Methods of purification of native or mutant forms of diphtheria toxin
OwnerAaron R. Goerke, Thomas Svab +2inventors
InventorsAaron R. Goerke, Thomas Svab, Patrick McHugh, Kristin Valente
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Abstract
The present invention relates to the use of hydroxyapatite chromatography and multimodal chromatography, for punfication of diphtheria toxin, or a mutant form thereof, from a mixture, for example, a host cell fermentation mixture containing impurities such as host cell proteins and DNA. This invention further relates to the integration of such a method into a multi-step procedure with other fractionation methods for purification of diphtheria toxin suitable for in vitro and in vivo applications.
Claims28
A method of purifying diptheria toxin, or a mutant form thereof, from a mixture containing the diptheria toxin, or mutant form thereof, comprising:
- a) contacting the mixture with a first separation agent under conditions such that the diphtheria toxin, or mutant form thereof, binds to the first separation agent;
- b) eluting the diphtheria toxin, or mutant form thereof, from the first separation agent;
- c) contacting the eluted material obtained from step a) with a second separation agent under conditions such that the diphtheria toxin, or mutant form thereof, binds to the second separation agent;
- and
- d) eluting said diptheria toxin, or mutant form thereof, from the second separation agent;
- wherein either 1) the first separation agent is hydroxyapatite and the second separation agent is a multimodal resin;
- or 2) the first separation agent is a multimodal resin and the second separation agent is hydroxyapatite;
- and when the first separation agent or second separation agent is hydroxyapatite, prior to eluting from the hydroxyapatite, the hydroxyapatite undergoes a washing step under conditions such that impurities are removed.
Dependencies are inferred from the claim text.