Method of purifying botulinum toxin
Disclosed is a method of purifying a botulinum toxin including (a) pre-treating a culture solution containing a botulinum toxin, (b) purifying the pre-treated botulinum toxin using anion exchange chromatography and (c) purifying the botulinum toxin using cation exchange chromatography. The method is capable of purifying a botulinum toxin with high purity and activity using a simple process including anion exchange chromatography and cation exchange chromatography, and is thus useful for botulinum toxin production.
1 . A two column chromatography method of purifying a 900 kDa botulinum toxin complex, comprising:
(a) pre-treating a culture solution containing a botulinum toxin;
(b) preparing the pre-treated botulinum toxin from step (a) by dissolving it in 30-70 mM sodium phosphate buffer at pH 5.5-6.5 and injecting it into an anion exchange chromatography column to bind the botulinum toxin to the anion exchange chromatography medium at a pH higher than an isoelectric point (PI) of botulinum toxin, said anion exchange chromatography column comprising a column packed with a resin containing a methacrylic bead with a particle size of 65 μm comprising a quaternary ammonium (Q) functional group, wherein the ion exchange capacity of the column is 0.25±0.05 meq/mL, and the DBC (dynamic binding capacity) of the column is 149 mg/mL based on BSA, and separating the botulinum toxin, which is bound to the anion exchange chromatography medium, by elution with a 30 to 70 mM sodium phosphate buffer having a pH of 5.5 to 6.5 comprising 0.4 to 0.6M sodium chloride; and
(c) preparing the botulinum toxin from step (b) by dissolving it in 15 to 25 mM sodium citrate buffer at pH 4.0 to 5.0 and injecting it into a cation exchange chromatography column to bind the botulinum toxin to the cation exchange chromatography medium, said cation exchange chromatography column comprising a column packed with a resin that contains a sulfopropyl functional group, has a 6% spherical, cross-linked agarose matrix form, and has a DBC of 55 mg/mL based on ribonuclease A and a particle size of 34 μm, and separating 900 kDa botulinum toxin complex from the solution which is bound to the cation exchange chromatography medium, by elution with a 15 to 25 mM sodium citrate buffer having a pH of 4.0 to 5.0 comprising 0.4 to 0.6M sodium chloride.
2 . The method according to claim 1 , wherein the purified botulinum toxin is botulinum toxin A having a purity of 95% or more.