# US 9,040,058 B2 · Fermentation media free of animal-derived components for production of diphtheria toxoids suitable for human vaccine use

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- Number: US 9,040,058 B2
- Document ID: US9040058B2
- Status: Granted
- Kind: B2
- Application number: 13/673,978
- Filed: 2012-11-09
- Published: 2013-05-16
- Granted: 2015-05-26
- Earliest claimed: 2011-11-11
- Assignees: Glaxosmithkline Biologicals SA
- Applicants: Novartis AG
- Inventors: Friedrich Blackkolb; Bernd Becker; Martha Reith; Manfred Isenberg; Anne Katrin Hilbert
- Web page: https://patentgrep.com/patent/US9040058B2

## CPC

- C12N 1/20: Bacteria; Culture media therefor
- A61K 39/05: {Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella}, Corynebacterium; Propionibacterium {(Mycobacterium A61K39/04)}
- A61P 31/04: Antibacterial agents
- C07K 14/34: from Corynebacterium (G)
- A61K 2039/522: {avirulent or attenuated}
- A61K 2039/55505: {Inorganic adjuvants}
- A61K 2039/6037: {Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]}
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to a fermentation medium for cultivating Corynebacterium diphtheriae. The present invention also relates to the use of the fermentation medium in processes for obtaining diphtheria toxin from the Corynebacterium diphtheriae bacteria being cultivated and the preparation of vaccines using the diphtheria toxin obtained in the processes. The present invention further relates to a purification and detoxification processes specifically adapted for preparing a diphtheria toxoid for inclusion into a vaccine.

## Claims

### Claim 1

A composition suitable for human vaccination, comprising a formaldehyde-linked diphtheria toxoid which is free from formaldehyde-linked animal-derived components, wherein the diphtheria toxoid was inactivated by formaldehyde crosslinking of diphtheria toxin that was at least 90% pure during detoxification, and wherein the diphtheria toxoid has (i) a specific purity of at least 1500 Lf/mg diphtheria toxin protein nitrogen during detoxification, and (ii) a potency of at least 60 IU/ml.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the monomer:dimer ratio of the diphtheria toxoid in the range of 3:1 to 8:1.

### Claim 3 (depends on 1, 2)

The composition of claim 1 or 2, comprising a protective antigen from at least one pathogen other than Corynebacterium diphtheriae.

### Claim 4 (depends on 3)

The composition of claim 3, wherein the non-diphtheria protective antigen is selected from hepatitis B virus surface antigen (HBsAg), tetanus toxoid (T), a pertussis antigen, a conjugated Haemophilus influenzae type B capsular saccharide (Hib), a conjugated Neisseria meningitidis capsular saccharide, a conjugated Streptococcus pneumoniae capsular saccharide, and/or an inactivated poliovirus.

### Claim 5 (depends on 4)

The composition of claim 4, wherein the HBsAg is free from animal-derived components.

### Claim 6 (depends on 4)

The composition of claims 4, wherein the composition is composed of:
the formaldehyde-linked diphtheria toxoid (D), T, HBsAg;
D, T, a cellular Pertussis antigen or a whole cell Pertussis antigen (Pw), HBsAg;
D, T, Pw, HBsAg, Hib;
D, T, Pw, HBsAg, Hib, an N. meningitidis serogroup A capsular saccharide (MenA), an N. meningitidis serogroup C capsular saccharide (MenC);
D, T, Pw, HBsAg, Hib, MenA, MenC, an N. meningitidis serogroup W135 capsular saccharide (MenW135);
D, T, Pw, HBsAg, Hib, MenA, MenC, an N. meningitidis serogroup Y capsular saccharide (MenY);
D, T, Pw, HBsAg, Hib, MenA, MenC, MenW135, MenY;
D, T, an acellular Pertussis antigen (Pa), HBsAg;
D, T, Pa, HBsAg, Hib;
D, T, Pa, HBsAg, poliovirus;
D, T, Pa, HBsAg, poliovirus, Hib;
D, T, Pa, HBsAg, poliovirus, Hib, MenC;
D, T, Pa, HBsAg, poliovirus, Hib, MenC, MenA;
D, T, Pa, HBsAg, poliovirus, Hib, MenC, MenY;
D, T, Pa, HBsAg, poliovirus, Hib, MenC, MenW135; or
D, T, Pa, HBsAg, poliovirus, Hib, MenC, MenA, MenW135, MenY.

### Claim 7

A composition suitable for human vaccination, comprising (i) a formaldehyde-linked diphtheria toxoid which is free from formaldehyde-linked animal-derived components, wherein the diphtheria toxoid was inactivated by formaldehyde crosslinking of diphtheria toxin that was at least 90% pure during detoxification, and (ii) a protective antigen from at least one pathogen other than Corynebacterium diphtheriae, wherein the diphtheria toxoid has (a) a specific purity of at least 1500 Lf/mg diphtheria toxin protein nitrogen, and (b) a potency of at least 60 IU/ml.

### Claim 8 (depends on 7)

The composition of claim 7, wherein the non-diphtheria protective antigen is selected from hepatitis B virus surface antigen, tetanus toxoid, a pertussis antigen, a conjugated H. influenzae type B capsular saccharide, a conjugated N. meningitidis capsular saccharide, a conjugated S. pneumoniae capsular saccharide, and/or an inactivated poliovirus.

### Claim 9 (depends on 8)

The composition of claim 8, wherein the HBsAg is free from animal-derived components.

### Claim 10 (depends on 8)

The composition of claims 8, wherein the composition is composed of:
D, T, HBsAg;
D, T, Pw, HBsAg;
D, T, Pw, HBsAg, Hib;
D, T, Pw, HBsAg, Hib, MenA, MenC;
D, T, Pw, HBsAg, Hib, MenA, MenC, MenW135;
D, T, Pw, HBsAg, Hib, MenA, MenC, MenY;
D, T, Pw, HBsAg, Hib, MenA, MenC, MenW135, MenY;
D, T, Pa, HBsAg;
D, T, Pa, HBsAg, Hib;
D, T, Pa, HBsAg, poliovirus;
D, T, Pa, HBsAg, poliovirus, Hib;
D, T, Pa, HBsAg, poliovirus, Hib, MenC;
D, T, Pa, HBsAg, poliovirus, Hib, MenC, MenA;
D, T, Pa, HBsAg, poliovirus, Hib, MenC, MenY;
D, T, Pa, HBsAg, poliovirus, Hib, MenC, MenW135; or
D, T, Pa, HBsAg, poliovirus, Hib, MenC, MenA, MenW135, MenY.

### Claim 11

A diphtheria toxoid obtainable by a process comprising:
(i) growing a culture of a strain of Corynebacterium diphtheriae expressing a diphtheria toxin in a fermentation medium free of animal-derived components;
(ii) purifying the diphtheria toxin from the fermentation medium to obtain a diphtheria toxin solution that was at least 90% pure;
(iii) adjusting the concentration of diphtheria toxin in the diphtheria toxin solution to at least 3000 Lf/mL to obtain a concentrated solution;
(iv) adding to the concentrated solution (a) an amino acid at a final concentration of no more than 0.025 M and (b) formaldehyde at a final concentration in the range of 0.75-1% to obtain a detoxification solution; and
(v) incubating the detoxification solution to obtain the diphtheria toxoid.

### Claim 12 (depends on 11)

A composition suitable for human vaccination, comprising the diphtheria toxoid of claim 11.

### Claim 13

A diphtheria toxoid for use in human vaccination, obtained by a process comprising:
(i) growing a strain of Corynebacterium diphtheriae expressing a diphtheria toxin or a derivative thereof in at least 100 L of a fermentation medium that is free from animal-derived components, optionally wherein the fermentation medium comprises yeast extract;
(ii) purifying the diphtheria toxin or derivative from the fermentation medium to obtain a purified diphtheria toxin or derivative, wherein the purified toxin or derivative is at least 90% pure;
(iii) adding formaldehyde to the purified diphtheria toxin or derivative; and
(iv) incubating the purified diphtheria toxin or derivative from step (iii) to obtain the diphtheria toxoid,
wherein the diphtheria toxoid is cross-linked by formaldehyde to at least one component of the fermentation medium.

### Claim 14

A diphtheria toxoid for use in human vaccination, obtained by a process comprising:
(i) growing a culture of a strain of Corynebacterium diphtheriae expressing a diphtheria toxin in a fermentation medium free of animal-derived components;
(ii) purifying the diphtheria toxin from the fermentation medium to obtain a diphtheria toxin solution that was at least 90% pure;
(iii) adjusting the concentration of diphtheria toxin in the diphtheria toxin solution to at least 2000 Lf/mL to obtain a concentrated solution;
(iv) adding to the concentrated solution (a) an amino acid at a final concentration of no more than 0.025 M and (b) formaldehyde at a final concentration in the range of 0.75-1% to obtain a detoxification solution; and
(v) incubating the detoxification solution to obtain the diphtheria toxoid,
wherein the diphtheria toxoid is cross-linked by formaldehyde to at least one component of the fermentation medium.

### Claim 15

A diphtheria toxoid obtainable by a process comprising:
(i) growing a strain of Corynebacterium diphtheriae expressing a diphtheria toxin or a derivative thereof in at least 100 L of a fermentation medium that is free from animal-derived components, optionally wherein the fermentation medium comprises yeast extract;
(ii) purifying the diphtheria toxin or derivative from the fermentation medium to obtain a purified diphtheria toxin or derivative, wherein the purified toxin or derivative is at least 90% pure;
(iii) adding formaldehyde to the purified diphtheria toxin or derivative; and
(iv) incubating the purified diphtheria toxin or derivative from step (iii) to obtain the diphtheria toxoid.

### Claim 16 (depends on 1)

The composition of claim 1, wherein the diphtheria toxoid has an isoelectric point in the range of 4.0 to 5.0.

### Claim 17 (depends on 1)

The composition of claim 1, wherein at least 70% of the diphtheria toxoid is in monomeric form.

### Claim 18 (depends on 1)

The composition of claim 1, wherein the formaldehyde-linked diphtheria toxoid is not conjugated to a saccharide as a carrier protein.

### Claim 19 (depends on 7)

The composition of claim 7, wherein the formaldehyde-linked diphtheria toxoid is not conjugated to a saccharide as a carrier protein.

### Claim 20 (depends on 11)

The diphtheria toxoid of claim 11, wherein the diphtheria toxoid is not conjugated to a saccharide as a carrier protein.

### Claim 21 (depends on 13)

The diphtheria toxoid of claim 13, wherein the diphtheria toxoid is not conjugated to a saccharide as a carrier protein.

### Claim 22 (depends on 15)

The diphtheria toxoid of claim 15, wherein the diphtheria toxoid is not conjugated to a saccharide as a carrier protein.

### Claim 23 (depends on 15)

The diphtheria toxoid of claim 15, wherein the diphtheria toxoid is not conjugated to a saccharide.