquid preparation does not contain albumin.

7. The syringe formulation of claim 1, wherein the botulinum toxin liquid preparation does not contain any animal component.

8. The syringe formulation of claim 6, wherein the botulinum toxin liquid preparation comprises botulinum toxin, an amino acid, a surfactant, and an isotonic agent.

9. The syringe formulation of claim 8, wherein the amino acid is methionine.

10. The syringe formulation of claim 8, wherein the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or poloxamer.

11. The syringe formulation of claim 6, wherein the botulinum toxin liquid preparation further contains a buffer to maintain a pH of 5.5 to 7.5.

12. The syringe formulation of claim 11, wherein the buffer is citrate, histidine, HEPES, arginine, acetic acid, phosphoric acid, a salt thereof, or a mixture thereof.

13. The syringe formulation of claim 1, wherein, upon storage for 2, 4, or 6 months at 25° C., which is an accelerated test condition, the botulinum toxin liquid preparation shows an LD.sub.50 titer recovery rate of 80% to 125% relative to an initial value.

14. The syringe formulation of claim 1, wherein, upon storage for 2, 4, or 6 months at 25° C., which is an accelerated test condition, the botulinum toxin liquid preparation shows a protease activity recovery rate of 80% to 125% relative to an initial value.

15. The syringe formulation of claim 1, wherein, upon storage for 2, 4, or 6 months at 25° C., which is an accelerated test condition, the botulinum toxin liquid preparation shows a pH change of ±1.0 or less relative to an initial value.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0062] FIG. 1 is a view showing an embodiment of a pre-filled syringe including a needle.

[0063] FIG. 2 is a view showing an embodiment of a pre-filled syringe including a tip cap.

[0064] FIG. 3 is a view showing an embodiment of a pre-filled syringe including a needle assembly and a needle shield.

MODE OF DISCLOSURE

[0065] Hereinafter, the present disclosure will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the present disclosure is not intended to be limited by these Examples.

Example 1: Stability of Botulinum Toxin Pre-Filled Liquid Syringe Formulation: Syringe Having a Barrel Made of COC or COP Material

[0066] 1. Preparation of Botulinum Toxin Pre-Filled Liquid Preparation

[0067] In this Example, a syringe (hereinafter referred to as an ‘HVD syringe’) was filled with a liquid botulinum toxin preparation for use in a highly viscous drug (HVD), and stability according to the storage period was measured. The HVD syringe had an inner diameter of 5.00 mm, an outer diameter of 9.40 mm, a barrel length of 80.0 mm, and a volume filled with a liquid preparation of 0.8 mL. For reference, as an example of a known syringe, TopPac™ syringe available from SCHOTT has an inner diameter of 6.50 mm, an outer diameter of 9.40 mm, a barrel length of 64.5 mm, and a volume filled with a liquid preparation of 1 mL. That is, the HVD syringe had, as compared with TopPac™ syringe, a small inner diameter and a long length. TopPac™ syringe included a barrel, a plunger, a plunger rod, and a tip cap, and the barrel was made of a COC material, and a luer-lock was formed. The inner surface of the barrel was siliconized with a reactive silicone mixture and then crosslinked by curing. The tip cap was made of rubber (Datwyler Corporation). The HVD syringe included a COC material made of TOPAS 6015 material and had improved heat resistance.

[0068] In addition, in the HVD syringe, stability according to the material of the barrel, the material of the plunger stopper and the material used for the coating thereof, and the composition of the aqueous liquid botulinum toxin preparation filled in the barrel were measured. Here, the syringe included a plastic syringe barrel, a capping device, and a plunger load assembly. The barrel included a proximal end and a distal end, and a wall that may extend therebetween and was generally cylindrical to form a barrel lumen. The barrel had a tip protruding to the distal having a fluid passage extending therethrough and in communication with the barrel lumen, and the wall which was generally cylindrical optionally had an interior surface coated with a barrier layer. In this embodiment, the inner surface of the wall was coated with silicone, that is, the barrier layer was a silicone layer. In addition, in this embodiment, the barrel has a COC or COP material.

[0069] The capping device had an outlet coupling portion that sealingly couples and closes the open outlet of the distal end of the barrel. The capping device for the distal end of the barrel consists of a cap and a rubber tip cap, the cap was made of polycarbonate, and the rubber tip cap was made of the same material as the plunger. Here, the rubber tip cap corresponds to the outlet coupling portion. The outlet coupling portion was made of an elastomer material optionally having a coating on the surface. The outlet coupling portion and the capping device are coupled in the form of a lure-lock.

[0070] The plunger rod assembly included a plunger stopper extending into the proximal end of the barrel and having sliding fluid-tight engagement with the cylindrical wall of the barrel lumen, wherein the stopper was made of an elastomer material and optionally had a coating on at least a portion of the stopper in contact with the aqueous liquid botulinum toxin preparation during storage and/or injection. In this embodiment, the material of the stopper was bromo butyl rubber (BIIR) or chlorobutyl rubber (CIIR), which is optionally coated with Teflon.

[0071] Table 1 shows a botulinum toxin pre-filled syringe preparation of a liquid botulinum toxin preparation in water pre-filled used in this Example.

TABLE-US-00001 TABLE 1 Formulation name A B C D E F G H Barrel COC COC COC COC COC COC COP COP material Plunger BIIR BIIR BIIR BIIR CIIR CIIR CIIR CIIR stopper material Plunger Not Not Teflon Teflon Not Not Teflon Teflon stopper progressed progressed progressed progressed coating L-Met (g/L) 0.1 0.2 0.1 0.2 0.1 0.2 0.1 0.2 T20 (g/L) 0.3 0.15 0.30 0.15 0.3 0.15 0.3 0.15 NaCl (g/L) 9 9 9 9 9 9 9 9 Toxin 40 40 40 40 40 40 40 40 concentration (U/mL) Water-filling 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 volume (mL)

[0072] In Table 1, COC represents cyclic olefin copolymer, and COP represents a cyclic olefin polymer. BIIR and CIIR represent bromobutyl rubber and chlorobutyl rubber (IIR), respectively. The plunger stopper material represents the material of the rubber part. Here, the butyl rubber (IRR) is a copolymer of isobutylene and isoprene. Teflon coating indicates a coating of FluroTecR film on the BIIR or CIIR surface. L-Met, T20, NaCl, and toxin respectively represent the amount of L-methionine, polysorbate 20, NaCl, and botulinum toxin type A component dissolved in water, i.e., injectable water.

[0073] The syringe preparation was to couple the plunger rod assembly into the lumen of the barrel such that the stopper of the plunger sealed the liquid preparation while in contact with the liquid preparation. In addition, the outlet coupling portion of the distal end of the barrel was sealed with the capping device to close the open outlet. The syringe and the capping device were integral, and the capping device was removed immediately before use. At this time, the liquid comes into contact with the rubber tip cap inside the capping device.

[0074] That is, the pre-filled syringe botulinum liquid preparation was in a state in which the syringe was not coupled to the needle and bound to the capping device.

[0075] As a result, the material of the syringe barrel was selected from COC and COP, and the stopper of the plunger was selected from bromo butyl rubber and chlorobutyl rubber and a Teflon coating thereof in 4 syringes in which concentrations of methionine and polysorbate 20 were varied twice, to prepare a total of 8 pre-filled syringe botulinum liquid preparations.

[0076] 2. Long-Term Stability Test of Pre-Filled Syringe Formulation

[0077] (1) Titer Test

[0078] The eight prepared formulations, i.e., Formulations A to H, were stored at 4° C. in a stable sample storage room to measure half lethal doses (LD.sub.50) of the mice at 0, 3, 6, 9, 12, 18, and 24 months.

[0079] The liquid samples of the 8 formulations were each serial diluted and administered to the abdominal cavity of 4-week-old female ICR mice weighing 17 g to 22 g. The fatality rate for each diluted sample was observed for 3 days after administration, and the lethal doses 50 were obtained using the statistical program PROBIT. The half lethal doses at 0 months was set to 100%, and the tendency to change in half lethal doses obtained at each point in time was expressed in %. Table 2 shows half lethal doses according to the storage period of Formulations A, B, C, D, E, F, G, and H.

TABLE-US-00002 TABLE 2 LD.sub.50 (recovery lethal dose with respect to initial value, %) Tested periods (months) Name 0 3 6 9 12 18 24 A 100 99 94 89 83 77 75 B 100 96 90 90 92 89 85 C 100 96 75 95 86 76 73 D 100 102 75 84 86 77 77 E 100 100 73 91 97 79 77 F 100 96 76 80 80 78 78 G 100 97 99 — — — — H 100 79 92 — — — —

[0080] As shown in Table 2, the recovery lethal dose of Formulations A to F at 24 months was measured at least 73%, but the recovery lethal dose of Formulations G and H were not measured from 9 months. This indicates that when the barrel is made of a COC material and coated with silicone, regardless of whether the plunger stopper material is BIIR or CIIR or whether the surface of the plunger stopper is Teflon-coated, the botulinum toxin is stable for 24 months. When the barrel material is COP, the recovery titer was not measured in the syringe after 9 months.

[0081] (2) Botulinum Toxin Endopeptidase Titer Analysis

[0082] The eight pre-filled syringe Formulations A to H were stored in a stability chamber to measure activity of BoNTA/LC at 0, 3, 6, 9, 12, 18, and 24 months.

[0083] 100 μL of SNAP25 (self-produced) fragments consisting of 70 amino acid sequences were added to each well of the 96-well plate and incubated at 2° C. to 8° C. for 16 hours. The SNAP25 fragment includes 10 or more consecutive amino acid sequences, including the cleavage site between amino acids No. 197 and No. 198.

[0084] BoNTA samples, which are standard samples from which quantitative curves may be obtained using BoNTA standards (Innotox available from Medytox), were prepared, and the test samples were prepared by diluting BoNTA in HEPES solution (available from Sigma) so that the result values of the Formulations A to H samples could be included in the quantitative curve range. The standard sample and each of the test samples A to H were placed in a well containing SNAP25 and reacted at room temperature for 1 hour. Next, an anti-SNAP25 polyclonal antibody (self-produced) was added to each well to react at room temperature for 1 hour. The HRP-bound secondary antibody was reacted at room temperature for 1 hour, and then the color reagent 3,3′,5,5′-tetramethylbenzidine (TMB) was added and incubated to induce a color reaction.

[0085] The absorbance was measured at 450 nm in a plate reader (Microplate Reader available from TECAN) to quantify the degree of color development according to the activity of BoNT/LC, and the quantitative curve was obtained using the absorbance value of the standard sample. The absorbance values of Samples A to H were substituted into the quantitative curve to calculate the activity of BoNT/LC of each sample. The activity of BoNT/LC at 0 months was set to 100%, and the tendency to change in activity of BoNT/LC obtained at each point in time was expressed in %. Table 3 shows the endopeptidase activity of BoNTA included in Formulations A to H according to the storage period.

TABLE-US-00003 TABLE 3 Protease activity (recovered activity relative to initial value, %) Tested periods (months) Name 0 3 6 9 12 18 24 A 100 106 109 103 121 123 141 B 100 102 105 103 119 115 130 C 100 107 111 101 127 107 124 D 100 100 104 94 121 101 118 E 100 99 107 92 119 99 111 F 100 108 104 96 130 111 130 G 100 107 112 — — — — H 100 100 109 — — — —

[0086] As shown in Table 3, the recovery titers of Formulations A to F at 24 months was maintained 111% to 141%, but the recovery titers of Formulations G and H were not measured from 9 months.

[0087] (3) pH Stability

[0088] At 0, 3, 6, 9, 12, 18, and 24 months of the eight pre-filled syringe Formulations A to H, each sample was placed in a 15 mL disposable tube to be at least 3 mL, and the pH of each sample was measured by using a pH meter (available from Mettler Toledo). Table 4 shows the pH changes of Formulations A to H with the storage period.

TABLE-US-00004 TABLE 4 pH Tested periods (months) Name 0 3 6 9 12 18 24 A 6.7 6.9 7.0 7.3 7.2 7.3 7.4 B 6.8 7.1 7.1 7.3 7.3 7.3 7.3 C 6.0 6.5 6.6 6.6 6.5 6.5 6.8 D 6.1 6.5 6.5 6.5 6.5 6.6 6.8 E 6.3 6.6 6.5 6.6 6.5 6.7 6.8 F 6.2 6.6 6.5 6.6 6.7 6.7 6.6 G 5.9 5.7 5.5 — — — — H 5.9 5.6 5.3 — — — —

[0089] As shown in Table 4, the pH of Formulations A to F were maintained between 6.8 and 7.4 at 24 months, but the pH of Formulations G and H were maintained between 5.3 to 5.9 at 6 months when activity was measured.

Example 2: Stability of Botulinum Toxin Pre-Filled Liquid Syringe Formulation: Syringe Having a Barrel Made of Glass or COP Material

[0090] 1. Preparation of Botulinum Toxin Pre-Filled Liquid Preparation

[0091] Botulinum toxin pre-filled liquid preparations as shown in Table 5 were prepared. Here, the syringe was the HVD syringe shown in Table 1, which had an inner diameter of 5.00 mm, an outer diameter of 9.40 mm, a barrel length of 80.0 mm, and a volume filled with a liquid preparation of 0.8 mL.

TABLE-US-00005 TABLE 5 Formulation name I J K L M N Barrel material Glass Glass COP COP COP COP Plunger stopper BIIR BIIR BIIR BIIR i-coating i-coating material L-Met (g/L) 0.05 0.1 0.05 0.1 0.05 0.1 T20 (g/L) 0.3 0.3 0.3 0.3 0.3 0.3 NaCl (g/L) 9 9 9 9 9 9 Toxin 40 40 40 40 40 40 concentration (U/mL) Water-filling 0.8 0.8 0.8 0.8 0.8 0.8 volume (mL/syringe)

[0092] In Table 5, the barrel of Formulations I and J was available from POONGLIM Pharmatech Inc. (catalog number, Art. 69, Korea), and the barrel of Formulations K and L was SiO.sub.2 Medical Products Inc. (Catalog No., 850009-100-04, USA), and the barrel of Formulations M and N was a product of Terumo Corporation (Catalog No., PJ-B1L9BFTF1, Japan). The botulinum toxin was BTX1301 (API batch number) from Medytox, BoNTA. In Table 5, COC represents cyclic olefin copolymer, and COP represents a cyclic olefin polymer. BIIR and CIIR represent bromobutyl rubber and chlorobutyl rubber (IIR), respectively. Here, the butyl rubber (IRR) is a copolymer of isobutylene and isoprene. L-Met, T20, NaCl, and toxin respectively represent the amount of L-methionine, polysorbate 20, NaCl, and botulinum toxin type A component dissolved in water. The liquid containing these components had a pH of 6.0 to 7.0.

[0093] The preparation was to couple the plunger rod assembly into the lumen of the barrel such that the stopper of the plunger sealed the liquid preparation while in contact with the liquid preparation. In addition, the outlet coupling portion of the distal end of the barrel was sealed with the capping device to close the open outlet. That is, the pre-filled syringe botulinum liquid preparation was in a state in which the syringe was not coupled to the needle and bound to the capping device.

[0094] As a result, the material of the syringe barrel was selected from glass and COP, and the stopper of the plunger was selected from bromo butyl rubber and i-coating in 3 syringes in which concentrations of methionine and polysorbate 20 were varied twice, to prepare a total of 6 pre-filled syringe botulinum Liquid Preparations I to N.

[0095] 2. Stability Test of Pre-Filled Syringe Formulation I to N

[0096] (1) Titer Test

[0097] The prepared 6 formulations, i.e., Formulations I to N samples, were stored at 25° C. in a thermohygrostat (available from Binder Inc.) for experimentation under accelerated test conditions. For long-term storage tests, the sample was stored at 4° C. in a stable sample storage room. The half lethal dose of mice at 0, 2, 3, 4, and 6 months after storage (LD.sub.50) was measured.

[0098] The liquid samples of the 6 formulations were each serial diluted and administered to the abdominal cavity of 4-week-old female ICR mice weighing 17 g to 22 g. The fatality rate for each diluted sample was observed for 3 days after administration, and the lethal doses 50 were obtained using the statistical program PROBIT. The half lethal doses at 0 months was set to 100%, and the tendency to change in half lethal doses obtained at each point in time was expressed in %. Table 6 shows half lethal doses according to the storage period of Formulations I, J, K, L, M, and N.

TABLE-US-00006 TABLE 6 LD.sub.50 (recovery lethal dose with respect to initial value, %) Tested periods (months) Name 0 2 3 4 6 I Accelerated 100  97 — 87 89 Long-term 100 — 93 — 94 J Accelerated 100  98 — 86 89 Long-term 100 — 89 — 94 K Accelerated 100 100 — 90 97 Long-term 100 — 92 — 97 L Accelerated 100 105 — 97 102 Long-term 100 — 100  — 108 M Accelerated 100 100 — 92 91 Long-term 100 — 96 — 102 N Accelerated 100  93 — 85 86 Long-term 100 — 90 — 93

[0099] As shown in Table 6, at 6 months, the titer of each of the six pre-filled syringe Formulations I to N maintained in the range of 86% to 108%, as compared with the initial titer.

[0100] (2) Botulinum Toxin Endopeptidase Titer Analysis

[0101] The six pre-filled syringe Formulations I to N were stored in a stability chamber to measure activity of BoNTA/LC at 0, 2, 3, 4, and 6 months.

[0102] 100 μL of SNAP25 (self-produced) fragments consisting of 70 amino acid sequences were added to each well of the 96-well plate and incubated at 2° C. to 8° C. for 16 hours. The SNAP25 fragment includes 10 or more consecutive amino acid sequences, including the cleavage site between amino acids No. 197 and No. 198.

[0103] BoNTA samples, which are standard samples from which quantitative curves may be obtained using BoNTA standards (Innotox available from Medytox), were prepared, and the test samples were prepared by diluting BoNTA in HEPES solution (available from Sigma) such that the resulting values of the Formulations A to H samples were included in the quantitative curve range. The standard sample and each of the test samples A to H were placed in a well containing SNAP25 and reacted at room temperature for 1 hour. Next, an anti-SNAP25 polyclonal antibody (self-produced) was added to each well to react at room temperature for 1 hour. The HRP-bound secondary antibody was reacted at room temperature for 1 hour, and then the color reagent 3,3′,5,5′-tetramethylbenzidine (TMB) was added and incubated to induce a color reaction.

[0104] The absorbance was measured at 450 nm in a plate reader (Microplate Reader available from TECAN) to quantify the degree of color development according to the activity of BoNT/LC, and the quantitative curve was obtained using the absorbance value of the standard sample. The absorbance values of Samples I to N were substituted into the quantitative curve to calculate the activity of BoNT/LC of each sample. The activity of BoNT/LC at 0 months was set to 100%, and the tendency to change in activity of BoNT/LC obtained at each point in time was expressed in %. Table 7 shows the endopeptidase activity of BoNTA included in Formulations I to N according to the storage period.

TABLE-US-00007 TABLE 7 Protease activity (recovered activity relative to initial value, %) Tested periods (months) Name 0 2 3 4 6 I Accelerated 100 81 — 54 53 Long-term 100 — 79 — 80 J Accelerated 100 68 — 67 65 Long-term 100 — 73 — 83 K Accelerated 100 76 — 63 70 Long-term 100 — 84 — 83 L Accelerated 100 78 — 82 78 Long-term 100 — 85 — 90 M Accelerated 100 82 — 79 81 Long-term 100 — 92 — 96 N Accelerated 100 86 — 83 84 Long-term 100 — 92 — 94

[0105] As shown in Table 7, the recovery titers of Formulations I to N at 6 months remained at 53% to 94%, as compared to the initial value.

[0106] (3) pH Stability

[0107] At certain points in time of the six pre-filled syringe Formulations I to N, each sample was placed in a 15 mL disposable tube to be at least 3 mL, and the pH of each sample was measured by using a pH meter (available from Mettler Toledo). Table 8 shows the pH changes of Formulations I to N with the storage period. The point in time was 0, 2, 4, and 6 months for the accelerated condition experiment, and 0, 3, and 6 months for the long-term preservation experiment.

TABLE-US-00008 TABLE 8 pH Tested periods (months) Name 0 2 3 4 6 I Accelerated 6.5 7.8 7.3 7.9 Long-term 6.5 7.6 7.7 J Accelerated 6.7 7.8 7.4 7.8 Long-term 6.7 7.6 7.8 K Accelerated 6.6 7.6 7.2 7.6 Long-term 6.6 7.4 7.5 L Accelerated 6.5 7.5 7.1 7.4 Long-term 6.5 7.2 7.4 M Accelerated 6.2 7.4 6.7 7.3 Long-term 6.2 7.2 7.3 N Accelerated 5.9 7.3 6.7 7.1 Long-term 5.9 7.1 7.1

[0108] As shown in Table 8, the pH of Formulations I to N was maintained at 7.1 to 7.9 until 6 months.

Example 3: Stability of Botulinum Toxin Pre-Filled Liquid Syringe Formulation: Effects of Albumin

[0109] 1. Preparation of Botulinum Toxin Pre-Filled Liquid Preparation

[0110] As shown in Table 9, Formulation O containing human serum albumin and not containing L-Met and polysorbate 20 and Formulation P containing L-Met and polysorbate 20 and not containing human serum albumin were prepared. In Table 9, the solvent was water, and Toxin was a BoNTA type available from Medytox Corporation.

[0111] The HVD syringes were filled with Formulations O and P. Here, the HVD syringe is Syringe B in Table 1, the barrel was made of a COC material, and the plunger material was made of siliconized BIIR.

TABLE-US-00009 TABLE 9 NaCl L-Met Tween20 HSA Toxin Name 0.9% 0.02% 0.015% 0.02% 30 Unit) O + − − + + P + + + − +

[0112] 2. Titer Test: LD50 (Accelerated Test)

[0113] Syringe formulations with which a syringe was pre-filled with the two prepared types, i.e., samples of Formulations O and P, were stored in a stability chamber, and the half lethal dose (LD.sub.50) of mice at 0, 2, 3, 7, 14, 28, and 56 days was measured.

[0114] Samples 1 and 2 were each serial diluted in water and administered to the abdominal cavity of 4-week-old female ICR mice weighing 17 g to 22 g. The fatality rate for each diluted sample was observed for 3 days after administration, and the lethal doses 50 were obtained using the statistical program PROBIT. The half lethal doses at 0 months was set to 100%, and the tendency to change in half lethal doses obtained at each point in time was expressed in %. The shelf life was calculated using the Arenius equation (https://met.uk.com/medical-device-packaging-testing/4a-medical-accelerated-ageing), which shows the relationship between the reaction rate constant and temperature.

[0115] Table 10 shows the storage stability of Formulation O containing albumin and the storage stability of Formulation P not containing albumin are shown in LD.sub.50 values.

TABLE-US-00010 TABLE 10 Stability test period (days) 0 2 5 7 14 28 56 LD50 (%) STD 97 97 103 101 103 105 96 O 92 68 50 48 25 N/A N/A P 109 98 91 94 86  86 52 Normalization O 100 74 55 53 28 N/A N/A P 100 90 84 87 79  79 48 Valid period (days) 0 30 60 90 170 340 680 

[0116] In Table 10, STD represents Meditoxin (available from Medytox) as the standard sample, normalization represents relative titers when the data value is set to 100 at day 0, and N/A represents that the experiment was not performed because the test result LD.sub.50 value in the previous cycle was very low at 20%, and it is not meaningful to proceed with the test. At 56 days of testing, that is, about 2 months, of three formulations prepared, i.e., the standard sample (STD), Formulation O, and Formulation P, under accelerated conditions, the recovery titer was lost at 28 days of testing in the case of Formulation O containing albumin, whereas the recovery titer of Formulation P not containing albumin was maintained at 86% at 28 days and at 52% at 56 days. The accelerated conditions were by storing the samples at 40° C. and recovering the samples at the time of 0, 2, 5, 7, 14, 28, and 56 days.

[0117] 3. Injection force test

[0118] Formulation O containing albumin and Formulation P not containing albumin as shown in Table 9 were respectively filled in 1 mL of SHOTT TopPac syringe and HVD syringe, i.e., B syringe in Table 1, as described in Section 1 of Example 1, thereby preparing four types of formulations, i.e., pre-filled syringe botulinum Liquid Preparations Q, R, S, and T.

[0119] For the four types of formulations, a MultiTest 2.5 (Mecmesin, UK) tensile compression apparatus was used to measure a plunger injection force according to the plunger movement rate of the syringe. The injection force is an average injection force.

[0120] Specifically, 30 G needles were first coupled to a syringe containing each of the 4 formulations. Next, the syringe was fixed to the jig of the tensile compression apparatus and adjusted such that the plunger rod was centered on the load cell of the apparatus. The jig is a syringe fixing part of the apparatus.

[0121] After setting the measuring distance in consideration of the length of each syringe, the test was performed by entering the speed value in the program embedded in the apparatus and pressing the start button to activate the apparatus. In case of the HVD syringe, due to a small inner diameter and a long length, when filling with 1 mL, the contents were filled to a height of about 40 mm, and in the case of the TopPac syringe, when filling with 1 mL, the contents were filled to a height of about 30 mm. Thus, when measuring the injection force, the measuring distance was set to have a margin of 5 mm. Thus, the measuring distance for the HVD syringe was from 0 mm to 35 mm, and the measuring distance for the TopPac syringe was from 0 mm to 25 mm, to press the plunger. The sample that was measured was removed from the jig and measured 3 times repeatedly per sample to obtain the injection force result. Table 11 shows the injection force according to the plunger movement rate.

TABLE-US-00011 TABLE 11 Name Q R S T Preparation O O P P Syringe type TopPac HVD TopPac HVD 1 mlL 1 mL 1 mL 1 mL Average  10 mm/min 1.45 1.68 1.13 1.35 plunger  50 mm/min 2.72 2.72 2.52 2.53 injection 100 mm/min 4.24 3.79 3.08 3.30 force (N) 200 mm/min 6.25 4.72 4.84 4.39

[0122] As shown in Table 11, Formulations R and T including the HVD syringes had the same or higher injection force, as compared with Formulations Q and S including the TopPac syringes at a low plunger movement rate of 10 and 50 mm/min, but the injection forces of Formulations R and T were lower than those of Formulations Q and S including the TopPac syringes at a high plunger movement rate of 100 and 200 mm/min. This indicates that, as compared with Formulations Q and S including the TopPac syringes, Formulations R and T including the HVD syringes have a high plunger compression force at a low plunger movement rate and have a low plunger compression force at a high plunger movement rate. Therefore, formulations including the HVD syringes may be finely manipulated at low speeds and easier to inject at high speeds.