US 12,441,991 B2
Method of purifying botulinum toxin
Ulf Stahl, Uppsala (SE); Peter Frank, Uppsala (SE); Anders Jarstad, Uppsala (SE); Sebastiaan Mul, Uppsala (SE); John Nolin, Storvreta (SE); Lena Nodqvist, Uppsala (SE); and Simon Aberg, Uppsala (SE)
Assigned to Galderma Holding SA, Zug (CH)
Appl. No. 17/757,734
Filed by GALDERMA HOLDING SA, Zug (CH); and IPSEN BIOPHARM LIMITED, Wrexham (GB)
PCT Filed Dec. 19, 2020, PCT No. PCT/IB2020/062249
§ 371(c)(1), (2) Date Jun. 20, 2022,
PCT Pub. No. WO2021/124295, PCT Pub. Date Jun. 24, 2021.
Claims priority of provisional application 62/951,828, filed on Dec. 20, 2019.
Prior Publication US 2023/0029456 A1, Feb. 2, 2023
Int. Cl. C12N 9/52 (2006.01); A61K 38/48 (2006.01); C07K 1/18 (2006.01); C07K 1/34 (2006.01)
CPC C12N 9/52 (2013.01) [C12Y 304/24069 (2013.01)] 14 Claims
 
1. A method for purifying a botulinum toxin from a solution comprising the toxin, comprising:
(a) filtering the solution comprising the toxin, wherein the toxin comprises botulinum neurotoxin serotype A;
(b) contacting a first chromatography column with the filtered solution comprising the toxin from (a) at a pH of 5.5 to 7, wherein the first chromatography column is an anion exchange chromatography column comprising an agarose bead-based medium;
(c) collecting a toxin-containing fraction, wherein the toxin-containing fraction flows through the first chromatography column without adsorbing to a stationary phase of the first chromatography column;
(d) contacting a second chromatography column with the toxin-containing fraction from (c) at a pH of 4 to 5, wherein the second chromatography column is a cation exchange chromatography column comprising an agarose bead-based medium;
(e) eluting the botulinum toxin from the second chromatography column to produce a first toxin-containing eluant;
(f) filtering the first toxin-containing eluant to produce a toxin-containing retentate;
(g) contacting a third chromatography column with the toxin-containing retentate from the filtering (f) at a pH of 7.5 to 8.5, wherein the third chromatography column is an anion exchange chromatography column comprising an agarose bead-based medium;
(h) eluting the botulinum toxin from the third chromatography column to produce a second toxin-containing eluant;
(i) contacting a fourth chromatography column with the second toxin-containing eluant at a pH of 6 to 7, wherein the fourth chromatography column is a gel filtration column comprising an agarose bead-based medium; and
(j) eluting the botulinum toxin from the fourth chromatography column, thereby producing a purified botulinum toxin,
wherein the method does not comprise precipitating, centrifuging or lyophilizing the botulinum toxin.