# US 2014/0193876 A1 · Methods of purification of native or mutant forms of diphtheria toxin

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- Number: US 2014/0193876 A1
- Document ID: US20140193876A1
- Status: Published application
- Kind: A1
- Application number: 14/125,695
- Filed: 2012-06-08
- Published: 2014-07-10
- Earliest claimed: 2011-06-13
- Inventors: Aaron R. Goerke; Thomas Svab; Patrick McHugh; Kristin Valente
- Web page: https://patentgrep.com/patent/US20140193876A1

## CPC

- C12N 9/1077: {Pentosyltransferases (2.4.2)}
- C07H 21/04: with deoxyribosyl as saccharide radical
- C07K 14/34: from Corynebacterium (G)
- Y02P 20/582: Recycling of unreacted starting or intermediate materials
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## 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.

## Claims

### Claim 1

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.

### Claim 2 (depends on 1)

The method of claim 1, wherein the washing is with a wash solution comprising from 0.01 to 1.0 M potassium chloride or sodium chloride at a pH from 6.5 to 8.0.

### Claim 3 (depends on 2)

The method of claim 2, wherein the wash buffer further comprises 0.1 to 20 mM potassium phosphate or sodium phosphate.

### Claim 4 (depends on 1)

The method of claim 1, wherein said eluting from hydroxyapatite comprises
a) step elution with an elution buffer comprising ≧about 30 mM potassium chloride or sodium chloride or about ≧15 mM potassium phosphate or sodium phosphate;
b) gradient elution comprising from about 10 to about 25 mM potassium phosphate or sodium phosphate or from about 100 mM to 2 M potassium chloride or sodium chloride; or
c) a pH change of ≧0.3 pH units.

### Claim 5 (depends on 1)

The method of claim 1, wherein the mixture or eluted material contacted with the multimodal resin comprises Tris, MES, MOPS, HEPES, phosphate, acetate, chloride or sulfate.

### Claim 6 (depends on 1)

The method of claim 1, wherein said eluting from the multimodal resin comprises
a) step elution with an elution buffer comprising ≧about 125 mM potassium chloride or sodium chloride at pH 6.8 to 9.5;
b) gradient elution comprising from about 0.2 to about 0.3 M sodium chloride, potassium chloride, sodium sulfate, ammonium sulfate, lithium sulfate, lithium chloride, or ammonium chloride;
c) a pH change of ≧0.5 pH units within a pH range of 6.5 to 9.5; or
d) a temperature change of ≧1° C. within a temperature of 2 to 30° C.

### Claim 7 (depends on 5)

The method of claim 5, wherein the mixture or eluted material contacted with the multimodal resin comprises EDTA or a protease inhibitor.

### Claim 8 (depends on 6)

The method of claim 6, wherein said eluting occurs in the presence of EDTA or a protease inhibitor.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first separation agent is hydroxyapatite and the second separation agent is a multimodal resin.

### Claim 10 (depends on 1)

The method of claim 1, wherein the multimodal resin contains ligands which comprise a charged part and a hydrophobic part.

### Claim 11 (depends on 10)

The method of claim 10, wherein the charged part is a negatively charged part.

### Claim 12 (depends on 11)

The method of claim 11, wherein said negatively charged part is an anionic carboxylate group or anionic sulfo group for cation exchange.

### Claim 13 (depends on 12)

The method of claim 12, wherein said multimodal resin is Capto-MMC™.

### Claim 14 (depends on 10)

The method of claim 10, wherein the charged part is a positively charged part.

### Claim 15 (depends on 14)

The method of claim 14, wherein said multimodal resin is Capto Adhere™.

### Claim 16 (depends on 1)

The method of claim 1, wherein, prior performing step (a), the mixture is subject to one or more of the following: centrifugation, flocculation, clarification, or anion-exchange chromatography.

### Claim 17 (depends on 16)

The method of claim 16, wherein the mixture is subject to two or three passes on anion-exchange chromatography.

### Claim 18 (depends on 16)

The method of claim 16, wherein the mixture is subject to centrifugation, flocculation, clarification and anion-exchange chromatography.

### Claim 19 (depends on 18)

The method of claim 18, wherein said clarifying is through centrifugation and depth filtration.

### Claim 20 (depends on 1)

The method of claim 1, wherein the mixture is obtained from cultured host cells.

### Claim 21 (depends on 20)

The method of claim 20, wherein the cultured host cells are osmotically shocked.

### Claim 22 (depends on 20)

The method of claim 20, wherein said fermentation cells are recovered by centrifugation or microfiltration.

### Claim 23 (depends on 1)

The method of claim 1, wherein the mixture is obtained from a cell-free production system.

### Claim 24 (depends on 1)

The method of claim 1, wherein the diphtheria toxin, or mutant form thereof, is subject to one or more of the following step (d): centrifugation, ultrafiltration, microfiltration, filtration and anion-exchange membrane chromatography.

### Claim 25 (depends on 24)

The method of claim 24, wherein the diphtheria toxin, or mutant form thereof, is subject to ultrafiltration, microfiltration, filtration and anion-exchange membrane chromatography.

### Claim 26 (depends on 1)

The method of claim 1, wherein the mutant diphtheria toxin is CRM197.

### Claim 27 (depends on 1)

A formulation comprising the diptheria toxin, or a mutant form thereof, obtained according to the method of claim 1 and in liquid form.

### Claim 28 (depends on 27)

The formulation of claim 27, wherein the purity of the diptheria toxin, or a mutant form thereof, is greater than 90% when maintained at a concentration of 10 g/L or more at 2° C. for at least six months.