Methods and systems for purifying non-complexed botulinum neurotoxin

摘要

Methods and systems for chromatographically purifying a botulinum neurotoxin are provided. These methods and systems allow for efficient purification of a non-complexed form of the botulinum neurotoxin in high purity and yield that can be used as an active ingredient in pharmaceutical preparations.

主权项

1. A method for purifying a non-complexed botulinum toxin type A (botulinum toxin A), the method comprising:
(a) providing a mixture comprising a crude non-complexed botulinum toxin A, in which said crude non-complexed botulinum toxin A is dissociated from native non-toxin proteins; wherein said mixture is obtained by:
(i) subjecting a fermentation culture comprising botulinum toxin A to acid precipitation to produce an insoluble acid precipitate;(ii) concentrating the acid precipitate from step (i) to produce a concentrated sample;(iii) subjecting the sample to nuclease digestion under conditions which reduce host cell nucleic acid content and which maintain a complex of botulinum toxin A and non-toxin proteins;(iv) removing cell debris from the sample of step (iii) to produce a clarified sample;(v) loading a hydrophobic interaction column with the clarified sample from step (iv) under conditions to permit capture of the botulinum toxin A complex by the hydrophobic interaction column and impurities to flow through the column;(vi) eluting the botulinum toxin A complex from the hydrophobic interaction column of step (vi); and(vii) dissociating the botulinum toxin A complex obtained from step (vi) under conditions which disrupt the complex and produce a mixture comprising crude non-complexed botulinum toxin A dissociated from the native non-toxin proteins; (b) loading the mixture containing the crude non-complexed botulinum toxin A dissociated from native non-toxin proteins from step (a) on an anion exchange column under conditions permitting capture of the non-complexed botulinum toxin A by the anion exchange column; (c) eluting the non-complexed botulinum toxin A from the anion exchange column of step (b) to give an eluent comprising the non-complexed botulinum toxin A; (d) loading a cation exchange column with the eluent from the anion exchange column of step (c) under conditions permitting capture of the non-complexed botulinum toxin A by the cation exchange column; and (e) eluting purified non-complexed botulinum toxin A from the cation exchange column of step (d).