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EP0576294A2
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EP
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Chondroitinase, process for preparing the same, and pharmaceutical composition comprising the same
Assignee: Seikagaku Corp
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Filing: 1993-06-25
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Granted: —
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Expires: 2013-06-25
Abstract
Abstract A crystallizable, purified chondroitinase ABC having a molecular weight of about 100,000 dalton by the measurement of the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the measurement by the gel permeation chromatography method, having alanine as the N-terminal amino acid and proline as the C-terminal amino acid. A process for the purification of the crystallizable purified chondroitinase ABC comprising removing nucleic acid from an surfactant solution extract obtained from cells of chondroitinase ABC-producing microorganisms and chromatographically treating by concentration gradient elution using a weak cation exchange resin or a strong cation exchange resin. A composition comprising a chondroitinase and serum albumin, gelatin, or a nonionic surfactant.
Inventors
- 👤 Nobukazu Hashimoto
- 👤 Akio Hamai
- 👤 Hideo Mochizuki
- 👤 Shinichi Ishikawa
- 👤 Misako Torikai
- 👤 Isao Miyachi
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-30 06:37
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/88; A; A61; A61K; A61K9/00; A61K9/20; A61K9/2004; A61K9/2022; A61K9/2063; C07; C07K; C07K1/00; C07K1/14; C07K1/16; C07K1/18; A61K38/00; C12Y; C12Y402/00; C12Y402/02; C12Y402/02004; G; G03; G03C; G03C1/00; G03C1/005; G03C1/04; G03C1/047; G03C2001/0471; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/873
Claims Analysis
Total Claims: 28
Claim 1
Independent Claim
171 words
A purified chondroitinase ABC having the following characteristics, (i) has a molecular weight of about 100,000 dalton both by the measurement of the SDS-polyacrylamide gel electrophoresis (SDS-PAGE), in either the reduced state or the unreduced state, and the measurement by the gel permeation chromatography method, (ii) has an isoelectric point of about pH 8.2 and about pH 8.5, (iii) has an optimum pH of 8.0 to 8.2 (substrate: chondroitin sulfate C, buffer solution: a Tris-HCl buffer solution) and an optimum reaction temperature of 37°C, (iv) the activity is inhibited by Zn²⁺, Ni²⁺, Fe³⁺, and Cu²⁺, (v) has alanine as the N-terminal amino acid residue and proline as the C-terminal amino acid residue, (vi) exhibits a single band in SDS-PAGE and a single peak in either HPLC (GPC; gel permeation chromatography) or cation exchange HPLC, (vii) does not contain a substantial amount of endotoxin and contain nucleic acid and protease of an amount below the detectable limit, (viii) can be crystallized, and (ix) has a specific activity of 300 U/mg or more.
Claim 2
Dependent Claim
29 words
The process for preparing the purified chondroitinase ABC as defined in Claim 1 comprising extracting chondroitinase ABC from cells of a microorganism producing chondroitinase ABC and purifying the extract.
Claim 3
Dependent Claim
22 words
The process for preparing the purified chondroitinase ABC according to Claim 2, wherein said extraction is carried out using a surfactant solution.
Claim 4
Dependent Claim
150 words
A process for preparing the purified chondroitinase ABC as defined in Claim 1 comprising, (i) a step of obtaining an enzyme-containing extract from cells of microorganism producing chondroitinase ABC (step 1), (ii) a step of removing nucleic acid from the enzyme-containing extract (step 2), and (iii) a step of chromatographic treatment which comprises, (a) absorbing the chondroitinase ABC by a chromatographic treatment of the enzyme-containing extract obtained by step 2 using a weak cation exchange resin, eluting the absorbed enzyme, absorbing the enzyme in the eluate, and eluting the absorbed enzyme by chromatography using a strong cation exchange resin (step 3-1), or (b) absorbing the chondroitinase ABC by a chromatographic treatment of the enzyme-containing extract obtained by step 2 using a strong cation exchange resin, eluting the absorbed enzyme, absorbing the enzyme in the eluate, and eluting the absorbed enzyme by chromatography using a weak cation exchange resin (step 3-2).
Claim 5
Dependent Claim
54 words
The process for preparing the purified chondroitinase ABC according to Claim 4, wherein the exchange group in said weak cation exchange resin used in said step 3-1 or 3-2 is a carboxyalkyl group and the exchange group in said strong cation exchange resin used in said step 3-1 or 3-2 is a sulfoalkyl group.
Claim 6
Dependent Claim
34 words
The process for preparing the purified chondroitinase ABC according to Claim 4, wherein said enzyme-containing extract in step 1 is obtained by extraction of cells of microorganism producing chondroitinase ABC using a surfactant solution.
Claim 7
Dependent Claim
89 words
A process for preparing the purified chondroitinase ABC as defined in Claim 1 comprising, (i) a step of obtaining an enzyme-containing extract from cells of microorganism producing chondroitinase ABC by extraction using a surfactant solution, (ii) a step of subjecting the enzyme-containing extract to a chromatographic treatment using a weak cation exchange resin or a strong cation exchange resin to absorb the chondroitinase ABC to said resin, and (iii) a step of eluting the chondroitinase ABC absorbed to said resin while continuously changing the ionic strength of the eluent.
Claim 8
Dependent Claim
22 words
Chondroitinase ABC in the form of needle-like or prismatic crystals and having the characteristics (i) to (ix) as defined in Claim 1.
Claim 9
Dependent Claim
21 words
Chondroitinase ABC in the form of crystals of Claim 8 prepared by crystallization in polyether having hydroxy groups at both ends.
Claim 10
Dependent Claim
55 words
Needle-like or prismatic crystals of chondroitinase ABC having characteristics as defined in Claim 1, which is of rhombic or monoclinic system and having the following crystal parameters. Rhombic system Monoclinic system Space group P222₁ P2₁ Lattice constant a=214 Å a=214 Å b=92 Å b=56 Å c=56 Å c=92 Å α=90° α=90° β=90° β≧90° γ=90° γ=90°
Claim 11
Dependent Claim
10 words
A composition comprising a chondroitinase and serum albumin or gelatin.
Claim 12
Dependent Claim
19 words
The composition according to Claim 11 having pH of 5 to 9 when it is made into a solution.
Claim 13
Dependent Claim
22 words
The composition according to Claim 11, wherein said chondroitinase is a purified chondroitinase having a specific activity of 300 U/mg or more.
Claim 14
Dependent Claim
22 words
The composition according to Claims 11 comprising serum albumin or gelatin in an amount of at least 0.001 time of the chondroitinase.
Claim 15
Dependent Claim
41 words
The composition according to Claim 11, wherein said chondroitinase is a purified chondroitinase ABC having a specific activity of 300 U/mg or more, containing no substantial amount of endotoxin and an amount of nucleic acid and protease below the detection limit.
Claim 16
Dependent Claim
14 words
The composition according to Claim 11, which is a solution or a dried product.
Claim 17
Dependent Claim
41 words
The composition according to Claim 11, which comprises a physiologically acceptable buffer agent and is adjusted so as to show pH of 5 to 9 and a concentration of the buffer of 1 to 100 mM when it is a solution.
Claim 18
Dependent Claim
67 words
The composition according to Claim 17, wherein said buffer agent is a buffer agent comprising one or more compounds selected from the group consisting of hydrochloric acid, sodium hydroxide, sodium carbonate, sodium hydrogencarbonate, phosphoric acid, potassium dihydrogenphosphate, dipotassium hydrogenphosphate, sodium dihydrogenphosphate, disodium hydrogenphosphate, aminoacetic acid, sodium benzoate, citric acid, sodium citrate, acetic acid, sodium acetate, tartaric acid, sodium tartarate, lactic acid, sodium lactate, ethanolamine, arginine, and ethylenediamine.
Claim 19
Dependent Claim
11 words
A pharmaceutical composition comprising a chondroitinase and serum albumin or gelatin.
Claim 20
Dependent Claim
11 words
An injection composition comprising a chondroitinase and serum albumin or gelatin.
Claim 21
Dependent Claim
19 words
Use of injection composition comprising a chondroitinase and serum albumin or gelatin for the treatment of intervertebral disc displacement.
Claim 22
Dependent Claim
8 words
A composition comprising chondroitinase and a nonionic surfactant.
Claim 23
Dependent Claim
41 words
The composition according to Claim 22, which comprises a physiologically acceptable buffer agent and is adjusted so as to show pH of 5 to 9 and a concentration of the buffer of 1 to 100 mM when it is a solution.
Claim 24
Dependent Claim
18 words
The composition according to Claim 22, wherein said chondroitinase has a specific activity of 300 U/mg or more.
Claim 25
Dependent Claim
16 words
The composition according to Claim 22, wherein said nonionic surfactant is polyoxyethylene sorbitan fatty acid ester.
Claim 26
Dependent Claim
58 words
The composition according to Claim 22, comprising a nonionic surfactant in an amount of 0.6 to 300 times by weight for 1 unit weight of the chondroitinase having a specific activity of 300 U/mg or more, and adjusted to pH 6 to pH 8 with a 10 to 50 mM phosphate buffer agent when it is a solution.
Claim 27
Dependent Claim
12 words
The composition according to Claim 22, which is in the liquid state.
Claim 28
Dependent Claim
44 words
A pharmaceutical composition comprising a sterile solution containing as an active ingredient chondroitinase in an admixture of serum albumin, gelatin or nonionic surfactant and sterile water without preservatives, said solution being adapted to be injected into a patient for the treatment of intervertebral displacement.
References Cited (Backward Citations)
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