WO2011152618A9 - 국소 근마비 효과를 갖는 비확산형 보툴리눔 독소와 그의 정제방법 - Google Patents
국소 근마비 효과를 갖는 비확산형 보툴리눔 독소와 그의 정제방법 Download PDFInfo
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- WO2011152618A9 WO2011152618A9 PCT/KR2011/003547 KR2011003547W WO2011152618A9 WO 2011152618 A9 WO2011152618 A9 WO 2011152618A9 KR 2011003547 W KR2011003547 W KR 2011003547W WO 2011152618 A9 WO2011152618 A9 WO 2011152618A9
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- botulinum toxin
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- 0 C**C*CO[N+](****O)[O-] Chemical compound C**C*CO[N+](****O)[O-] 0.000 description 1
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- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
- A61K38/4893—Botulinum neurotoxin (3.4.24.69)
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- A61K9/00—Medicinal preparations characterised by special physical form
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/24—Metalloendopeptidases (3.4.24)
- C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
Definitions
- the present invention relates to a method for purifying non-diffusion botulinum toxin and a non-diffusion botulinum toxin having a local myopic effect obtained thereby.
- Botox is widely used in the treatment of cerebral palsy patients, as well as in the treatment of rigid neck muscles, pain caused by facial muscle stiffness and vocal cord paralysis. In the field of beauty treatment, it is used for wrinkle removal and skin anti-aging treatment. Recently, as anti-aging medical treatment has become popular, botox usage is increasing every year.
- botulinum toxin purified by the above method has spread to the body other than the treatment site, causing paralysis of surrounding organs and respiratory muscles, and in severe cases, it is known to have serious side effects leading to death.
- the present invention is proposed in order to solve the above problems, three different heterogeneous subunits of the conventional botulinum toxin type A purified by acid chromatography after acid precipitation or after acid precipitation using anion ion exchange resin chromatography using sodium chloride. It was confirmed that the fraction exists, one of the subfractions were confirmed to be non-diffusion type in the body while having myopic activity.
- an object of the present invention is to provide a non-diffusion botulinum toxin and a method for isolating the non-diffusion type botulinum toxin having local myopic activity with local limitation.
- the method of purifying the non-diffusion botulinum toxin of the present invention the pH of the purified botulinum toxin type A formulation in the range of 4.5 to 6.5, by adjusting the concentration of sodium chloride (NaCl) to 0.02 to 0.2M Separating the subfraction by performing anion ion exchange resin chromatography added; And obtaining a non-diffusion botulinum toxin fraction in which the value of A260 / A280 is between 0.4 and 0.6 in the subfraction.
- NaCl sodium chloride
- the non-diffusion type botulinum toxin of the present invention has been separated and purified by the method described above, and at least 150 ppb of Zn ions, at least 80 ppb of Fe ions, and at least 140 ppb of Mg ions per 100 U / ml of the toxin. Characterized in that included.
- the non-diffusion botulinum toxin is injected into the lower right mouse mouse in an amount of 1.5 to 3 times its half lethal dose, Characterized by determining whether the respiratory muscles paralyzed and lethal.
- a non-diffusion botulinum toxin having a local myopathic effect is generated quickly and continuously, while the myopic effect is generated in a desired portion and the toxin is not diffused in other portions. have.
- anion chromatography conditions can be adjusted to obtain a large amount of non-diffusion botulinum toxin that is not mixed with the diffusive toxin.
- Example 1 is an ion resin chromatography result showing the result of separating the subfraction in Example 1.
- Figure 2 is a graph showing the change in subfraction peak% by the pH change of the buffer.
- 3 is a graph showing the change in each subfraction peak% according to the sample amount of the sample.
- FIG. 5 is a photograph comparing the body diffusivity of the non-diffusion active fraction (pII) in the subfraction separated by Example 1 with the conventional commercial product in the lower right of the mouse after 12 hours.
- Figure 6 shows the survival rate according to the dose (A) and time after administration (B) after administration of the non-diffusion active fraction (pII) in the subfraction separated by Example 1 and the existing commercial product in the lower right of the mouse It is a graph.
- Example 7 is a graph comparing the start time of myopathic effect after administration to the lower extremity of mice by non-diffusion active fraction (pII) in the subfraction separated by Example 1 and 1U of a conventional commercial product.
- pII non-diffusion active fraction
- FIG. 8 is a graph comparing the duration of myopathic effect after administration to the lower extremity of mice by non-diffusion active fraction (pII) in the subfraction separated by Example 1 and 0.5U of conventional commercial products.
- the present invention is to perform the anion chromatography in which the purified botulinum toxin type A preparation is adjusted by adjusting the concentration of sodium chloride (NaCl) to separate the subfraction and the value of A260 / A280 in the subfraction is 0.4 to 0.6 level. And obtaining a non-diffusion botulinum toxin fraction that maintains.
- NaCl sodium chloride
- Purification method of the non-diffusion botulinum toxin of the present invention is applied to the new anion ion exchange resin chromatography method proposed by the inventor of the present invention to the botulinum toxin obtained by using conventional acid precipitation method or ion precipitation chromatography after acid precipitation. To obtain a fraction of the non-diffusion botulinum toxin.
- the method for purifying the non-diffusion botulinum toxin of the present invention includes botulinum obtained by acid-precipitating a botulinum toxin type A preparation from Clostridium botulinum type A strain culture or by chromatography after acid precipitation and chromatography. Toxin type A preparations are used.
- the buffer used to perform the anion ion exchange resin chromatography is sodium acetate buffer, and each fraction is separated by sequentially increasing the sodium chloride concentration added to obtain a subfraction.
- sodium chloride was not used (concentration 0M NaCl)
- the fraction separated in the body was confirmed to be a diffusion type active fraction (peak I, pI)
- the fraction separated when the concentration of sodium chloride was 0.02 to 0.2 M was determined as non-diffusion in the body. It was confirmed that the active fraction (peak II, PII), the fraction separated when the concentration of sodium chloride to 1 M was confirmed to be a diffusion inactive fraction (peak III, pIII) in the body.
- the concentration of sodium chloride used to obtain the above pII is in the range of 0.02 to 0.2M, and when no sodium chloride is used or the concentration of sodium chloride is higher or lower than the above range, the pI or pIII fraction is mixed with the obtained subfraction. As such, it is difficult to obtain a non-diffusing active fraction having a local myopathic effect desired in the present invention.
- the amount increases. That is, when the volume of the column is 1 ml and the amount of purified botulinum toxin type A preparation sample is 0.2 to 1 ml, the fraction of pII obtained is expressed in the range of 20% to 50%.
- the pII fraction obtained by the method of purifying the non-diffusion botulinum toxin of the present invention was confirmed to be stable, such as the total lethal dose (total LD50) of 1 X 10 5 to 5 X 10 5 U / ml. It became.
- total lethal dose total LD50
- the onset of myotropic effect is significantly faster and the duration of myoplegia is long.
- the non-diffusion botulinum toxin (pII fraction) obtained by the purification method of the non-diffusion botulinum toxin of the present invention includes Zn ions of at least 150 ppb, Fe ions of at least 80 ppb, and Mg ions of at least 140 ppb per 100 U / ml of the toxin. do.
- the purification method of the non-diffusion botulinum toxin of the present invention was confirmed that the content of Mg, Fe and Zn is significantly higher than the botulinum toxin type A purified by the conventional method.
- the non-diffusion botulinum toxin obtained by the purification method of the present invention was found to have no paralysis or lethal activity of the respiratory muscles while paralyzing the lower limb muscles when injected into the lower limbs of mice in an amount of 1.5 to 3 times its half lethal dose. Thus, it is possible to determine whether or not the non-diffusion botulinum toxin of the present invention.
- Botox preparations of Allergan, Inc. (Lot #: manufactured by the method disclosed in USP 7,354,740 [Animal product free system and process for purifying a botulinum toxin]).
- C2066 C2, C2481 C2) or the present invention does not apply only to the botulinum toxin type A preparations purified by the commercially available products or methods provided herein, and may be sufficiently used as botulinum toxin type A preparations purified by acid precipitation or the like.
- C2066 C2, C2481 C2 or the present invention does not apply only to the botulinum toxin type A preparations purified by the commercially available products or methods provided herein, and may be sufficiently used as botulinum toxin type A preparations purified by acid precipitation or the like.
- FIG. 1 Three subfractions were separated by anion chromatography using a purified product of botulinum toxin culture by conventional acid precipitation method, and the separation results are shown in FIG. 1 [pI, Peak I (Diffusive activity in the body) Fractions); pII, Peak II (non-diffuse active fraction in the body); pIII, PeakIII (inactive fraction)].
- the instrument used for anion ion exchange chromatography is AKTA FPLC (GE Healthcare), the column used is Hitrap DEAE FF 1ml (GE healthcare, Cat # 17-5055-01), and the buffer used is a running buffer ( As a running buffer, sodium acetate buffer (pH 5.5) and an elution buffer.
- the peak% of the subfraction according to the pH change of the buffer used in the anion ion exchange chromatography is shown in FIG. 2. As shown in FIG. 2, it can be seen that the peak% of pII increases when the pH of the buffer ranges from 4.5 to 5.5.
- the subfraction A260 / A280 according to the pH of the buffer is shown in Table 1 below, and the total lethal dose (Total LD50 (Unit)) is shown in Table 2 below.
- the semi-aridal dose may be 0.8 X 10 5 to 3.5 X 10 5 U / ml, indicating that the recovery of pII is high.
- a subfraction was obtained in the same manner as in Example 1, but the peak characteristic change of each subfraction (pI, pII, and pIII) due to the change in the amount of the sample was shown in FIG. 3, and the change of A260 / A280 for each subfraction Is shown in Table 3, and the total lethal dose (Total LD50 (Unit)) is shown in Table 4 below.
- A260 / A280 appears as 0.4 to 0.6, and thus may be determined to be suitable as the administered sample.
- non-diffuse active fraction pII
- the half lethal dose may be 0.8 X 10 5 to 3.5 X 10 5 U / ml, indicating that the recovery of pII is high.
- Example 1 In order to confirm the paraplegic effect of the subfraction separated by Example 1, the lower extremity muscle injection was performed using a mouse.
- mice used (ICR mouse, female, 4week, 18-22g) is 10 per experimental group, the weight of the mouse was accurately measured and recorded.
- the subfraction was filled in 50 ⁇ l Hamilton syringes without air at a concentration of 1 U / 20 ⁇ l, and then injected into the muscle by inserting a needle about 3 cm from the right ankle of an ICR mouse.
- the paralysis index and mortality were observed according to the paralysis index and measurement criteria shown in Table 5 below, and the results are shown in FIG. 4.
- Table 5 Score Measure 0 Foot Shape and Gait of Normal Rats One The toes are not collected, but when you walk, drag your legs. 2 Toes are collected with the criteria of 1. 3 With the criterion of 2, the foot is bent inward. 4 With the criterion of 3, the foot is completely attached towards the leg muscles and the legs cannot be used.
- the non-diffusion active fraction (pII) in the subfraction separated from Example 1 showed a significant increase in survival rate compared to the diffuse active fraction (pI) (A) and administration. It can be seen that the survival rate over time at the dose 2U shows an extreme difference of 0% pI compared to 100% of pII (B).
- mice used (ICR mouse, female, 4week, 18-22g) is 10 per experimental group, the weight of the mouse was accurately measured and recorded.
- the sample and subfraction pII were pre-purified without air at a concentration of 1 U / 20 ⁇ l, and then injected into the muscle by inserting a needle about 3 cm from the right ankle of an ICR mouse.
- the degree of paralysis of the mouse was visually checked, and the state of the mouse 12 hours after the injection was shown in FIG. 5, and there was death according to the administration time and dose. The results of the observation are shown in FIG. 6.
- the onset time of the muscle paralysis effect is shown in FIG. 7, and the duration of the muscle paralysis effect is shown in FIG. 8.
- pII had a longer duration of myopathic effect than 80 days at a dose of 0.5 U compared to a commercial product.
- lyophilization was performed by diluting the pII fraction with the same amount of botulinum toxin (100U) and ancillary additives. After confirming, the ion content analysis was performed. After dissolving the two lyophilisates using 10 ml of distilled water, the ion content was measured using an inductively coupled plasma mass spectrometer. The results are shown in Table 7 below.
- the freeze-dried lyophilisate of the non-diffusion botulinum toxin (pII fraction) purified by the present invention was found to have a significantly higher content of Mg, Fe and Zn ions than commercially available products.
- the present invention it is possible to obtain a large amount of non-diffusion botulinum toxin having a local myopathic effect, which occurs quickly and continuously, while the myopic effect is generated in a desired portion and the toxin is not diffused in other portions. have.
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Abstract
Description
A260/A280 | ||||
pH 4.5 | pH 5.0 | pH 5.5 | pH 6.0 | |
pI | 0.76± 0.15 | 0.69± 0.09 | 0.67± 0.05 | 0.63± 0.08 |
pII | 0.50± 0.05 | 0.55± 0.04 | 0.55± 0.03 | 0.81± 0.19 |
pIII | 0.76± 0.10 | 0.82± 0.13 | 0.84± 0.12 | 0.79± 0.09 |
Total LD50(Unit) | ||||
pH 4.5 | pH 5.0 | pH 5.5 | pH 6.0 | |
기존 산침전법 정제물(전시료) | 5± 0.8 | 5± 1 | 5± 0.8 | 5± 1.2 |
pI | 1.5± 0.4 | 3± 0.5 | 2± 0.4 | 2± 0.8 |
pII | 0.2± 0.2 | 1± 0.2 | 3± 0.4 | 0.4± 0.2 |
pIII | - | 0.2± 0.1 | 0.2± 0.1 | 2± 1 |
(단위: 105 U) |
A260/A280 | ||||
0.25 ml | 0.5ml | 1ml | 2ml | |
pI | 0.67± 0.08 | 0.64± 0.07 | 0.68± 0.09 | 0.67± 0.13 |
pII | 0.55± 0.02 | 0.56± 0.01 | 0.55± 0.04 | 0.55± 0.03 |
pIII | 0.85± 0.14 | 0.84± 0.09 | 0.82± 0.18 | 0.84± 0.24 |
Total LD50(Unit) | ||||
0.25 ml | 0.5 ml | 1 ml | 2 ml | |
기존 산침전법 정제물(전시료) | 2.5± 0.5 | 5± 1 | 10± 4 | 20± 5 |
pI | 0.8± 0.2 | 1.6± 0.4 | 5± 2 | 15± 4.8 |
pII | 1.6± 0.2 | 4± 0.6 | 4± 0.9 | 4± 1.5 |
pIII | 0.2± 0.1 | 0.4± 0.1 | 0.8± 0.2 | 0.6± 0.3 |
(단위: 105 U) |
Score | 측정 기준 |
0 | 정상 쥐의 발 모양새와 걸음걸이 |
1 | 발가락은 모아지지 않았으나 걸을 때 다리를 끈다. |
2 | 1의 기준과 함께 발가락이 모아져 있다. |
3 | 2의 기준과 함께 발마디가 안쪽으로 굽어있다. |
4 | 3의 기준과 함께 발이 다리근육쪽으로 완전히 붙어있으며 다리를 못 쓴다. |
분획 | 마우스 우하지주사시 마비부위 |
체내 확산형 활성 분획 | 우하지, 횡경막, 좌하지 등 체내 여러 부위 |
체내 비확산형 활성 분획 | 우하지 |
체내 확산형 비활성 분획 | - |
Mg | Fe | Zn | |
pII | 183.0 ± 19.3* | 105.4 ± 22.1** | 332.6 ± 140.8*** |
시판제품 | 78.1 ± 7.4* | 39.6 ± 3.3 | 65.6 ± 25.9 |
* p<0.0015, ** p<0.011, ***p<0.045 |
Claims (3)
- 정제된 보툴리눔 독소 A 형 제제를 pH는 4.5 내지 6.5 범위, 염화나트륨(NaCl)의 농도를 0.02 내지 0.2M로 조절하여 첨가한 음이온 이온교환수지 크로마토그래피를 수행하여 아분획을 분리하는 단계; 및상기 아분획 중 A260/A280의 값이 0.4 내지 0.6 수준을 유지하는 비확산성 보툴리눔 독소 분획을 수득하는 단계를 포함하여 이루어지는 것을 특징으로 하는 비확산형 보툴리눔 독소의 정제방법.
- 청구항 1에 의하여 분리 정제된 것으로,독소 100U/ml당 Zn 이온이 적어도 150ppb, Fe 이온이 적어도 80ppb, Mg 이온이 적어도 140ppb로 포함되는 것을 특징으로 하는 비확산형 보툴리눔 독소.
- 청구항 2의 비확산형 보툴리눔 독소를 그의 반수치사량의 1.5 내지 3배의 양으로 마우스 우하지에 주사하고, 우하지 근육과 호흡근의 마비 여부와 치사여부를 판단하는 것을 특징으로 하는 비확산형 보툴리눔 독소를 판별하는 방법.
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/700,867 US9598683B2 (en) | 2010-05-31 | 2011-05-13 | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
EP11789976.5A EP2578228B1 (en) | 2010-05-31 | 2011-05-13 | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
AU2011262499A AU2011262499B2 (en) | 2010-05-31 | 2011-05-13 | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
JP2013513102A JP5919608B2 (ja) | 2010-05-31 | 2011-05-13 | 局所筋麻痺効果を有する非拡散型ボツリヌス毒素とその精製方法 |
ES11789976.5T ES2659820T3 (es) | 2010-05-31 | 2011-05-13 | Toxina botulínica no difusiva que provoca una parálisis muscular localizada y procedimiento de purificación de la misma |
CN201180028634.2A CN102985102B (zh) | 2010-05-31 | 2011-05-13 | 引起局部肌麻痹的非扩散型肉毒杆菌毒素及其精制方法 |
US15/422,083 US20170145399A1 (en) | 2010-05-31 | 2017-02-01 | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
US15/422,129 US10369235B2 (en) | 2010-05-31 | 2017-02-01 | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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KR10-2010-0051076 | 2010-05-31 | ||
KR1020100051076A KR101134146B1 (ko) | 2010-05-31 | 2010-05-31 | 국소 근마비 효과를 갖는 비확산형 보툴리눔 독소와 그의 정제방법 |
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---|---|---|---|
US13/700,867 A-371-Of-International US9598683B2 (en) | 2010-05-31 | 2011-05-13 | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
US15/422,129 Division US10369235B2 (en) | 2010-05-31 | 2017-02-01 | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
US15/422,083 Division US20170145399A1 (en) | 2010-05-31 | 2017-02-01 | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
Publications (4)
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WO2011152618A2 WO2011152618A2 (ko) | 2011-12-08 |
WO2011152618A9 true WO2011152618A9 (ko) | 2012-03-22 |
WO2011152618A3 WO2011152618A3 (ko) | 2012-05-24 |
WO2011152618A4 WO2011152618A4 (ko) | 2012-07-12 |
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PCT/KR2011/003547 WO2011152618A2 (ko) | 2010-05-31 | 2011-05-13 | 국소 근마비 효과를 갖는 비확산형 보툴리눔 독소와 그의 정제방법 |
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US (3) | US9598683B2 (ko) |
EP (1) | EP2578228B1 (ko) |
JP (2) | JP5919608B2 (ko) |
KR (1) | KR101134146B1 (ko) |
CN (1) | CN102985102B (ko) |
AU (1) | AU2011262499B2 (ko) |
ES (1) | ES2659820T3 (ko) |
WO (1) | WO2011152618A2 (ko) |
Families Citing this family (2)
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KR101134146B1 (ko) | 2010-05-31 | 2012-04-19 | 메덱스젠 주식회사 | 국소 근마비 효과를 갖는 비확산형 보툴리눔 독소와 그의 정제방법 |
KR101775682B1 (ko) * | 2015-11-30 | 2017-09-06 | 주식회사 대웅 | 보툴리눔 독소의 제조방법 |
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US5939070A (en) * | 1996-10-28 | 1999-08-17 | Wisconsin Alumni Research Foundation | Hybrid botulinal neurotoxins |
CA2319113A1 (en) * | 1998-01-26 | 1999-07-29 | University Of Massachusetts | Biologically active hemagglutinin from type a clostridium botulinum and methods of use |
DE19925739A1 (de) * | 1999-06-07 | 2000-12-21 | Biotecon Ges Fuer Biotechnologische Entwicklung & Consulting Mbh | Therapeutikum mit einem Botulinum-Neurotoxin |
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JP2003009897A (ja) * | 2001-07-03 | 2003-01-14 | Keiji Oguma | ボツリヌス毒素の分離・精製法 |
KR20030060150A (ko) * | 2002-01-07 | 2003-07-16 | (주)메디톡스 | 클로스트리디움 보툴리눔 a형 독소를 정제하는 방법 |
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KR101134146B1 (ko) * | 2010-05-31 | 2012-04-19 | 메덱스젠 주식회사 | 국소 근마비 효과를 갖는 비확산형 보툴리눔 독소와 그의 정제방법 |
SG10201506030RA (en) * | 2010-07-30 | 2015-09-29 | Medimmune Llc | Method for purifying active polypeptides or immunoconjugates |
-
2010
- 2010-05-31 KR KR1020100051076A patent/KR101134146B1/ko active IP Right Grant
-
2011
- 2011-05-13 US US13/700,867 patent/US9598683B2/en not_active Expired - Fee Related
- 2011-05-13 EP EP11789976.5A patent/EP2578228B1/en not_active Revoked
- 2011-05-13 WO PCT/KR2011/003547 patent/WO2011152618A2/ko active Application Filing
- 2011-05-13 JP JP2013513102A patent/JP5919608B2/ja not_active Expired - Fee Related
- 2011-05-13 CN CN201180028634.2A patent/CN102985102B/zh not_active Expired - Fee Related
- 2011-05-13 AU AU2011262499A patent/AU2011262499B2/en not_active Ceased
- 2011-05-13 ES ES11789976.5T patent/ES2659820T3/es active Active
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2015
- 2015-05-07 JP JP2015094777A patent/JP5998419B2/ja not_active Expired - Fee Related
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2017
- 2017-02-01 US US15/422,083 patent/US20170145399A1/en not_active Abandoned
- 2017-02-01 US US15/422,129 patent/US10369235B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
JP5919608B2 (ja) | 2016-05-18 |
US10369235B2 (en) | 2019-08-06 |
KR20110131572A (ko) | 2011-12-07 |
EP2578228A4 (en) | 2013-10-02 |
CN102985102B (zh) | 2016-06-08 |
US20170145399A1 (en) | 2017-05-25 |
EP2578228B1 (en) | 2017-11-15 |
US20170143849A1 (en) | 2017-05-25 |
JP2013533856A (ja) | 2013-08-29 |
JP2015200659A (ja) | 2015-11-12 |
US20130071331A1 (en) | 2013-03-21 |
WO2011152618A2 (ko) | 2011-12-08 |
KR101134146B1 (ko) | 2012-04-19 |
WO2011152618A4 (ko) | 2012-07-12 |
EP2578228A2 (en) | 2013-04-10 |
ES2659820T3 (es) | 2018-03-19 |
WO2011152618A3 (ko) | 2012-05-24 |
AU2011262499A1 (en) | 2013-01-24 |
AU2011262499B2 (en) | 2015-01-22 |
JP5998419B2 (ja) | 2016-09-28 |
CN102985102A (zh) | 2013-03-20 |
US9598683B2 (en) | 2017-03-21 |
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