WO2006042542A2

WO2006042542A2 - Production of tetanus, diphtheria, and pertussis toxins and toxoids using fermentation media containing no components of animal or soy origin

Info

Publication number: WO2006042542A2
Application number: PCT/DK2005/000636
Authority: WO
Inventors: Susanne Aamand; Jesper Westphal Petersen
Original assignee: Statens Serum Institut
Priority date: 2004-10-19
Filing date: 2005-10-05
Publication date: 2006-04-27
Also published as: WO2006042542A3

Abstract

The present invention discloses that non-animal and non-soy based result in increased growth of C.tetani, C.diphteriae, and B.pertussis and result in equivalent or increased yield of tetanus toxin, diphteria toxin and pertussis toxin, respectively, compared to cultivation of the bacteria in media containing animal-derived or soy-derived proteinaceous material. Preferably the proteinaceous hydrolysates are derived from wheat, a mixture of rice and wheat, from potato, or from yeast.

Description

Production of tetanus, diphtheria, and pertussis toxins and toxoids using fermentation media containing no components of animal or soy origin.

Field of invention

The present invention discloses a fermentation medium for producing bacterial toxins, where the protein source is non-animal and non-soy derived protein e.g. potato peptone, wheat peptone, rice peptone, rice-wheat peptone, cotton peptone, pea peptone, or yeast peptone.

General background

Tetanus, diphtheria and pertussis toxoid vaccines are based on toxins produced by the bacteria Clostridium tetani, Corynebacterium diphtheriae and Bordetella pertussis, re¬ spectively.

Toxoid production usually involves the following steps: i) in one or more successive pre- culture steps the bacteria are cultivated in a seed medium sustaining good growth, of the bacteria; ii) the seed culture is used for inoculation of a fermentor where formation of toxin occurs in a medium which is designed for good toxin yield; iii) and finally the toxin is separated from the bacteria/culture medium and inactivated by treatment with formal¬ dehyde¹⁴'²⁵ or hydrogen peroxide²⁰. The detoxified toxins are designated toxoids.

In conventional production methods, C. tetani, C. diphtheriae and B. pertussis are culti¬ vated in media containing nutrients of animal origin (e.g. casein digests or meat extracts) fulfilling the nutritional requirement for protein, peptides and/or amino acids and essen¬ tial growth factors⁷'⁹' ^11>16>21. However, the content of animal-derived components in the culture media implies a risk of carry-over to the final toxoid preparation of undesirable contaminants or impurities, such as allergens, vira, prions, or other adventitious xmdesired biological effectors that are difficult to control. It has become a requirement of the health authorities to ensure that the final product is free from substances that may cause toxic or allergic reactions in humans²⁶, and therefore manufactures of medicinal products are en- couraged if possible not to use material of animal origin at all ' . Accordingly a shift from animal-based to vegetable-based nutrients in the manufacturing process is recom¬ mended¹⁰.

Today, meat-derived components have probably been eliminated from many growth me¬ dia used in the manufacturing of toxoids. C. tetani, C. diphtheriae and B. pertussis can grow and produce toxin in semi-synthetic media containing enzymatic digests of casein as the only source of proteinaceous nutrients⁹'¹¹'²¹, and for B. pertussis even a chemically defined medium can be used²². Casein is derived from milk, and as regards Bovine Spongiform Encephalopathies (BSE) milk is so far considered unlikely to present any risk of contamination⁶. Casein hydrolysates used for vaccine manufacturing, however, are generally manufactured by use of animal-derived enzymes (e.g. trypsin, pancreatin, or even raw pancreas) in the hydrolysis process, and these animal-derived enzyme prepara¬ tions may amount to a considerable part of the final batch size of the casein hydrolysate. The hydrolysis may last for several days during which microbial contamination of the product is not unusual and necessitates the addition of large volumes of undesirable bac¬ teriostatic remedies like toluene. In addition to the obvious disadvantage connected with the content of animal-derived materials in the final casein hydrolysate product, these fac¬ tors also contribute significantly to the lot-to-lot variability of many casein hydrolysates.

Vegetable extracts can be used in media for growth of pathogenic bacteria and production of their toxins. Vegetable extract are aqueous extracts of plants containing amino acids and low molecular weight peptides, relatively high concentrations of carbohydrates, vi¬ tamins and other growth factors¹. Results published long ago in patent GB512196 showed that B. pertussis could be grown in any suitable vegetable extract in place of soluble starch. This growth medium, however, was not further specified and it most probably contained additional, probably proteinaceous, components as the source of ni¬ trogen. Likewise, C. diphtheriae could be cultivated in a proteinaceous medium supple¬ mented with soybean extract²³. Today, the use of vegetable extracts have been widely replaced by protein hydrolysates (peptones) formed by controlled enzymatic or acid di¬ gestion of proteinaceous material derived from vegetables. Recently it has been shown that vegetable protein hydrolysates derived from soy not only could supplement animal derived proteinaceous components such as beef heart infusion (BHI) and casein hydro- Iy sate, but even could replace these components in media for growth of pathogenic bacte¬ ria and production of their toxins ^>4>1 .

For producers of tetanus toxin, however, it is the general experience that replacement of 5 the casein hydro lysate in the production medium is difficult. The production process is very delicate in the sense that growth of C. tetani is relatively unaffected by changes in the proteinaceous nutrient whereas the production of toxin, which is tightly regu¬ lated¹²'¹⁹'²⁷, is very sensitive to even small alterations in the medium composition such as the content of certain amino acids or peptides²'¹³'^15>17-²⁴. For that reason toxin formation is 0 not simply correlated with the general growth process ^17>19'²⁷ ₅ as frequently experienced by producers of tetanus toxoid.

In patent US6558926³ a process for producing tetanus toxin by using soy hydrolysate instead of casein hydrolysate and BHI in the fermentation medium was disclosed. Ac- .5 cording to this work the preferred soy product was Hy-Soy (Quest, now Kerry Bio- Science). Furthermore yeast extract and/or soy hydrolysate could replace BHI and animal derived peptone in the seed medium for C. tetani.

In patent application WO9854296¹⁸ the use of the soy hydrolysates for similar processes !0 was disclosed. The soy hydrolysates Hy-Soy (Kerry Bio-Science) and Soytone (Difco) could replace casein hydrolysate and BHI in the fermentation media for production of tetanus toxin by C. tetani. Soy hydrolysate (Soytone, Difco) or yeast extract (HyYest, Kerry Bio-Science) could replace casein hydrolysate in the fermentation media for pro¬ duction of diphtheria toxin, and finally soy hydrolysate (NZ-Soy, Kerry Bio-Science) .5 could replace casein hydrolysate in the fermentation media for production of pertussis toxin by B. pertussis. In addition yeast extract could be used, together with soy hydrolys¬ ate, in seed media for C. tetani.

The present invention provides a method for production of tetanus toxin and toxoid, diph- 0 theria toxin, and pertussis toxin based on non-animal and non-soy protein hydrolysates. Surprisingly these protein hydrolysates meets or exceeds the functionality of casein hy¬ drolysate and of soy hydrolysate in the manufacturing of tetanus, diphtheria, and pertussis toxins. Summary of the invention

The present invention comprises the finding that animal-derived and soy-derived compo¬ nents in the fermentation media for production of bacterial toxins can be replaced by components derived from vegetables of non-soy origin or from yeast.

In accordance with the present invention non soy- vegetable peptones derived from po- tato, wheat, rice, a mixture of wheat and rice, cotton, or pea can substitute for casein hy- drolysate and support growth of the bacteria. For C. tetani growth the preferred peptone is potato peptone which results in far better growth yield than different soy peptones tested and even better than casein hydrolysate, whereas wheat peptone and rice-wheat peptone meet the functionality of casein hydrolysate. For C. diphtheriae growth the pre- ferred peptone is potato peptone which results in far better growth yield than casein hy¬ drolysate and which meets the functionality of soy peptone, whereas rice peptone, rice- wheat peptone, and pea peptone meet the functionality of casein hydrolysate. For B. per¬ tussis growth the preferred peptone is derived from yeast, whereas rice-wheat peptone, wheat peptone, cotton peptone, and pea peptone meet or exceed the functionality of ca- sein hydrolysate.

The present invention also encompasses the finding that non-soy vegetable peptones de¬ rived from wheat, a mixture of wheat and rice, cotton, or pea can substitute for casein hydrolysate in the production medium and support toxin production by the bacteria. For toxin production by C. tetani the preferred peptones are wheat peptone and rice-wheat peptone which meet or exceed the functionality of casein hydrolysate and are far better than soy peptone, whereas yeast-derived peptone can partly replace casein hydrolysate. For toxin production by C. diphtheriae the preferred peptones are wheat peptone and rice- wheat peptone which meet or exceed the functionality of casein hydrolysate and are far better than soy peptone, whereas yeast-derived peptone can partly replace casein hy¬ drolysate. For toxin production by B. pertussis the preferred peptones are yeast peptone, cotton peptone, pea peptone, wheat peptone, and rice-wheat peptone which meet or ex¬ ceed the functionality of casein hydrolysate and are far better than soy peptone. The present invention also encompasses the finding that the quality (based on measure¬ ments of purity and toxicity) of the purified tetanus toxin produced on the basis of vege¬ table peptone derived from a mixture of wheat and rice is similar to the quality of purified toxin produced on the basis of casein hydrolysate.

Finally, the present invention encompasses the finding that the quality (based on meas¬ urements of purity and the potency) of a vaccine preparation using tetanus toxoid pro¬ duced on the basis of vegetable peptone derived from a mixture of wheat and rice is simi¬ lar to the quality of a vaccine preparation using tetanus toxoid produced on the basis of casein hydrolysate.

Detailed disclosure of the invention

The present invention disclose fermentation media for production of bacterial toxins, where the proteinaceous nutrient is derived from non-animal and non-soy protein.

The fermentation media disclosed encompasses i) seed media where the proteinaceous nutrient is a potato peptone, a rice-wheat peptone, or a wheat peptone and ii) fermentation media for growth (seed medium) and/or toxin production (production medium) where the proteinaceous nutrient is a wheat peptone, a rice peptone, a rice-wheat peptone, a potato peptone, a cotton peptone, a pea peptone, or a yeast peptone.

The disclosed fermentation media are used for production of tetanus, diphtheria or per¬ tussis toxin.

Also disclosed is the preferred peptone concentration in the fermentation medium as re¬ spectively between 5 - 50 g/1, preferably between 10 - 40 g/1 most preferably between 15 — 30 g/1 for tetanus toxin production, a peptone concentration between 10 - 50 g/1, pref¬ erably between 20 - 40 g/1, most preferably app. 33 g/1 for diphtheria toxin production, and a total concentration of peptone and glutamate between 10 - 30 g/1, preferably be¬ tween 15 - 25 g/1 most preferably app. 20 g/1 for production of pertussis toxin.

The invention also discloses the use of the above mentioned toxins produced with above mentioned fermentation media for the manufacture of a toxoid Finally the invention discloses a vaccine comprising a toxoid derived from a toxin pro¬ duced with above mentioned fermentation medium.

Definitions

The term "extract" means aqueous extracts of vegetable or microbial material, e.g. vege¬ table extract, yeast extract. Extracts are usually prepared by boiling a given amount of material and then using the liquid, or drying the broth and using the solids. The extracts are considered a complex material which contain both proteins, lipids, carbohydrates and micronutrients.

The term "vegetable extract" means aqueous extracts of any vegetable, containing amino acids and low molecular weight peptides, carbohydrates, vitamins and other growth fac- tors.

The term "yeast extract" means any yeast-derived proteinaceous material obtained by aqueous extraction or by enzymatic hydrolysis.

The term "meat extract" means any animal-derived proteinaceous material obtained by aqueous extraction or by enzymatic hydrolysis.

The term "infusion" means aqueous extract of animal tissues or plants.

The term "hydrolysate" or "peptone" means hydro lysed proteinaceous material formed by enzymatic or acid digestion.

The term "vegetable peptone" means proteinaceous material, derived from vegetables, which has been hydrolysed by use of microbial or vegetable enzymes, or by acid hy- drolysis. The protein substrate for forming peptones may be any proteinaceous material derived from vegetables, or protein concentrate isolated from flour of e.g. rice, wheat, or soy. The term "yeast peptone" means proteinaceous material derived from yeast cells, which has been hydrolysed by autolysis, or by use of microbial or vegetable enzymes, or by acid hydrolysis.

The term "fermentation medium" means any medium for cultivating bacteria either for growth in order to produce a seed culture to be used for inoculation of the production medium, or the production medium in which the bacteria grow and produce their toxin.

The term "seed medium" means any fermentation medium for cultivating bacteria in or- der to produce a seed culture to be used for inoculation of the production medium.

The term "production medium" means any fermentation medium in which the bacteria grow and produce their toxin.

Currently, standard media for toxin production contain animal-derived proteinaceous nutrients. By replacement of animal-derived components in media for vaccine produc¬ tion, the risk of carry-over to the final toxoid vaccine of undesirable contaminants from animals is reduced. The identification of a variety of alternatives to animal components ensures a wide spectrum of possibilities in attempts to avoid undesirable inherent con- stituents or impurities.

The present invention provides methods for using media based on non-animal and non- soy protein hydro lysates for growth of C. tetani, C, diphtheriae, or B. pertussis and pro¬ duction of tetanus toxin, diphtheria toxin and pertussis toxin, respectively, for use in for- mulations of toxoids and toxoid-based vaccines. The methods comprise growth media that contain no components of animal or soy origin.

In accordance with the present invention animal-derived and soy-derived components in the media for production of bacterial toxins can. be replaced by components derived from vegetables of non-soy origin or from yeast. The media include any standard media used for production of stock cultures (lyophilized or frozen), for any other long-term storage of cultures, for growth of seed cultures in a number of successive steps, and for production of toxin in any scale of fermentation. Non-animal and non-soy derived components in¬ clude hydrolysed proteinaceous materials derived from any vegetable or from yeast. The non-animal and non-soy peptones in the fermentation medium of the present invention may comprise two or more different materials, such as a mixture of the peptones.

In accordance with the present invention the toxins produced by the methods disclosed may be isolated, purified, detoxified to toxoids, and used for formulation of vaccines by methods well known to those skilled in the art.

The present invention surprisingly revealed that non-animal and non-soy based media result in increased growth of C. tetani, C, diphtheriae, and B. pertussis and result in in- creased yield of tetanus toxin, diphtheria toxin and pertussis toxin, respectively, com¬ pared to cultivation of the bacteria in media containing animal-derived or soy-derived proteinaceous material. Preferably in accordance with the present invention, the proteina- ceous hydro lysates are derived from -wheat, rice, a mixture of rice and wheat, pea, cotton, potato, or from yeast.

The present invention also reveal the finding that the quality (purity and toxicity) of puri¬ fied tetanus toxin produced on the basis of vegetable peptone derived from a mixture of wheat and rice is similar to the quality of purified tetanus toxin produced on the basis of casein hydrolysate.

Finally, the present invention disclose the finding that the quality (purity and the potency) in a vaccine preparation of tetanus toxoid produced on the basis of vegetable peptone derived from a mixture of wheat and rice is similar to the quality of tetanus toxoid pro¬ duced on the basis of casein hydrolysate.

Seed media.

The present invention provides a method for the production of seed cultures for inocula¬ tion of the production medium. For seed culture the bacteria are grown in a seed medium in one or more successive steps depending on the volume of the production medium. The seed media generally contain animal-based or soy-based hydrolysates as the source of proteinaceous nutrient. In accordance with the present invention the hydrolysates in the seed media may be replaced by non-animal and non-soy based proteinaceous hydrolys¬ ates derived from potato, wheat, rice, a mixture of rice and wheat, pea, cotton, or from yeast. Preferably the bacteria are cultured in the seed medium under suitable conditions for growth. The suitable conditions for growth and the culture period will vary depending on the bacterium being cultured.

For C. tetani the seed medium may be any standard medium for cultivation of anaerobic microorganisms where the animal-derived or soy-derived proteinaceous material has been replaced by proteinaceous hydrolysate derived from non-soy vegetables. Any source of non-soy vegetable may be used. In accordance with the present invention casein hy¬ drolysate is replaced by peptones derived from potato, wheat, or a mixture of rice and wheat. In accordance with the present invention the concentration of the vegetable hydro¬ lysate in the seed medium ranges between 5 — 50 g/1, preferably between 10 - 40 g/1, most preferably between 15 — 30 g/1. According to the present invention C. tetani is sus¬ pended in the seed medium and incubated at a temperature permitting growth of the bac¬ terium, preferably 35 ± 1 ⁰C, for 24 - 72 h, preferably 24 ± 4 h, and subsequently used for inoculation of the production medium or for a subsequent seed culture.

For C. diphtheriae the seed medium may be any standard medium for cultivation of aero¬ bic microorganisms where the animal-derived or soy-derived proteinaceous material has been replaced by proteinaceous hydrolysate derived from non-soy vegetables. Any source of non-soy vegetable may be used. In accordance with the present invention casein hy¬ drolysate is replaced by peptones derived from potato, wheat, rice, a mixture of rice and wheat, pea, cotton, or yeast. In accordance with the present invention the concentration of the vegetable hydrolysate in the seed medium is in the range between 10 - 50 g/1, pref¬ erably 20 - 40 g/1, most preferably app. 33 g/1. According to the present invention C diphtheriae is suspended in the seed medium and incubated at a temperature permitting growth of the bacterium, preferably 35 ± 1 ⁰C, for 24 - 72 h, preferably 24 ± 4 h, and subsequently used for inoculation of the production medium or for a subsequent seed culture.

For B. pertussis the seed medium may be any standard medium for cultivation of aerobic microorganisms where the animal-derived or soy-derived proteinaceous material has been replaced by proteinaceous hydrolysate derived from non-soy vegetables, or where the amino acid nutrients has been supplemented with proteinaceous hydrolysate derived from non-soy vegetables. Any source of non-soy vegetable may be used. In accordance with the present invention casein hydrolysate is replaced by peptones derived from wheat, a mixture of rice and wheat, pea, cotton, potato, or yeast. In accordance with the present invention the total concentration of vegetable peptone and glutamate in the seed medium is in the range between 10 — 30 g/1, preferably between 15 - 25 g/1, most pref- erably app. 20 g/1. According to the present invention B. pertussis is suspended in the seed medium and incubated at a temperature permitting growth of the bacterium, prefera¬ bly 36 ± 2 ⁰C, for 24 - 72 h, preferably 24 ± 4 h, and may subsequently be used for in¬ oculation of the production medium or for a subsequent seed culture.

Production media.

For the production of toxins by C. tetani, C. diphtheriae or B. pertussis the bacteria are grown in a production medium. Production media generally contain animal-based or soy- based hydrolysates as the source of proteinaceous nutrient. In accordance with the present invention the animal-based or soy-based hydrolysates in the production media may be replaced by non-animal and non-soy based proteinaceous hydrolysates derived from a mixture of rice and wheat, from wheat, from cotton, from pea, from potato, or from yeast. Preferably the bacteria are cultured in the production medium under suitable conditions for toxin production. The suitable conditions for toxin production and the culture period will vary depending on the bacterium being cultured.

For C. tetani, production media are based on media containing animal-derived proteina¬ ceous nutrients like casein hydrolysate, or alternatively non-animal soy hydrolysate may be used. According to the present indention the animal-derived or soy-derived proteina- ceous hydrolysate is replaced by proteinaceous hydrolysate derived from non-soy vegeta¬ bles or from yeast. In accordance with the present invention casein hydrolysate is re¬ placed by peptones derived from wheat, a mixture of rice and wheat, pea, cotton, potato, or yeast. In accordance with the present invention the concentration of the vegetable hy¬ drolysate in the fermentation medium in test-tube cultures is in the range between 10 — 30 g/1, preferable between 12 — 18 g/1, most preferably app. 15 g/1, and in fermentor cultures between 5 - 50 g/1, preferably between 10 - 40 g/1, most preferably between 15 - 30 g/1. According to the present invention C. tetani is grown in the production medium until cell lysis is complete, at a temperature permitting growth of the bacterium, preferably 35 ±= 1 ⁰C. For C. diphtheriae, production media are based on media containing animal-derived pro- teinaceous nutrients like casein hydrolysate, or alternatively vegetable soy hydrolysate may be used. According to the present invention the animal-derived or soy-derived pro- teinaceous hydrolysate has been replaced by proteinaceous hydrolysate derived from noji- soy vegetables or from yeast. In accordance with the present invention casein hydrolysate is replaced by peptones derived from wheat, rice, a mixture of rice and wheat, pea, cot¬ ton, potato, or from yeast. In accordance with the present invention the concentration of the vegetable hydrolysate in the fermentation medium is in the range between 10 - 50 g/1, preferable between 25 — 35 g/1, most preferably app. 33 g/1. According to the present in¬ vention C. diphtheriae is grown in the production medium until cell growth ceases, at a temperature permitting growth of the bacterium, preferably 35 ± 1 ⁰C.

For B. pertussis, production media may be based on media containing glutamate and animal-derived proteinaceous nutrients like casein hydrolysate, or alternatively vegetable soy hydrolysate may be used. According to the present invention the animal-derived or soy-derived proteinaceous hydrolysate has been replaced by proteinaceous hydrolysate derived from non-soy vegetables or from yeast. In accordance with the present invention casein hydrolysate is replaced by peptones derived from wheat, a mixture of rice and wheat, pea, cotton, potato, or from yeast. In accordance with the present invention the total concentration of vegetable peptone and glutamate in the fermentation medium is in the range between 10 - 30 g/1, preferable between 15 - 25 g/1, most preferably app. 20 g/1. According to the present irrvention B. pertussis is grown in the production medium until cell growth ceases, at a temperature permitting growth of the bacterium, preferably 36 ± 2 ⁰C.

Experimental results

C. tetani:

The medium components for production of tetanus toxin by C. tetani are shown in Tables 1 - 3. Experiments were performed to test the ability of a range of vegetable peptones and yeast peptone to replace the casein hydrolysate in the fermentation media. The results of the experiments are summarized in Tables 4 - 6. For growth of C. tetani in seed medium vegetable peptones derived from potato, wheat, or a mixture of rice and wheat, can substitute for casein hydrolysate (Table 4). The best peptone for growth in seed medium was a potato peptone (Plant peptone El 19025, Or- ganotechnie) which resulted in far better growth yield (as measured by optical density) than several soy peptones tested and even better than casein hydrolysate, whereas wheat peptones (HyPep 4601, Kerry Bio-Science and El 19555, Organotechnie) and rice-wheat peptone (HyPep 5603, Kerry Bioscience) met the functionality of casein peptone.

For production of tetanus toxin by C. tetani vegetable peptones derived from a mixture of rice and wheat, from wheat, or from yeast can substitute for casein hydrolysate in the production medium. Screening experiments in test tubes (Table 5) indicated that the best peptones for production of tetanus toxin was a rice-wheat peptone (HyPep 5603, Kerry Bio-Science), which was far better than soy peptones and even gave a higher yield of tetanus toxin than casein hydrolysate. The test-tube experiments showed that peptones in the concentration range of 12 - 27.5 g/1 support production of tetanus toxin. The results, however, also revealed that the optimal concentration of vegetable or yeast peptone for toxin production may be specific for each peptone and different from the optimal concen¬ tration of casein hydrolysate in trie control medium. Experiments in lab-scale fermentor cultures (Table 6) showed that the toxin yields were also dependent on the culture condi¬ tions, thus the highest yields of tetanus toxin with the rice-wheat peptone were obtained at app. 15 g peptone per litre in the test-tube experiments (Table 5) and at 30 g/1 in fer¬ mentor cultures (Table 6). Furthermore, the experiments showed that in fermentor cul¬ tures the rice-wheat peptone (HyPep 5603, Kerry Bio-Science) and the wheat peptone (HyPep 4601 , Kerry Bio-Science) resulted in far better toxin yields than casein peptone.

C. diphtheriae:

The medium components for production of diphtheria toxin by C. diphtheriae are shown in Tables 1 and 7. Experiments were performed to test the ability of a range of vegetable peptones and yeast peptone to replace the casein hydrolysate in the media. The results of the experiments are summarized in Table 8. The results reveal that vegetable peptones derived from wheat, rice, a mixture of wheat and rice, pea, potato, or yeast can substitute for casein hydrolysate and support growth and/or toxin production in the medium for C. diphtheriae. For growth of C. diphtheriae potato peptone (Plant peptone El 19025, Organotechnie) met the functionality of soy peptones and was far better than casein hydrolysate (as measured by optical density) (Ta¬ ble 8). For production of diphtheria toxin by C. diphtheriae vegetable peptones derived from a mixture of rice and wheat, from wheat, or from yeast can substitute for casein hy¬ drolysate (Table 8). The best peptones for production of diphtheria toxin were a rice- wheat peptone (HyPep 5603, Kerry Bio-Science) or a wheat peptone (HyPep 4602, Kerry Bio-Science), which met the functionality of casein hydrolysate and were far better than the soy peptones. Peptone derived from yeast (19512, Organotechnie) could partly re¬ place casein hydrolysate.

B. pertussis:

The medium components for production of pertussis toxin by B, pertussis are shown in Tables 1 and 9. Experiments were performed to test the ability of a range of vegetable peptones and yeast peptone to replace the casein hydrolysate in the media. The results of the experiments are summarized in Table 10.

The results reveal that vegetable peptones derived from wheat, a mixture of wheat and rice, pea, cotton, potato, or yeast can substitute for casein hydrolysate and support growth and/or toxin production in the medium for B. pertussis. For growth of B. pertussis (Table 10) wheat peptone (HyPep 4601, Kerry Bio-Science), cotton peptone (HyPep 7504, Kerry Bio-Science), pea peptone (Hy-Pea 7404, Kerry Bio-Science) and yeast extract

(19512, Organotechnie) met or exceeded the functionality of casein hydrolysate and soy peptones (as measured by optical density). For production of pertussis toxin by B. pertus¬ sis (Table 10) vegetable peptones derived from wheat, a mixture of rice and wheat, cot¬ ton, or pea, or yeast peptone can substitute for casein hydrolysate. The best peptones for production of pertussis toxin were cotton peptone (HyPep 7504, Kerry Bio-Science), rice-wheat peptone (HyPep 5603, Kerry Bio-Science), and yeast peptone (HYP-A, Bio Springer) which greatly exceeded the functionality of casein hydrolysate and soy pep¬ tone, a pea peptone (Hy-Pea 7404, Kerry Bio-Science) which was far better than casein hydro Iy sate and met the functionality of soy peptone, and a wheat peptone (HyPep 4601, Kerry Bio-Science) which exceeded the functionality of casein hydrolysate.

Examples

In the following examples practicable methods and results of the present invention are illustrated. The presented methods are only a selection of possible ways of conducting these experiments. Similar results may be obtained by variety of standard methods which are encompassed by the present invention.

The range of peptones used in the experiments is shown in Table 1.

TABLE 1. Peptones in fermentation media

Soy peptone (Hy-Soy, Kerry Bio-Science)

Soy peptone (HyPep 1510, Kerry Bio-Science)

Soy peptone (A2 19549, Organotechnie)

Soy peptone (A3 19585, Organotechnie)

Wheat peptone (HyPep 4601, Kerry Bio-Science)

Wheat peptone (HyPep 46O2, Kerry Bio-Science)

Wheat peptone (HyPep 46O5, Kerry Bio-Science)

Wheat peptone (Hy- Wheat 4321, Kerry Bio-Science)

Wheat peptone (E 1 19559, Organotechnie)

Rice- wheat peptone (HyPep 5603, Kerry Bio-Science)

Rice peptone (Hy-Rice 53O3, Kerry Bio-Science)

Potato peptone (Plant peptone El 19025, Organotechnie)

Pea Peptone (HyPep 7401 dev, Kerry Bio-Science)

Pea Peptone (Hy-Pea 7404, Kerry Bio-Science)

Cotton Peptone (HyPep 7504, Kerry Bio-Science)

Cotton Peptone (Hy-Cotton, Kerry Bio-Science)

Yeast peptone (HYP-A, Bio Springer)

Yeast extract (19512, Organotechnie)

Casein hydrolysate (Pepticase, Kerry Bio-Science). Control (Tetanus)

Casein hydrolysate (N-Z- Amine A, Kerry Bio-Science). Control (Diphtheria) Casamino acids (Bacto). Control (Pertussis) Example 1

The purpose of these experiments was to investigate the ability of non-animal peptones derived from the non-soy vegetables rice, wheat, cotton, pea, or potato, or from yeast, to sustain growth in the seed medium and/or to replace casein hydrolysate in the production medium used for manufacture of tetanus toxin by C. tetani.

Microorganism,

Lyophilized culture of Clostridium tetani (Albany-Harvard strain 43415). The strain is maintained as frozen culture at ÷80 ⁰C in 10% (v/v) glycerol (working seed).

Media and culture conditions.

All media and solutions were prepared with water for injections (WFI).

Seed culture.

The composition of the seed medium is shown in Tables 1 and 2. The seed medium, was distributed in 10-ml aliquots in test tubes (13 x 150 mm). Test tubes with seed medium was inoculated with 1 ml working seed and incubated at 35 ± 1 ⁰C for 24 — 72 h. For use as inoculum of the production medium the seed cultures were incubated for 24 ± 4 b.

TABLE 2. Seed medium for C. tetani (pr. 1000 ml)

Peptone¹ 15 g

Yeast extract (19512, Organotechnie) 5 g

Glucose monohydrate 5.5 g

NaCl 2.5 g

Sodium thioglycollate 0.5 g

L-Cystine 0.5 g

FeSO₄-7H₂O 40 mg pH (adjusted with NaOH) 7.3

Peptones shown in Table 1. Peptone type as specified in the Results section. Production culture.

The composition of the production medium is shown in Tables 1 and 3. The medium was distributed in 20-ml aliquots in test tubes (18 x 180 mm); 20 ml production medium was inoculated with 0.5 ml seed culture. Alternatively, the medium was distributed in lab- scale fermentors (2.0 1 working volume); 2,0 1 production medium was inoculated with 6.0 ml seed culture. All cultivations were run at 35 ± 1 ⁰C for 4 - 9 days until cell autoly¬ sis had ceased. Each separate medium test included a control medium with casein hydro- lysate at 27.5 g/1.

TABLE 3. Production medium for C. tetani (pr. 1000 ml)

Peptone¹ 12 - 27.5 g

Glucose monohydrate (^TQ

NaCl 2.5 g

KH₂PO₄ 0.15 g

MgSO₄-7H₂O 0.15 g

Na₂HPO₄-2H₂O 2.5 g

Sodium citrate, dihydrate 0.4O g

L-Cystine 0.25

Tyrosine 0.50 g

Uracil 2.5 mg

Calcium pantothenate 2.00 mg

Thiamine hydrochloride 0.50 mg

Pyridoxine hydrochloride 0.50 mg

Riboflavin phosphate, sodium salt, dihydrate 0.64 mg

Biotin 5.0 μg

Nicotinic acid 0.25 mg

Cyanocobalamine 5.0 μg

FeSO₄-7H₂O 40 mg pH (adjusted with NaOH) 7.3

Peptones shown in Table 1. Peptone type and peptone concentration as specified in the Results section. Measurement of cell density.

Cell density was determined spectrophotometrically at 600 run.

Measurement of tetanus toxin. When cell autolysis had ceased (after 7 - 9 days) the concentration of tetanus toxin (in Lf/ml) was determined with Ramon's flocculation test. 1 Lf (limit of flocculation) is equivalent to 1 unit of reference antitoxin (K 1/67, Statens Serum Institut); the reference antitoxin is calibrated against an international reference toxoid (The 1^st International Ref¬ erence Reagent of Tetanus Toxoid for Flocculation Test, NIBSC, UK).

Manufacture of purified tetanus toxin and detoxified tetanus toxoid.

Tetanus toxin in fermentation culture filtrate was purified and detoxified by standard methods.

Measurement of purity of purified tetanus toxin and detoxified tetanus toxoid.

Total nitrogen (TN) and protein nitrogen (PN) were measured by Kjeldahl analyses ac¬ cording to European Pharmacopoeia. HPLC analyses were size-exclusion chromatogra¬ phy according to European Pharmacopoeia.

Measurement of toxicity of tetanus toxin.

Toxicity (L+/ 10/50) of purified tetanus toxin was measured according to "Manual for the production and control of vaccines. Tetanus toxoid" (WHO, BLG/UNDP/77.2).

Measurement of potency of tetanus toxoid. Potency of detoxified tetanus toxoid was measured according to European Pharmaco¬ poeia Assay of Tetanus Vaccine (adsorbed) 2.7.8.

Results:

The ability of vegetable peptones and casein hydrolysate to support growth of C. tetani in seed cultures is shown in Table 4. Good growth was obtained with all the tested peptones. Potato peptone (Plant peptone El 19025, Organotechnie) gave the highest growth yield after 24 h, which is the time of inoculation of the secondary seed culture or of the produc- tion medium. Potato peptone resulted in far better growth of C. tetani than a range of dif¬ ferent soy peptones and even better than casein hydrolysate.

TABLE 4. Growth of C. tetani in seed media with different peptones

Peptone in seed medium Growth Growth

(ODfiOnm) (OD_{600 nm}) after 24 h after 24 h

(%) ^l

Soy peptone (HyPep 1 510, Kerry Bio-Science) 1,00 79

Soy peptone (A2 19549, Organotechnie) 1,22 98

Soy peptone (A3 19585, Organotechnie) 1,27 103

Wheat peptone (HyPep 4601, Kerry Bio-Science) 1,44 105

Wheat peptone (El 19559, Organotechnie) 1,26 106

Rice-wheat peptone (HyPep 5603, Kerry Bio-Science) 1,55 106

Potato peptone (Plant peptone El 19025, Organotechnie) 1,69 127

Casein peptone (Pepticase, Kerry Bio-Science) 1,25 100

Growth is given as percentage of control medium with casein hydrolysate (Pepticase, Kerry Bio- Science). Data were corrected for the difference in absorbancy at time 0.

The ability of a range of different vegetable peptones and a yeast peptone to replace ca¬ sein hydrolysate in the production medium and support production of tetanus toxia by C. tetani in test-tube experiments is shown in Table 5. The vegetable peptones were tested at different concentrations as indicated in the table. All seed cultures were grown in seed medium with potato peptone.

The results presented in Table 5 reveal that vegetable protein hydrolysates obtained from non-soy vegetables or from yeast are able to replace casein hydrolysate in the medium for production of tetanus toxin by C. tetani in test-tube cultures. The best peptone for toxin production was the rice-wheat peptone HyPep 5603 (Kerry Bio-Science), which was far better than the soy peptones and even gave a higher toxin yield than casein hydrolysate. The results reveal the optimal concentration of peptone for production of toxin was spe- cific for each peptone and in most cases different from the concentration of casein hydro¬ lysate in the control medium. The highest yield of toxin with the rice-wheat peptone in test-tube experiments was obtained with 15 g peptone per litre. TABLE 5. Production of tetanus toxin by C. tetani in production media with different peptones. Test-tube cultures

Peptone type Concentration Concentration of peptone (g/1) of toxin C/o) ¹

Rice-wheat peptone 12 82

(HyPep 5603, Kerry Bio-Science) 15 104

17.5 91

22.5 44

27.5 36

Soy peptone 15 75

(Hy-Soy, Kerry Bio-Science) 27.5 50

Soy peptone 15 35

(HyPep 1510, Kerry Bio-Science) 27.5 75

Soy peptone 15 40

(A2 19549, Organotechnie) 27.5 45

Soy peptone 15 35

(A3 19585, Organotechnie) 27.5 45

Wheat peptone 15 45

(HyPep 4601, Kerry Bio-Science) 27.5 32

Wheat peptone 15 36

(HyPep 4602, Kerry Bio-Science) 27.5 32

Wheat peptone 15 30

(El 19559, Organotechnie) 27.5 22

Potato peptone 15 8

(Plant peptone El 19025, Organotechnie) 27.5 10

Pea peptone 27,5 10

(HyPep 7401 dev, Kerry Bio-Science)

Cotton peptone 27,5 28

(HyPep 7504, Kerry Bio-Science)

Yeast peptone 15 63

(HYP-A, Bio Springer) 27.5 42

Concentration of toxin in production medium is given as percentage of control medium with casein hydrolysate at 27.5 g/1 (Pepticase, Kerry Bio-Science). The ability of selected vegetable peptones to replace casein hydro Iy sate in the production medium and support production of tetanus toxin by C. tetani in fermentor cultures is shown in Table 6. The vegetable peptones were tested at the concentrations as indicated in the table. All seed cultures were grown in seed medium with potato peptone.

The results presented in Table 6 reveal that vegetable protein hydrolysates obtained from non-soy vegetables axe able to replace casein hydrolysate in the medium for production of tetanus toxin by C. tetani also in fermentor cultures. For production of tetanus toxin rice-wheat peptone Hy Pep 5603 (Kerry Bio-Science) and wheat peptone HyP ep 4601 (Kerry Bio-Science) were far better than casein hydrolysate or soy peptone. The results reveal the optimal concentration of vegetable peptone for production of toxin may be different than observed in the test-tube experiments, thus the highest yield of^" toxin with the rice-wheat peptone in fermentor experiments was obtained with 30 g peptone per li¬ tre. However, 30 g peptone per litre was the maximum concentration tested in these ex¬ periments and higher concentrations of peptone may support even higher toxin produc¬ tion.

TABLE 6. Production of tetanus toxin by C. tetani in production media with different peptones. Fermentor cultures

Peptone type Concentration Concentration of peptone (g/1) of toxin (%) ^l

Rice-wheat peptone 15 64

(HyPep 5603, Kerry Bio-Science) 20 120

25 125

30 150

Wheat peptone 27,5 150

(HyPep 4601, Kerry Bio-Science)

Soy peptone 27,5 88

(Hy-Soy, Kerry Bio-Science)

¹ Concentration of toxin in production medium is given as percentage of control medium with casein hydrolysate at 27.5 g/1 (Pepticase, Kerry Bio-Science). Comparison of the results in Table 5 and Table 6 illustrates that results obtained, from test-tube cultures should be taken only as an preliminary indication of whether the non- animal peptones support growth and toxin production. In fermentor cultures, where con¬ ditions generally are much more favourable for growth and toxin production, a larger selection of the peptones may have the ability to replace casein hydrolysate.

Tetanus toxins in fermentor culture filtrates from fermentations based on rice-wheat pep¬ tone and on casein hydrolysate (control), respectively, were purified. The specific purity (Lf/mg PN) and the purity judged by Lf/mg TN of purified tetanus toxin produced on the basis of rice- wheat peptone gave results which were in compliance with the current speci¬ fications, and were at the same levels as the purity of purified tetanus toxin produced on the basis of casein hydrolysate. Furthermore, qualitative judgement of the purity of puri¬ fied tetanus toxin by HPLC analyses showed no differences between purified tetanus toxin produced on the basis of rice-wheat peptone and purified tetanus toxin produced on the basis of casein hydrolysate.

The toxicity (L+/ 10/50) is an indirect measure of the integrity and biological function of the toxin molecule. The results of the measurements of the toxicity of purified tetanus toxin produced on the basis of rice-wheat peptone were in compliance with the current specifications and at the same level as the toxicity of purified tetanus toxin produced on the basis of casein hydrolysate.

Purified tetanus toxins produced on the basis of rice-wheat peptone and on casein hydro¬ lysate (control), respectively, were detoxified. The specific purity (Lf/mg PN) and the purity judged by Lf/mg TN of detoxified tetanus toxoid produced on the basis of rice- wheat peptone gave results which were in compliance with the current specifications, and were at the same levels as the purity of tetanus toxoid produced on the basis of casein hydrolysate. Furthermore, qualitative judgement of the purity of tetanus toxoid by HPLC analyses showed no differences between tetanus toxoid produced on the basis of rice- wheat peptone and tetanus toxoid produced on the basis of casein hydrolysate.

Potency is a measure of the ability of the toxoid to induce a immune response, thus an indirect measure of the integrity and biological function of the toxoid molecule. The re¬ sults of the measurements of the potency of a preparation of tetanus vaccine produced on the basis of rice-wheat peptone were in compliance with the current specifications and at the same level as the potency of tetanus vaccine produced on the basis of casein hydro- Iy sate.

Example 2

The purpose of these experiments was to investigate the ability of non-animal peptones derived from the non-soy vegetables rice, wheat, pea, cotton, or potato, or from yeast, to sustain growth and/or production of diphtheria toxin in media used for production of diphtheria toxin by C. diphtheήae.

Microorganism.

Lyophilized culture of Corynebacterium diphtheriae. The strain is maintained as frozen culture at ÷80 ⁰C in 10% (v/v) glycerol (working seed).

Media and culture conditions.

All media and solutions were prepared with WFI.

Fermentation medium. The composition of the medium is shown in Tables 1 and 7. The medium was distributed in 200-ml aliquots in 500-ml Erlenmeyer flasks. Flasks with medium were inoculated with 0.5 ml working seed and incubated at 35 ± 1 ⁰C in an orbital shaker at 150 rpm. When cell growth had ceased (after 24 - 72 h) growth and formation of diphtheria toxin was measured. Each separate medium test included a control medium with casein hydro- lysate.

TABLE 7. Fermentation medium for C. diphtheriae (pr. 1000 ml)

Peptone¹ 33 g

Na₂HP0₄'2H₂O 2.2 g

Maltose monohydrate 25 g

Sodium lactate 1.4 g

K₂HPO₄ 0.81 g

KH₂PO₄ 0.27 g

Cystine 0.22 g

MgSO₄-7H₂O 1.8 g

CaCl₂-2H₂O 5 g β-alanine 18.4 mg

CuSO₄- 5H₂O 4 mg

ZnSO₄-7H₂O 6.4 mg

MnCl₂4H₂O 1.9 mg

Pimelic acid 1.2 mg

Nicotinic acid 36.8 mg

FeSO₄-7H₂O 13.5 mg pH (adjusted with NaOH/acetic acid) 7.5

Peptones shown in Table 1. Peptone type specified in the Results section.

Measurement of cell density. Cell density was determined spectrophotometrically at 600 nm.

Measurement of diphtheria toxin.

The concentration of diphtheria toxin (in Lf/ml) was determined with Ramon's floccula- tion test. 1 Lf (limit of flocculation) is equivalent to 1 unit of reference antitoxin (1971, Statens Serum Institut); the reference antitoxin is calibrated against an international refer¬ ence toxoid (The 1^st International Reference Reagent for Diphtheria toxoid for Floccula¬ tion Test, NIBSC, UK). Results:

The ability of a range of vegetable peptones and a yeast peptone to replace casein hydro- lysate and support growth and toxin production with C. diphtheriae is shown in Table 8.

The results presented in Table 8 reveal that vegetable protein hydrolysates of non-soy or yeast origin were able to replace casein hydrolysate in the medium for growth of C. diph¬ theriae. Good growth was obtained with most of the tested peptones. Potato peptone (Plant peptone El l 9025, Organotechnie) met the functionality of soy peptones and was far better than casein hydrolysate for growth of C. diphtheriae (as measured by optical density). Rice peptone (Hy-Rice 5303, Kerry-Bio-Science), rϊce-wheat peptone (HyPep 5603, Kerry-Bio-Science), and pea peptone (Hy-Pea 7404, KLerry-Bio-Science) met the functionality of casein hydrolysate for growth of C. diphtheriae.

The results presented in Table 8 also reveal that vegetable protein hydrolysates of non- soy or yeast origin were able to replace casein hydrolysate for production of diphtheria toxin by C. diphtheriae. The best peptones for toxin production were rice-wheat peptone HyPep 5603 (Kerry Bio-Science) and wheat peptone HyPep 4602 (Kerry Bi-Science), which met or exceeded the functionality of casein hydrolysate and were far better than the soy peptones.

TABLE 8. Growth and production of diphtheria toxin by C. diphtheriae in fermenta- tion medium with different peptones

Peptone type Growth (OD₆₀O n_n.) Concentration (%) ^l of toxin C/o) ¹

Rice-wheat peptone 109 98 ² - 118 ³ (HyPep 5603, Kerry Bio-Science)

Rice peptone 92 36 (Hy-Rice 5303, Kerry Bio-Science)

Soy peptone 104 55 (Hy-Soy, Kerry Bio-Science)

Soy peptone 115 57 (HyPep 1 510, Kerry Bio-Science)

Soy peptone 138 42 (A2 19549, Organotechnie)

Soy peptone 136 28 (A3 19585, Organotechnie)

Wheat peptone 77 94 (HyPep 4602, Kerry Bio-Science)

Wheat peptone 61 84 (HyPep 4605, Kerry Bio-Science)

Wheat peptone 63 37 (HyPep 4601, Kerry Bio-Science)

Wheat peptone 34 17 (Hy- Wheat 4321, Kerry Bio-Science)

Wheat peptone 73 47 (El 19559, Organotechnie)

Cotton peptone 60 11 (Hy-Cotton7803, Kerry Bio-Science)

Pea peptone 97 6 (Hy-Pea 7404, Kerry Bio-Science)

Potato peptone

137 22 (Plant peptone El 19025, Organotechnie)

Yeast peptone

40 49 (HYP-A, Bio Springer)

Yeast extract

83 59 (19512, Organotechnie) Table 8: ' Growth and concentration of diphtheria toxin in production medium is given as percentage of control medium with casein hydrolysate (N-Z-Amine A, Kerry Bio-Science). Data given are from the time of cessation of cell growth where the concentration of toxin was maximal (after 24, 48 or 72 h). Growth data were corrected for the difference in absorbancy at time 0. ²48 h ³72 h

The range of peptones shown in Table 8 were tested only at one single concentration which was similar to the concentration of casein hydrolysate in the control medium. However, higher or lower concentrations of the peptones may support even higher growth of C. diphtheriae and/or production of diphtheria toxin.

Example 3

The purpose of these experiments was to investigate the ability of non-animal peptones derived from the non-soy vegetables rice, wheat, cotton, pea, or potato, or from yeast to replace the casamino acids in the media for growth and production of pertussis toxin by B. pertussis.

Microorganism.

Lyophilized cultures of Bordetella pertussis. Strains are maintained as lyophilized cul¬ tures (working seeds).

Media and culture conditions. All media and solutions were prepared with WFI.

Fermentation medium.

The fermentation medium was basically CL medium⁹, which was also used in patent ap¬ plication WO9854296¹⁸. The composition of the medium is shown in Tables 1 and 9. The medium was distributed in 50-ml aliquots in 250-ml Erlenmeyer flasks. Flasks with me¬ dium were inoculated with 6 ml working seed. After 24 - 72 h incubation at 36 ⁰C growth and formation of pertussis toxin (PT) was measured. Each separate medium test included a control medium with casamino acids. TABLE 9. Fermentation medium for B. pertussis (pr. 1000 ml)

L-Glutaniate 10.7 g

Peptone¹ 1,0 - 1O g

L-Proline 0,24 g

NaCl 2,5 g

KH₂PO₄ 2,69 g

K₂HPO₄-3H₂O 6,87

KCl 0,2 g

MgCl₂-OH₂O 0,I g

CaCl₂-2H₂O 0,02 g

L-cystine 0,04 g

L-ascorbic acid 0,02 g

Niacin 4 mg

Reduced glutathione O₅I g

FeSO₄-7H₂O O₅OI g

Trizma base 1,52 g

Heptakis(2,6-O-dimethyl)β-Cyclodextrin 1.00 g

Antifoam C emulsion 0,13 ml

Peptones shown in Table 1. Peptone type and concentration as specified in the Re¬ sults section.

Measurement of cell density. Cell density was determined spectrophotometrically at 650 nm.

Measurement of pertussis toxin.

Pertussis toxin (PT) was measured by a quantitative PT ELISA using monoclonal PT antibodies (20.6 Gl 2 H2-prot A-SSI, and 21.3 Di₁-SSI, Statens Serum Institut).

Results:

The ability of a range of vegetable peptones and a yeast peptone to replace casein hydro- lysate and support growth and toxin production with B. pertussis is shown in Table 10. The results presented in Table 10 reveal that vegetable protein hydrolysates of non-soy or yeast origin were able to replace casein hydrolysate in the medium for growth of B. per¬ tussis. Good growth was obtained with all of the tested peptones. The best peptones for growth were yeast extract (19512, Organotechnie) and yeast peptone (HYP-A, Bio Springer) which exceeded the functionality of casein hydrolysate for growth of B. pertus¬ sis (as measured by optical density). Rice-wheat peptone (HyPep 5603, Kerry-Bio- Science), wheat peptone (HyPep 4601, Kerry-Bio-Science), cotton peptone (HyPep 7504, Kerry-Bio-Science), and pea peptone (Hy-Pea 7404, Kerry-Bio-Science), met the func¬ tionality of casein hydrolysate for growth.

The results presented in Table 10 also reveal that vegetable protein hydrolysates of non- soy origin were able to replace casein hydrolysate for production of pertussis toxin by B. pertussis. The best peptones for toxin production were the cotton peptone (HyPep 7504, Kerry Bio-Science) and the yeast peptone (HYP-A, Bio Springer), which greatly ex- ceeded the functionality of casein hydrolysate and were far better than the soy peptones. Wheat peptone (HyPep 4601, Kerry Bio-Science), pea peptone (Hy-Pea 7404, Kerry Bi- Science), and rice-wheat peptone (HyPep 5603, Kerry Bio-Science) exceeded the func¬ tionality of casein hydrolysate, and the pea peptone (Hy-Pea 7404, Kerry Bio-Science) met the functionality of soy peptone.

TABLE 10. Growth and production of pertussis toxin by B. pertussis in fermentation medium with different peptones

Growth and concentration of pertussis toxin in production medium is given as percentage of control me¬ dium with casein hydrolysate 10 g/1 (Casamino acids, Bacto). Data given are maximum values within a growth period of 48 h). Growth data were corrected for the difference in absorbancy at time 0.

All peptones shown in Table 10 were tested at a concentration similar to the concentra¬ tion of casein hydrolysate in the control medium. A few selected peptones were tested at a lower concentration as well. For the control medium (casein hydrolysate) and the rice- wheat peptone medium a reduced peptone concentration resulted in improved growth and toxin yields, whereas for cotton peptone a reduced peptone concentration resulted in de¬ creased growth and toxin yields. These results indicate that the optimal concentration of peptone may be specific for each peptone, i.e. higher or lower concentrations of the pep¬ tones may support even higher growth of B. pertussis and/or production of pertussis toxin.

Reference List

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2. Bizzini, B. 1979. Tetanus toxin. Microbiological Reviews 43:224-240. 3. Demain, A. and Fang, A. Method for production of tetanus toxin using media sub¬ stantially free of animal products. [US 6558926], 1-39. 2003. Ref Type: Patent

4. Demain, A., D. F. Gerson, and A. Fang. 2005. Efective levels of tetanus toxin can be made in a production medium totally lacking both animal (e.g., brain heart infu- sion) and dairy proteins or digests (e.g., casein hydrolysates). Vaccine, in press xxx:xxx.

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Ref Type: Patent

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Claims

1. A fermentation medium for producing bacterial toxins where the protein source is non-animal and non-soy derived protein.

2. A fermentation medium according to claim I₅ where the medium is the seed me¬ dium and the protein source is potato peptone, wheat peptone, a rice peptone, a cotton peptone, a pea peptone, a yeast peptone or rice-wheat peptone.

3. A fermentation medium according to claim 1 , where the medium is the production medium and the protein source is a wheat peptone, a rice peptone, a rice-wheat peptone, a potato peptone, a cotton peptone, a pea peptone, or a yeast peptone.

4. A fermentation medium according to claim 1-3, where the bacterial toxin pro- duced is tetanus toxin.

5. A fermentation medium according to claim 1-3, where the bacterial toxin pro¬ duced is diphtheria toxin.

6. A fermentation medium according to claim 1-3, where the bacterial toxin pro¬ duced is pertussis toxin.

7. A fermentation medium according to claim 4, where the peptone concentration is between 5 - 50 g/1, preferably between 10 — 40 g/1 most preferably between 15 - 30 g/1.

8. A fermentation medium according to claim 5, where the peptone concentration is between 10 - 50 g/1, preferably between 20 - 40 g/1 most preferably app. 33 g/1.

9. A fermentation medium according to claim 6, where the total concentration of peptone and glutamate is between 10 - 30 g/1, preferably between 15 - 25 g/1 most preferably app. 20 g/1.

10. Use of the toxin produced with a fermentation medium according to any preced¬ ing claim for manufacturing a vaccine.

11. A vaccine comprising a toxoid derived from a toxin produced with a fermentation medium according to claim 1-9.