FERMENTATION PROCESS

专利摘要:
This invention relates to a chemically defined medium for the cultivation on an industrial scale of species of the genus Bordetella.
公开号:BE1022290B1
申请号:E2014/0685
申请日:2014-09-11
公开日:2016-03-14
发明作者:Philippe Marc Helene Dehottay；Philippe Goffin；dos Santo Filipe Branco；Bas Teusink
申请人:Glaxosmithkline Biologicals S.A.；
IPC主号:

专利说明:

FERMENTATION PROCESS
CONTEXT
The Bordetella genus is the causative agent of a number of bacterial diseases, for example Bordetella pertussis (also known as Haemophilus pertussis) is responsible for whooping cough, a respiratory disease that can be serious in infants and children. young children. The clinical course of the disease is characterized by rapid coughing followed by inspiratory effort, often associated with a characteristic "cock song". In severe cases, oxygen deprivation can lead to brain damage; however, the most common complication is secondary pneumonia.
Pertussis is generally considered to be caused by B. pertussis, but occasionally B. parapertussis is isolated from patients with typical signs and symptoms of whooping cough. With 5 to 10% of pertussis associated with B. parapertussis (Mertsola (1985) Eur J Clin Microbiol 4, 123, Lautrop (1971) Lancet 1 (7711) 1195-1198), B. parapertussis infection is less common than that to B. pertussis. B. parapertussis is associated with moderate clinical symptoms which, combined with serum cross-reactivity with B. pertussis, makes B. parapertussis difficult to diagnose.
First-generation pertussis vaccines against B. pertussis were whole cell vaccines consisting of whole killed bacteria. They were introduced in many countries in the 50s and 60s and were successful in reducing the incidence of pertussis. The problem with whole cell pertussis vaccines against B. pertussis is the high level of reactogenicity associated with them. Acellular vaccines containing purified B. pertussis proteins are less reactogenic and have been adopted for vaccination programs in many countries. Acellular vaccines typically containing pertussis toxoid (PT), filamentous haemagglutinin (FHA), and very often pertactin (PRN) are widely used and offer effective protection against severe forms of whooping cough.
Bordetella toxins for use in these vaccines are generated by fermentation of Bordetella and isolation of the virulence factors produced, although Bordetella species are fastidious organisms, difficult to cultivate at high concentrations (Doern Clin, Infected, 2000). 166-173), and it is furthermore difficult to express Bordetella virulence factors such as FHA (filamentous haemagglutinin), pertactin (PRN) and pertussis toxoid (PT) derived from Bordetella pertussis. has high levels.
Bordetella can be grown in chemically defined media. For example Stainer-Scholte (Journal of General Microbiology (1971), 63, 211-220) describe a simple chemically defined medium for the production of pertussis. Growth in chemically defined media offers advantages over undefined environments that may have a variable nutritional content leading to unpredictability of growth and expression.
The chemically defined medium may, however, be expensive and difficult to produce in large quantities, and designing chemically defined balanced media that support high levels of toxin production may also be difficult. Surprisingly, the present inventors have discovered that a number of modifications can be introduced into a chemically defined medium for Bordetella pertussis species to obtain simple media that support high levels of virulence factor production.
SHORT SUMMARY
According to a first aspect, the invention relates to a chemically defined medium for a species of the genus Bordetella in which the chemically defined medium comprises one or more of the following modifications: (i) the chemically defined medium comprises less than 0.035 mM, less than 0.030 mM less than 0.020 mM or less than 0.010 mM sulfate; (ii) the chemically defined medium comprises a source of cysteine selected from the group consisting of cysteine and cystine wherein the cysteine source is at a concentration of less than 0.50 mM, less than 0.30 mM, less than 0 25 mM, less than 0.20 mM, less than 0.15 mM, less than 0.10 mM, less than 0.05 mM or less than 0.03 mM; (iii) the chemically defined medium comprises an inorganic source of sulfur selected from the group consisting of thiosulfate, trithionate, tetrathionate, peroxodisulfate, sulfide and sulfite; (iv) the chemically defined medium does not include an organic source of sulfur; (v) the chemically defined medium comprises a buffer selected from the group consisting of MOPS buffers (3- (N-morpholino) propanesulfonic acid), MES (2- (N-morpholino) -ethanesulfonic acid), HEPES (4- (2-hydroxyethyl) -1-piperazine-ethanesulfonic acid) and PIPES (piperazine-N, N'-bis (2-ethanesulfonic acid)); (vi) the chemically defined medium comprises more than 2 μΜ, more than 3 μΜ, more than 4 μΜ, more than 5 μΜ, or more than 6 μΜ of copper; (vii) the chemically defined medium contains more than 2 μΜ, more than 5 μΜ, more than 10 μΜ, more than 50 μΜ, more than 100 μΜ or more than 400 μΜ magnesium; (viii) the chemically defined medium comprises a single source of amino acid; (ix) the chemically defined medium does not include an amino acid source; (x) the chemically defined medium comprises an additive selected from the group consisting of zinc, cobalt, thiamine, riboflavin and pantothenate; (xi) the chemically defined medium comprises an additive selected from the group consisting of more than 0.4 μΜ of biotin, more than 50 μΜ of calcium, more than 15 μΜ of niacin, and more than 25 μΜ of ascorbic acid; or (xii) the chemically defined medium comprises an amino acid selected from the group consisting of aspartate at a concentration greater than 1,000 μΜ, glycine at a concentration greater than 1,000 μΜ, methionine at a concentration greater than 500 μΜ and leucine at a concentration greater than 1500 μΜ.
According to a second aspect, the invention relates to a chemically defined medium for a species of the genus Bordetella in which the chemically defined medium comprises at least two components and wherein said at least two components are selected from the group consisting of: a) carbon and phosphorus in a ratio greater than 100: 1, greater than 125: 1, greater than 150: 1, greater than 175: 1 or greater than 200: 1 (carbon: phosphorus) (mol / mol); (b) glutamate and phosphorus in a ratio greater than 20: 1, greater than 22: 1, greater than 24: 1 or greater than 25: 1 (glutamate: phosphorus) · (mol / mol); (c) carbon and magnesium in a ratio of less than 600: 1, less than 500: 1, less than 400: 1 or less than 300: 1 (carbon: magnesium) (mol / mol); (d) glutamate and magnesium in a ratio of less than 115: 1, less than 110: 1, less than 105: 1 or less than 100: 1 (glutamate: magnesium) (mol / mol); (e) carbon and copper in a ratio greater than 3,000: 1, greater than 3,500: 1, or greater than 4,000: 1 (carbon: copper) (mol / mol); (f) glutamate and copper in a ratio greater than 170: 1, greater than 180: 1, greater than 200: 1 or greater than 250: 1 (glutamate: copper) (mol / mol); (g) carbon and iron greater than 9500: 1, greater than 1000: 1, greater than 1250: 1 or greater than 1500: 1 (carbon: iron) (mol / mol); (h) glutamate and iron in a ratio greater than 1600: 1, greater than 1800: 1, greater than 2000: 1 or greater than 2500: 1 (glutamate: iron) (mol / mol); (i) carbon and glycine in a ratio less than 500: 1, less than 400: 1, less than 300: 1 or less than 250: 1 (carbon: glycine) (mol / mol); (j) glutamate and glycine in a ratio less than 100: 1, less than 80: 1, less than 75: 1 or less than 60: 1 (glutamate: glycine) (mol / mol); (k) carbon and leucine in a ratio less than 440: 1, less than 400: 1, less than 350: 1 or less than 300: 1 (carbon: leucine) (mol / mol); (l) glutamate and leucine in a ratio of less than 75: 1, less than 70: 1, less than 60: 1 or less than 50: 1 (glutamate: leucine) (mol / mol); (m) carbon and methionine in a ratio of less than 1200: 1, less than 1000: 1, less than 800: 1 or less than 750: 1 (carbon: methionine) (mol / mol); (n) glutamate and methionine in a ratio of less than 200: 1, less than 175: 1, less than 150: 1 or less than 120: 1 (glutamate: methionine) (mol / mol); (o) carbon and calcium in a ratio greater than 3750: 1, greater than 4,000: 1, greater than 4,500: 1 or greater than 5,000: 1 (carbon: calcium) (mol / mol); (p) glutamate and calcium in a ratio greater than 620: 1, greater than 650: 1, greater than 675: 1 or greater than 750: 1 (glutamate: calcium) (mol / mol); (q) carbon and cobalt in a ratio greater than 3,000: 1, greater than 3,500: 1, greater than 4,750: 1 or greater than 5,000: 1 (carbon: cobalt) (mol / mol); (r) glutamate and cobalt in a ratio greater than 750: 1, greater than 1,000: 1, greater than 1,250: 1 or greater than 1,500: 1 (glutamate: cobalt) (mol / mol); (s) carbon and zinc in a ratio greater than 3,000: 1, greater than 3,500: 1, greater than 4,000: 1 or greater than 5,000: 1 (carbon: zinc) (mol / mol); (t) glutamate and zinc in a ratio greater than 750: 1, greater than 1,000: 1, greater than 1,250: 1 or greater than 1,500: 1 (glutamate: zinc) (mol / mol); (u) carbon and sulfate equivalents in a ratio greater than 750: 1, greater than 1,000: 1, greater than 1,250: 1 or greater than 1,500: 1 (carbon: sulfate equivalents) (mol / mol ); and (v) glutamate and sulfate equivalents in a ratio greater than 130: 1, greater than 150: 1, greater than 175: 1 or greater than 200: 1 (glutamate: sulfate equivalents) (mol / mol).
According to a third aspect, the invention relates to a fermentation method for growing a species of the genus Bordetella in a chemically defined medium (CDM) comprising (a) inoculating the chemically defined medium according to the invention with the species of the genus Bordetella; (b) maintenance of the Bordetella species in the chemically defined medium for a period of time sufficient to allow accumulation of biomass.
According to a fourth aspect, the invention relates to a virulence factor obtainable by the fermentation process according to the invention.
According to a fifth aspect, the invention relates to a virulence factor obtained by the fermentation process according to the invention.
According to a sixth aspect, the invention relates to an immunogenic composition comprising the virulence factor according to the invention.
According to a seventh aspect, the invention relates to a vaccine comprising the immunogenic composition according to the invention.
According to an eighth aspect, the invention relates to a use of the immunogenic composition according to the invention or of the vaccine according to the invention in the prevention or the treatment of the disease.
According to a ninth aspect, the invention relates to a use of the immunogenic composition according to the invention or the vaccine according to the invention in the preparation of a medicament for the treatment or prevention of a bacterial disease.
According to a tenth aspect, the invention relates to a method of preventing or treating the disease comprising administering the immunogenic composition or vaccine to a patient.
DETAILED DESCRIPTION OF CHEMICALLY DEFINED MEDIA
Chemically defined media (MDC) are often considered beneficial because, unlike chemically undefined media, they contain a precise concentration of each nutrient, thus reducing the variability of the medium and improving the quality of the fermented product. However, it may be difficult to create an optimal balanced chemically defined medium as it is difficult to predict the nutrients / environmental components required by the different bacteria. Ideally the chemically defined medium should be substantially equilibrated, ie at the end of fermentation there should be no excess of any particular component of the medium due to the presence of too much of this component of the medium. medium for the bacteria to metabolize it, since the balanced media support more efficient growth and are more profitable. A semi-synthetic medium for Bordetella pertussis was developed by Goldner (J. Gen. Microbiol. (1966), 44, 439-444), however it was too complicated and expensive to use on an industrial scale. Stainer and Schölte have attempted to design a simpler medium that is expected to be more suitable for industrial scale fermentation, but it has not proved optimal for the production of virulence factors (Journal of General Microbiology (1971), 63). , 211-220). The present inventors have discovered that certain modifications can be implemented to simplify the chemically defined media or to significantly increase the yield of virulence factors obtained from Bordetella grown in these media.
These modifications are as follows: (i) the chemically defined medium comprises less than 0.035 mM, less than 0.030 mM, less than 0.020 mM or less than 0.010 mM sulfate; (ii) the chemically defined medium comprises a source of cysteine selected from the group consisting of cysteine and cystine wherein the cysteine source is at a concentration of less than 0.50 mM, less than 0.30 mM, less than 0 25 mM, less than 0.20 mM, less than 0.15 mM, less than 0.10 mM, less than 0.05 mM or less than 0.03 mM; (iii) the chemically defined medium comprises an inorganic source of sulfur selected from the group consisting of thiosulfate, trithionate, tetrathionate, peroxodisulfate, sulfide and sulfite; (iv) the chemically defined medium does not include an organic source of sulfur; (v) the chemically defined medium comprises a buffer selected from the group consisting of MOPS, MES, HEPES and PIPES buffers; (vi) the chemically defined medium comprises more than 2 μΜ, more than 3 μΜ, more than 4 μΜ, more than 5 μΜ, or more than 6 μΜ of copper; (vii) the chemically defined medium contains more than 2 μΜ, more than 5 μΜ, more than 10 μΜ, more than 50 μΜ, more than 100 μΜ or more than 400 μΜ magnesium; (viii) the chemically defined medium comprises a single source of amino acids; (ix) the chemically defined medium does not include a source of amino acids; (x) the chemically defined medium comprises an additive selected from the group consisting of zinc, cobalt, thiamine, riboflavin and pantothenate; (xi) the chemically defined medium comprises an additive selected from the group consisting of more than 0.4 μΜ of biotin, more than 50 μΜ of calcium, more than 15 μΜ of niacin, and more than 25 μΜ of ascorbic acid; or (xii) the chemically defined medium comprises an amino acid selected from the group consisting of aspartate at a concentration greater than 1,000 μΜ, glycine at a concentration greater than 1,000 μΜ, methionine at a concentration greater than 500 μΜ and leucine at a concentration greater than 1500 μΜ.
Thus, according to a first aspect, the present invention relates to a chemically defined medium for a species of the genus Bordetella in which the chemically defined medium comprises one or more of the modifications described above.
In addition attempts to formulate new chemically defined media often involve taking a complex medium and replacing the complex medium component (such as an acid casein hydrolyzate) with equivalent amounts of individual chemically defined components. However, the present inventors have surprisingly found that the ratio of the components of the medium can be very important to ensure that the chemically defined medium is balanced and supports the high yield production of the virulence factors.
Therefore according to a second aspect the present invention relates to a chemically defined medium for a Bordetella species in which the chemically defined medium comprises at least two components selected from the group consisting of: a) carbon and phosphorus in a ratio greater than 100: 1, greater than 125: 1, greater than 150: 1, greater than 175: 1 or greater than 200: 1 (carbon: phosphorus) (mol / mol); (b) glutamate and phosphorus in a ratio greater than 20: 1, greater than 22: 1, greater than 24: 1 or greater than 25: 1 (glutamate: phosphorus) (mol / mol); (c) carbon and magnesium in a ratio of less than 600: 1, less than 500: 1, less than 400: 1 or less than 300: 1 (carbon: magnesium) (mol / mol); (d) glutamate and magnesium in a ratio of less than 115: 1, less than 110: 1, less than 105: 1 or less than 100: 1 (glutamate: magnesium) (mol / mol); (e) carbon and copper in a ratio greater than 3,000: 1, greater than 3,500: 1, or greater than 4,000: 1 (carbon: copper) (mol / mol); (f) glutamate and copper in a ratio greater than 170: 1, greater than 180: 1, greater than 200: 1 or greater than 250: 1 (glutamate: copper) (mol / mol); (g) carbon and iron greater than 9500: 1, greater than 1000: 1, greater than 1250: 1 or greater than 1500: 1 (carbon: iron) (mol / mol); (h) glutamate and iron in a ratio greater than 1600: 1, greater than 1800: 1, greater than 2000: 1 or greater than 2500: 1 (glutamate: iron) (mol / mol); (i) carbon and glycine in a ratio less than 500: 1, less than 400: 1, less than 300: 1 or less than 250: 1 (carbon: glycine) (mol / mol); (j) glutamate and glycine in a ratio less than 100: 1, less than 80: 1, less than 75: 1 or less than 60: 1 (glutamate: glycine) (mol / mol); (k) carbon and leucine in a ratio less than 440: 1, less than 400: 1, less than 350: 1 or less than 300: 1 (carbon: leucine) (mol / mol); (l) glutamate and leucine in a ratio of less than 75: 1, less than 70: 1, less than 60: 1 or less than 50: 1 (glutamate: leucine) (mol / mol); (m) carbon and methionine in a ratio of less than 1200: 1, less than 1000: 1, less than 800: 1 or less than 750: 1 (carbon: methionine) (mol / mol); (n) glutamate and methionine in a ratio of less than 200: 1, less than 175: 1, less than 150: 1 or less than 120: 1 (glutamate: methionine) (mol / mol); (o) carbon and calcium in a ratio greater than 3750: 1, greater than 4,000: 1, greater than 4,500: 1 or greater than 5,000: 1 (carbon: calcium) (mol / mol); (p) glutamate and calcium in a ratio greater than 620: 1, greater than 650: 1, greater than 675: 1 or greater than 750: 1 (glutamate: calcium) (mol / mol); (q) carbon and cobalt in a ratio greater than 3,000: 1, greater than 3,500: 1, greater than 4,750: 1 or greater than 5,000: 1 (carbon: cobalt) (mol / mol); (r) glutamate and cobalt in a ratio greater than 750: 1, greater than 1,000: 1, greater than 1,250: 1 or greater than 1,500: 1 (glutamate: cobalt) (mol / mol); (s) carbon and zinc in a ratio greater than 3,000: 1, greater than 3,500: 1, greater than 4,000: 1 or greater than 5,000: 1 (carbon: zinc) (mol / mol); (t) glutamate and zinc in a ratio greater than 750: 1, greater than 1,000: 1, greater than 1,250: 1 or greater than 1,500: 1 (glutamate: zinc) (mol / mol); (u) carbon and sulfate equivalents in a ratio greater than 750: 1, greater than 1,000: 1, greater than 1,250: 1 or greater than 1,500: 1 (carbon: sulfate equivalents) (mol / mol ); and (v) glutamate and sulfate equivalents in a ratio greater than 130: 1, greater than 150: 1, greater than 175: 1 or greater than 200: 1 (glutamate: sulfate equivalents) (mol / mol).
The term "chemically defined medium" refers to a medium that is essentially free of complex substance such as yeast, an acid hydrolyzate of casein, peptones, tryptones, yeast extract. See e.g., Jayme and Smith, Cytotechnology 33 (1-3): 27-36 (2000). In particular, as used herein, a chemically defined medium does not include casein acid hydrolyzate (CAA) as the source of amino acids in the medium. As used herein, "casein acid hydrolyzate" refers to a mixture of amino acids obtained by the hydrolysis of casein.
The CDMs according to the present invention are described both in positive terms (ingredient (s) or component (s) included in the medium) and in negative terms (ingredient (s) or component (s) excluded from the medium).
A "source" is a component of the medium that provides at least one medium-specific ingredient. Cystine is, e.g., a source of cysteine in that it provides a cysteine that will be used by the organisms grown on the medium. As used herein, the ingredient itself is considered a "source", e.g., sulfate is a source of sulfate, cysteine is a source of cysteine, etc. A "source" may provide more than one ingredient, e.g., an amino acid may be a source of carbon and a source of nitrogen, as well as an amino acid source.
The term "medium" refers to a sufficient source of nutrients to allow Bordetella to grow to relatively high densities (eg, to a biomass greater than 1.0 g / L greater than 1.5 g / L, greater than 2.0 g / L or greater than 2.5 g / L dry weight of cells).
The chemically defined medium according to the invention is intended for the culture on an industrial scale of a species of the genus Bordetella, the term "culture on an industrial scale" designating the culture in a fermenter, in one embodiment, the "Industrial scale" means culture in a fermenter with a working volume of between 5 and 10,000 liters, between 10 and 5,000 liters, between 20 and 2,000 liters, between 50 and 1,000 liters, greater than or equal to to 5 liters, greater than or equal to 10 liters, greater than or equal to 15 liters, greater than or equal to 20 liters, greater than or equal to 25 liters, greater than or equal to 50 liters, greater than or equal to 100 liters, less than or equal to 10,000 liters, less than or equal to 5,000 liters or less than or equal to 2,500 liters. In another embodiment, "commercial scale culture" is a culture suitable for the production of more than 10 mg / L, more than 15 mg / L or more than 20 mg / L of pertussis toxoid.
During the fermentation process, the chemically defined medium according to the invention is added to the fermenter at the beginning of the process, although eventually additional quantities of medium may be added during the fermentation process (for example in batch fermentation). ); alternatively a medium of different composition can be added later in the fermentation. This medium can also be added continuously in the culture medium in the case of systems such as chemostats or retentostats. Preferably, the fermentation is a batch fermentation.
The chemically defined medium according to the invention preferably supports a growth yield of the species of the genus Bordetella greater than that supported by the Stainer-Scholte medium (described in Journal of General Microbiology (1971), 63: 211-220). This can be determined by inoculation of a Bordetella strain, inoculation of a vial or fermentor containing Stainer-Scholte medium with a first sample of the Bordetella strain and inoculation of a vial or fermenter containing the medium. chemically defined to be tested with a second sample of the Bordetella strain (in the same volume as that chosen for Stainer-Scholte medium). The optical density at 650 nm, OD650nm is measured at multiple double time points for both samples, these time points to include a time point just after inoculation (designated time point A) and a time point corresponding to the end of the growth (designated time point B). Growth is considered complete when the cell concentration between two consecutive time points (separated by at least 24 hours) has not increased by more than 10%. If the difference of DO650nm between the time point B and the time point A is greater for the second sample than for the first one, which is inoculated in the Stainer-Scholte medium, the chemically defined medium tested supports a growth efficiency of species of the genus Bordetella superior to that supported by the medium Stainer-Scholte.
The chemically defined medium preferably supports an average generation time of the Bordetella species less than 15h, less than 12h, less than 10h or less than 9h. This can be verified using a method similar to that described in the previous paragraph, although the average generation time is obtained by dividing the time between the time point A and the time point B by the number of generations between these two time points, the number of generations between the time points A and B being itself obtained by calculating the ratio between the DO650nm at the second time point and the DC> 650nm at the first time point, converted into Log2.
The chemically defined medium preferably supports higher levels of pertussis toxoid production than those supported by Stainer-Scholte medium. This can be determined by inoculating a vial or fermentor containing Stainer-Scholte medium with a first sample of the Bordetella pertussis strain and inoculating BE201 with a vial or fermentor containing the chemically defined medium to be tested ( in the same volume as that chosen for the Stainer-Scholte medium) with a second sample of the Bordetella pertussis strain, incubation of the two samples until stopping growth and calculation of the level of production of pertussis toxoid in each sample . A method for determining the level of pertussis toxoid production is described in Example 1. If the level of pertussis toxoid production of the second sample is greater than that of the first sample, the chemically defined medium supports levels of production. pertussis toxoid higher than those supported by the Stainer-Scholte medium.
In a preferred embodiment, the chemically defined medium according to the invention supports the production of pertussis toxoid by the species of the genus Bordetella in a yield greater than 10 mg / l, or greater than 15 mg / l, the yield being more preferably greater than 20 mg / L. Whether or not the chemically defined medium supports the production of pertussis toxoid by Bordetella species at a certain yield can be determined by inoculation of the chemically defined medium with a sample of the species of the genus Bordetella, and incubation of the cells. until growth stops. At the end of the growth, the pertussis toxoid yield can be calculated by the method described in Example 1.
In one embodiment, the chemically defined medium is a substantially balanced medium. A substantially balanced medium is a medium in which, at the end of the fermentation, there is no significant excess of a particular nutrient. Whether a chemically defined medium is a substantially balanced medium or not can be determined by incubating the species of the genus Bordetella in the medium until the growth stops and study of
,. -,. ..,,. BCZU IH medium supernatant after stopping growth. If the metabolic sources (ie sources of nitrogen, phosphorus and sulfur) are used at a substantially similar rate (within 10% of each other), then the chemically defined medium is balanced. In a preferred embodiment, the final concentrations of all metabolic sources will be in the vicinity of 0 mM.
In general, the chemically defined media must contain at least one carbon source, one phosphorus source, one nitrogen source, one sulfur source, and one buffer. The nitrogen source can be organic or inorganic. The nitrogen source may be an amino acid or a peptide, alternatively, the nitrogen source may be a nitrogen source that is not an amino acid or a peptide in which case the chemically defined medium is a medium. chemically defined that does not include an amino acid. In one embodiment, the nitrogen source is inorganic. In one embodiment, the nitrogen source comprises or consists of a compound selected from the group consisting of amino acids, urea, polyamines, ammonium (such as ammonium chloride, sodium sulfate, and the like). ammonium or ammonium nitrate), nucleobases, nucleosides, and nucleotides. In another embodiment, the nitrogen source comprises or consists of ammonium chloride. The carbon source may comprise or consist of an amino acid or a peptide, or may comprise or consist of a carbon source which is neither an amino acid nor a peptide in which case the chemically defined medium does not comprise an acid. amine. As used herein, the term "does not include amino acid" means that the medium "does not include" peptides or proteins, since the peptides or proteins are sources of amino acids. In one embodiment, the carbon source comprises or consists of a compound selected from the group consisting of monosaccharides, disaccharides, polysaccharides, polyols (sugar alcohols), organic acids and amino acids. In another embodiment, the carbon source comprises or consists of a compound selected from the group consisting of glucose, fructose, sorbose, galactosamine, mannose, sucrose, rhamnose, sorbitol, mannitol, citrate, lactate, acetate, pyruvate, fumarate, succinate, proline and glutamate. In another embodiment, the carbon source comprises glutamate or proline. In another embodiment, the carbon source comprises or consists of an organic acid selected from the group consisting of citrate, lactate, acetate, pyruvate, fumurate and succinate.
The chemically defined medium according to the invention is intended for the cultivation on an industrial scale of a species of the genus Bordetella. In one embodiment, the medium comprises the species of the genus Bordetella. In one embodiment, the species of the genus Bordetella is a species selected from the group consisting of Bordetella petrii, Bordetella avium, Bordetella hinzii, Bordetella trematum, Bordetella holmesii, Bordetella parapertussis, Bordetella bronchiseptica and Bordetella pertussis (also known as Haemophilus pertussis). Preferably, the species of the genus Bordetella is selected from the group consisting of Bordetella parapertussis, Bordetella bronchiseptica and
Bordetella pertussis. More preferably, the species of the genus Bordetella is Bordetella pertussis.
SOURCES OF SULFUR
In a first embodiment, the chemically defined medium comprises less than 0.035 mM, less than 0.030 mM, less than 0.020 mM, less than 0.010 mM sulfate, preferably less than 0.005 mM, less than 0.0001 mM, less than 0.00005 mM, less than 0.00001 mM, between 0.035 and 0 mM, between
öbZUI 0.005 and 0 mH or between 0.00001 and 0 mM. Surprisingly, the present inventors have found that the removal of sulfate from the chemically defined medium significantly increases the yield of virulence factors such as PT when used in a chemically defined medium for Bordetella. WO0178462 and Lacey (1960, J. Hyg 58: 57-93) develop the idea that sulfate can be an inhibitor of production of virulence factors; however, the low sulphate media described in WO0178462 contained 0.001 g / L FeSCu added. It is therefore clear that the inventors in WO0178462 considered that the presence of at least some amount of FeSC> 4 was required to obtain a chemically defined medium for the growth of pertussis. It should be noted that to obtain high levels of virulence factors, the medium must support both virulence factor production and Bordetella growth until a suitable biomass is obtained, and although sulphate was known to inhibit virulence factor expression, it was not known that Bordetella could grow to a reasonable biomass in the absence of sulphate. It should also be noted that Jebb and Tomlinson (J. Gen. Microbiol 17, 59-68) indicate that sulfate was not sufficient to provide a source of sulfur, which contradicts other techniques, and subsequent documents citing Jebb and Tomlinson (such as Licary, Siber and Swartz Journal of Biotechnology 1 (1991) 117-130) continuing to add sulfate to media. This conclusion is supported by other media publications for Bordetella, all of which appear to generally require sulphate to be present (for example, the Stainer-Scholte medium described above contains some). The present inventors have, however, surprisingly found that FeSC> 4 may be replaced by Fe (III) citrate to remove sulfate.
BE201A (thereby reducing the inhibition of virulence factor expression), that an effective medium that supports Bordetella growth is still obtained, and that reducing the sulfate to an even lower content than that described in WO0178462 results in significant increase in the yield of virulence factors such as PT. In another embodiment, the chemically defined medium does not include sulfate. The term "does not include" a certain substrate such as sulphate means a medium in which the creator of the medium has not added a significant amount of said substance. Therefore, a medium may be considered as "not including" a certain substance if the medium comprises a small amount of that substance, which is, for example, a contaminant. Alternatively, a medium may be considered as "not including" a certain substance if the creator of the medium has added a very small amount of that substance which is not sufficient to alter the performance of a virulence factor such as pertussis toxoid. This can be determined by culturing the Bordetella species in the presence of the small amount of this substance and in the absence of the small amount of this substance and measuring the yield of this virulence factor in both cultures by ELISA. An appropriate ELISA is described later.
In one embodiment, the invention relates to a chemically defined medium that comprises a source of cysteine selected from the group consisting of cysteine and cystine wherein the cysteine source is at a concentration of less than 0.50 mM, lower at 0.30 mM, less than 0.25 mM, less than 0.20 mM, less than 0.15 mM, less than 0.10 mM, less than 0.05 mM, less than 0.03 mM, less than 0.01 mM, less than 0.005 mM,
DtZUI 4/1 less than 0.001 mM, less than 0.0005 mM, less than 0.0001 mM, less than 0.00005 mM or less than 0.00001 mM.
Cysteine is generally used for biomass synthesis by Bordetella, however when cysteine is present at high concentrations, it will be catabolized to sulfate (Bogdan et al ((2001) Infect Immun 69: 6823-6830)). This sulphate can not be assimilated because the sulfate assimilation pathway is not functional (Parkhill et al ((2003) Nat Genet 35: 32-40)). Therefore, the use of high concentrations of cysteine in a medium can provide sulfate ions which, as described above, inhibit the expression of virulence factor. However, Bogdan et al have admitted that cysteine is required for growth, and therefore the media described in Bogdan et al (even those believed to contain reduced amounts of cysteine) contain relatively high concentrations of cysteine. Similarly, Jebb and Tomlinson (J. Gen. Microbiol., 17, 59-68) describe the presence of cysteine as essential for growth. The present inventors have, however, demonstrated for the first time that Bordetella can grow in the absence of cysteine and therefore that cysteine concentrations even lower than those described in Bogdan et al. can be used.
Cystine is a cysteine dimer that can be similarly metabolized to cysteine by Bordetella, but provides twice as much cysteine to Bordetella.
In another embodiment, the chemically defined medium does not include cysteine or cystine. In a preferred embodiment, the chemically defined medium does not include sulfate, cysteine, or cystine.
In another embodiment, the chemically defined medium comprises an inorganic source of sulfur selected from the group consisting of thiosulfate, trithionate,
BtZU tetrathionate, peroxodisulfate, sulphide and sulphite. In another embodiment, the chemically defined medium does not include an organic source of sulfur.
The present inventors have demonstrated, for the first time, that inorganic sulfur can be used as a source of sulfur (rather than cysteine) to grow Bordetella.
It appears from the technique, for example Jebb and Tomlinson (J. Gen. Microbiol., 17, 59-68), that an organic source of sulfur is required for Bordetella growth, knowing that the route of synthesis of the Cysteine from sulfate and thiosulfate is not operational in members of the genus Bordetella (Parkhill et al ((2003) Nat Genet 35: 32-40)). However, the inventors have demonstrated for the first time that Bordetella can grow in the absence of an organic source of sulfur (as long as an inorganic source of sulfur such as a thiosulfate is present).
In one embodiment, the chemically defined medium comprises thiosulfate. In another embodiment, the chemically defined medium comprises more than 0.005 mM, more than 0.006 mM, more than 0.007 mM, more than 0.008 mM, more than 0.010 mM, more than 0.050 mM, more than 0.100 mM, between 0, 005 and 0.100 mM, between 0.005 and 0.050 mM, between 0.005 and 0.025 mM, about 0.120 mM or about 0.011 mM thiosulfate. In another embodiment, the chemically defined medium comprises trithionate. In another embodiment, the chemically defined medium comprises more than 0.003 mM, more than 0.004 mM, more than 0.005 mM, more than 0.008 mM, more than 0.010 mM, more than 0.020 mM, more than 0.050 mM, between 0, 003 and 0.500 mM, between 0.003 and 0.100 mM, between 0.005 and 0.010 mM, about 0.007 mM or about 0.080 mM trithionate. In one embodiment, the chemically defined medium comprises tetrathionate. In another embodiment, the chemically defined medium comprises more than 0.002 mM, more than 0.003 mM, more than 0.004 mM, more than 0.005 mM, more than 0.025 mM, more than 0.050 mM, between 0.002 and 1.000 mM, 0.010 and 0.100 mM, about 0.060 mM or about 0.0006 mM tetrathionate. In one embodiment, the chemically defined medium comprises peroxodisulfate. In another embodiment, the chemically defined medium comprises more than 0.005 mM, more than 0.006 mM, more than 0.007 mM, more than 0.008 mM, more than 0.010 mM, more than 0.050 mM, more than 0.100 mM, between 0.005 and 1,000 mM, between 0.005 and 0.200 mM, between 0.005 and 0.015 mM, about 0.120 mM or about 0.011 mM peroxodisulfate. In one embodiment, the chemically defined medium comprises sulfide. In another embodiment, the chemically defined medium comprises more than 0.010 mM, more than 0.012 mM, more than 0.014 mM, more than 0.016 mM, more than 0.020 mM, more than 0.100 mM, more than 0.200 mM, between 0.010 mM and 1,000 mM, between 0.010 and 0.300 mM, between 0.010 and 0.100 mM, about 0.240 mM or about 0.022 mM sulfide. In one embodiment, the chemically defined medium comprises sulfite. In another embodiment, the chemically defined medium comprises greater than 0.010 mM, more than 0.012 mM, more than 0.014 mM, more than 0.016 mM, more than 0.020 mM, more than 0.100 mM, more than 0.200 mM, approximately 0.240 mM or about 0.022 mM sulphite.
In one embodiment, the chemically defined medium comprises thiosulfate and trithionate, thiosulfate and tetrathionate, thiosulfate and peroxodisulfate, thiosulfate and sulfide, thiosulfate and sulfite, trithionate and tetrathionate, trithionate and peroxodisulfate, trithionate and sulphide, trindate and sulphite, tetrathionate and peroxodisulphate, tetrathionate and sulphide, tetrathionate and sulphite, peroxodisulphate and sulphide, peroxodisulphate and sulphite or sulphide and sulphite . In another embodiment, the chemically defined medium comprises 2, 3, 4, 5, 6 or more inorganic sources of sulfur selected from the group consisting of thiosulfate, trithionate, tetrathionate, peroxodisulfate, sulphide and sulphite.
In a preferred embodiment, the chemically defined medium does not include sulfate, cysteine or cystine and comprises more than 0.005 mM, more than 0.006 mM, more than 0.007 mM, more than 0.008 mM, more than 0.010 mM , more than 0.050 mM, greater than 0.100 mM, 0.005 to 0.100 mM, 0.005 to 0.050 mM, 0.005 to 0.025 mM, approximately 0.120 mM or approximately 0.011 mM thiosulfate.
BUFFER
In another embodiment, the chemically defined medium comprises a buffer selected from the group consisting of MOPS, MES, HEPES and PIPES.
Surprisingly, the present inventors have found that a chemically defined medium comprising buffers other than tris and β-glycerophosphate, particularly an MOPS buffer, gives improved growth rates for Bordetella pertussis compared to other media. Other buffers that can be used in chemically defined media for Bordetella pertussis have been explored by Lothe et al (Journal of Biological Standardization (1985) 13, 129-134), who however concluded that β-glycerophosphate was a superior buffer. The present inventors have found, however, that not only other buffers may be effective, but also that MOPS buffer has advantages over β-glycerophosphate. This is the reason why the present invention relates to a chemically defined medium comprising a MOPS buffer. In one embodiment, the buffer is MOPS at a concentration greater than 2 mM, greater than 5 mM, greater than 7 mM, greater than 9 mM, greater than 10 mM, greater than 11 mM, between 2 and 100 mM, between 2 and 50 mM, between 5 and 20 mM or about 12 mM.
HIGH CONCENTRATIONS OF COPPER
It has been shown that copper is not required in Bordetella medium (Stainer and Schölte, Journal of General Microbiology (1971), 63: 211-220), however the present inventors surprisingly discovered that the addition a relatively high concentration of copper to a chemically defined medium for Bordetella results in a significant increase in the amount of toxin produced by Bordetella (eg the expression of pertussis toxoid from Bordetella pertussis).
Therefore in another embodiment, the chemically defined medium comprises more than 2 μΜ, more than 3 μΜ, more than 4 μΜ, more than 5 μΜ, more than 6 μΜ, more than 7 μΜ, more than 8 μΜ, less 200 μΜ, less than 150 μΜ, less than 100 μΜ, between 4 and 10 μΜ, between 2 and 200 μΜ, between 3 and 150 μΜ or between 5 and 100 μΜ of copper. In one embodiment, the copper source is selected from the group consisting of copper chloride, copper sulfate, copper acetate, copper chlorate, and copper carbonate. In another embodiment, the copper is in the form of copper chloride.
HIGH CONCENTRATIONS OF MAGNESIUM
The high magnesium concentrations are known to modulate Bordetella, and induce the conversion of Bordetella to a state where the bacterium is less likely to express virulence factors such as pertussis toxoid and FHA (Idigbe et al J. MED. MICROBIOL (1981) 409-418) and Lacey et al ((1960) J. Hyg 58: 57-93)). As explained above, growing Bordetella in an environment that induces high levels of toxin expression is advantageous, and the addition of magnesium was known to reduce the expression of virulence factor, hence its elimination of the media. intended for the production of pertussis vaccines. However, the present inventors have surprisingly found that the addition of high concentrations of magnesium can be used in a chemically defined medium having high levels of expression of virulence factors such as PT.
For these reasons, in one embodiment the chemically defined medium comprises more than 2 μΜ, more than 5 μΜ, more than 10 μΜ, more than 25 μΜ, more than 50 μΜ, more than 75 μΜ, more than 100 μΜ, more 200 μΜ, more than 300 μΜ, more than 400 μΜ, between 2 and 6,000 μΜ, between 1,000 and 6,000 μΜ, or about 5,000 μΜ of magnesium.
SOURCE OF AMINO ACIDS
It is generally known that media must include a source of nitrogen and a source of carbon; and that in many cases, certain amino acids are required for growth (essential amino acids). Stainer and Schölte (Stainer and Schölte, Journal of General Microbiology (1971), 63: 211-220) have attempted to create a simplified chemically defined medium but have come to the conclusion, however, that at least two amino acids are required, know glutamic acid, proline and cystine.
However, the present inventors have surprisingly found that Bordetella can grow in media comprising a single type of amino acid. In particular, the inventors have demonstrated that Bordetella can grow on media which comprise only one amino acid and do not comprise cysteine, this being all the more surprising since, as described above, it was previously thought that Cysteine was essential as a source of sulfur. This discovery is advantageous because, as described above, the commercial media must be as simple as possible, to reduce the difficulties of manufacturing the medium, its cost and potential sources of variability from one batch to another . This is why, in one embodiment, the chemically defined medium comprises only one source of amino acid. The term "single source of amino acid" refers to a compound which provides to the medium a source of a single type of amino acid (such as a source of glutamine, or asparagine or other amino acid), a compound since cystine can be considered as a single source of amine acid since, although it is a dipeptide, it contains only cysteine and therefore only one amino acid is provided. In particular, a medium will be considered as comprising only one source of amino acid if both cysteine and cystine are present, since these two components bring only cysteine (only amino acid) in the middle. This term includes the enantiomers D and L of the amino acids. In one embodiment, the amino acid source is an enantiomer D, in another embodiment, the amino acid source is an L enantiomer, and in another embodiment, the amino acid source can be be either an L enantiomer or a D enantiomer. A medium comprising a "single source of amino acid" does not include other amino acids, for example, a medium comprising cysteine as the only source of amino acid does not include glutamate, alanine, aspartate, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, serine, valine, tyrosine or other amino acid. As explained above, the term "does not include" a certain substrate such as certain amino acids means a medium in which the creator of the medium has not added a significant amount of said substance. Therefore, a medium may be considered as "not including" a certain substance if the medium comprises a small amount of that substance, which is, for example, a contaminant. Alternatively, a medium may be considered as "not including" a certain substance if the creator of the medium has added a very small amount of that substance which is not sufficient to alter the performance of a virulence factor such as pertussis toxoid. This can be determined by culturing the Bordetella species in the presence of the small amount of this substance and in the absence of the small amount of this substance and measuring the yield of this virulence factor in both cultures by ELISA ( as described above). In another embodiment, the only source of amino acid is a single source of nitrogen.
In one embodiment, the only source of amino acid is selected from the group consisting of cysteine, cystine, alanine, glycine, glutamate, proline, serine, glutamine, aspartate, leucine, isoleucine, valine, tyrosine, phenylalanine, tryptophan, histidine, arginine, ornithine, lysine, threonine, asparagine and methionine. In one embodiment, the only source of amino acid is cysteine at a concentration greater than 75 mM, greater than 100 mM, greater than 125 mM, between 75 and 250 mM, between 100 and 150 mM, or about 125 mM mM. In one embodiment, the only source of amino acid is proline at a concentration greater than 75 mM, greater than 100 mM, greater than 125 mM, 75 to 250 mM, 100 to 150 mM, or approximately 125 mM. mM. In one embodiment, the only source of amino acid is glutamate at a concentration greater than 75 mM, greater than 100 mM, greater than 125 mM, 75 to 250 mM, 100 to 150 mM, or approximately 125 mM. mM. In one embodiment, the only source of amino acid is glutamine at a concentration greater than 75 mM, greater than 100 mM, greater than 125 mM, between 75 and 250 mM, between 100 and 150 mM, or approximately 125 mM mM. In one embodiment, the only source of amino acid is DL 4 I 4 aspartate at a concentration greater than 10 mM, greater than 20 mM, greater than 30 mM, 10 mM to 100 mM, 20 mM to 50 mM, or about 30 mM. In one embodiment, the only source of amino acid is asparagine at a concentration greater than 75 mM, greater than 100 mM, greater than 125 mM, between 75 and 250 mM, between 100 and 150 mM or approximately 125 mM. In one embodiment, the only source of amino acid is serine at a concentration greater than 75 mM, greater than 100 mM, greater than 125 mM, between 75 and 250 mM, between 100 and 150 mM, or about 125 mM. mM. In one embodiment, the only source of amino acid is alanine at a concentration greater than 75 mM, greater than 100 mM, greater than 125 mM, between 75 and 250 mM, between 100 and 150 mM or greater. 125 mM.
The inventors have furthermore demonstrated that, although it is advantageous to use a single source of amino acid in a chemically defined medium for Bordetella in order to support a high production of toxins, it is also possible to create a medium which does not does not include an amino acid source at all. This provides a medium in which the carbon and nitrogen sources are constituted by separate components, thereby manipulating said sources of carbon and nitrogen separately. In this regard, Thalen et al. (Journal of Biotechnology (1999) 75: 147-159) reported a nitrogen-to-carbon ratio of 1: 5 (as in Stainer and Schölte's medium (Journal of General Microbiology (1971), 63: 211-220) ) is not optimal for the growth of Bordetella, and generates an accumulation of ammonia. Thalen et al. showed that ammonia accumulation could be significantly reduced by using a 1:10 nitrogen to carbon ratio. This ratio, however, can not be reached with natural amino acids, for which this ratio is determined by the molecular composition, and ranges from 1: 1.5 (Arginine) to 1: 9 (Tyrosine and Phenylalanine). To work around this limitation, BE20
Thalen et al. have manipulated the carbon to nitrogen ratio by adding a second carbon source that does not contain nitrogen (lactate, organic acid). However, this solution is complex in terms of metabolic flux, which in turn complicates the monitoring and understanding of the method, as well as obtaining a balanced medium (Neeleman et al., Applied Microbiology and Biotechnology (2001), 57: 489-493)).
Completely eliminating amino acids offers an alternative solution to the precise manipulation of the carbon to nitrogen ratio, by careful adjustment of the relative concentrations of a non-nitrogen-containing carbon source, on the one hand, and a source of nitrogen. nitrogen containing no carbon, on the other hand. For this reason, in another embodiment, the chemically defined medium does not include an amino acid source.
The medium should contain a source of carbon if the medium does not contain an amino acid source, the carbon source preferably being an organic acid. In one embodiment, the organic acid is selected from the group consisting of citrate, lactate, acetate, pyruvate, fumarate, and succinate. The present inventors have demonstrated that organic acids are suitable substitutes for glutamate as a carbon source for Bordetella supporting reasonable levels of growth.
In one embodiment, if the chemically defined medium comprises a single amino acid source, or does not include an amino acid source, the chemically defined medium comprises at least one of the chemically defined medium components comprising the hydrogenphosphate. potassium, potassium chloride, magnesium, calcium, Fe (III) citrate, MOPS buffer, niacin, dimethyl-β-cyclodextrin, copper, or cobalt, preferably the medium comprises 2, 3 , 4, 5, 6, 7, 8, or 9 of these components.
In a preferred embodiment, the chemically defined medium comprises all of these components. In another embodiment, the chemically defined medium may also include sodium, zinc, biotin, riboflavin, calcium panthothenate. Preferably the medium comprises sodium, zinc, biotin, riboflavin and calcium panthothenate.
In another embodiment, the chemically defined medium comprises a single amino acid source or does not include an amino acid source, and the chemically defined medium comprises between 250 and 750 mg / L KH2PO4, between 100 and 300 mg / L of KC1, between 500 and 1500 mg / L of MgCl2.6H20, between 50 and 150 mg / L of CaCl2.2H20, and between 10 and 30 mg / L of Fe (III) citrate. 3H2O, between 1000 and 5000 mg / L of MOPS, between 4 and 8 mg / L of niacin, between 500 and 2000 mg / L of dimethyl-β-cyclodextrin, between 0.5 and 2 mg / L of CUCI2 .2H2O and between 0.1 and 1 mg / L of CoCl 2 H 2 O. In another embodiment, the medium further comprises between 1 and 25 mg / L of ZnCl 2, between 0.01 and 1.00 mg / L of biotin, and between 0.01 and 1.00 mg / L of riboflavin. between 1 and 10 mg / L of calcium panthothenate and between 5 000 and 1 500 mg / L of NaCl.
OTHER BENEFICIAL ADDITIVES
As described above, it is important that a chemically defined medium contain at least one carbon source, a nitrogen source, a phosphorus source, a sulfur source and a buffer. In general, it is advantageous to design a chemically defined medium that is simple (not containing too many components) to reduce the cost and complexity of manufacturing. However, the present inventors have demonstrated that the addition of an additive selected from the group consisting of zinc, cobalt, thiamine, riboflavin, pantothenate, more than 0.4 μl of biotin, more than 50 μl of calcium , more than 15 μΜ of niacin, and more than 25 μΜ of ascorbic acid can significantly improve the expression efficiency of virulence factors such as pertussis toxoid. For this reason, in one embodiment the chemically defined medium comprises an additive selected from the group consisting of zinc, cobalt, thiamine, riboflavin and pantothenate. In another embodiment, the chemically defined medium comprises an additive selected from the group consisting of more than 0.4 μΜ of biotin, more than 50 μΜ of calcium, more than 15 μΜ of niacin, and more than 25 μΜ of ascorbic acid.
In one embodiment, the chemically defined medium comprises at least 2, 3, 4, 5, 6, 7, 8, or 9 of these additives. In a preferred embodiment, the chemically defined medium comprises all additives of zinc, cobalt, riboflavin, thiamine, pantothenate, more than 0.4 μΜ of biotin, more than 0.05 mM of calcium, more than 15 μΜ of niacin , and more than 25 μΜ of ascorbic acid. In one embodiment, the concentration of the additive in the chemically defined medium is sufficient for the additive to increase the level of virulence factor production by Bordetella (which can be verified using the assay previously described for measure whether the addition of an additive alters the yield of pertussis toxoid or not).
In one embodiment, the chemically defined medium comprises more than 0.1 μΜ, more than 1 μΜ, more than 5 μΜ, more than 10 μΜ, more than 20 μΜ, more than 30 μΜ, more than 40 μΜ, more than 50 μΜ, more than 60 μΜ, more than 70 μΜ, more than 100 μΜ, more than 200 μΜ, more than 400 μΜ, more than 600 μΜ, more than 700 μΜ, between 10 and 2,000 μΜ, between 20 and 1,000 μΜ, between 30 and 100 μΜ, or about 75 μΜ of zinc. In one embodiment, the chemically defined medium comprises more than 0.05 μΜ, more than 0.10 μΜ, more than 0.15 μΜ, between 0.10 and 0.30 μ, between 0.10 and 0.20. μΜ, or about 0.18 μΜ of cobalt. In one embodiment, the chemically defined medium comprises more than 0.05 μΜ, more than 0.10 μΜ, more than 0.15 μΜ, between 0.05 and 5.00 μΜ, between 0.10 ^ ¾ ^ 1 , 00 μΜ, or between 0.15 and 0.50 μΜ of thiamine. In one embodiment, the chemically defined medium comprises more than 0.1 μΜ, more than 0.2 μΜ, more than 0.3 μΜ, more than 0.4 μΜ, more than 0.5 μΜ, more than 0, 6 μΜ, more than 0.8 μΜ, between 0.1 and 10 μΜ, between 0.5 and 1.0 μΜ, or about 0.8 μΜ of riboflavin. In one embodiment, the chemically defined medium comprises more than 0.10 μΜ, more than 0.5 μΜ, more than 1.0 μΜ, more than 1.5 μΜ, more than 2.0 μΜ, more than 5, 0 μΜ, more than 8.0 μΜ, between 0.5 and 100 μΜ, between 0.5 and 25.0 μΜ, between 5.0 and 10.0 μΜ, or approximately 8.0 μΜ of pantothenate. In one embodiment, the chemically defined medium comprises more than 0.4 μΜ, more than 0.5 μΜ, more than 0.6 μΜ, more than 0.8 μΜ, between 0.5 and 100 μΜ, between 0, 5 and 25.0 μΜ, between 5.0 and 10.0 μΜ, or about 8.0 μΜ of biotin. In one embodiment, the chemically defined medium comprises more than 100 μΜ, more than 120 μΜ, more than 140 μΜ, between 50 and 1,000 μΜ, between 50 and 500 μΜ, between 100 and 200 μΜ, or about 140 μΜ of calcium. In one embodiment, the chemically defined medium comprises more than 20 μΜ, more than 30 μΜ, more than 35 μΜ, between 15 and 500 μΜ, between 15 and 100 μΜ, between 25 and 75 μΜ, or about 50 μΜ of niacin. . In one embodiment, the chemically defined medium comprises more than 50 μΜ, more than 75 μΜ, more than 100 μΜ, more than 1,000 μΜ, more than 2,000 μΜ, more than 3,000 μΜ, between 25 and 10,000. μΜ, between 10,000 and 5,000 μΜ, or approximately 3,500 μΜ of ascorbic acid.
In another preferred embodiment, the chemically defined medium comprises more than 0.01 mM zinc, more than 0.0005 mM cobalt, more than 0.005 mM thiamine, more than 0.0001 mM riboflavin, more than 0.005 mM mM pantothenate, more than 0.4 μΜ of biotin, more than 0.05 mM of calcium, more than 15 μΜ of niacin and more than 25 μΜ of ascorbic acid.
In another preferred embodiment, the chemically defined medium comprises more than 700 μΜ of zinc, more than
DCZU I 0.15 μΜ of cobalt, more than 29 μΜ of thiamine, more than 0.8 μΜ of riboflavin, more than 8.0 μΜ of pantothenate, more than 0.8 μΜ of biotin, more than 140 μΜ of calcium, more than 35 μΜ of niacin, and more than 3,000 μΜ of ascorbic acid.
In another preferred embodiment, the chemically defined medium comprises between 10 and 150 μl of zinc, between 0.10 and 0.30 μl of cobalt, between 25 and 200 μl of thiamine, and between 0.1 and 10 μl of riboflavin. , between 0.5 and 100 μΜ of pantothenate, between 0.5 and 100 μΜ of biotin, between 50 and 1000 μΜ of calcium, between 1 and 500 μΜ of niacin, and between 25 and 10,000 μΜ of ascorbic acid.
In another preferred embodiment, the chemically defined medium comprises between 30 and 80 μΜ of zinc, between 0.10 and 0.20 μΜ of cobalt, between 25 and 50 μΜ of thiamine, between 0.5 and 1.0 μΜ of riboflavin, between 5.0 and 10.0 μΜ of pantothenate, between 5.0 and 10.0 μΜ of biotin, between 100 and 200 μΜ of calcium, between 25 and 75 μΜ of niacin, and between 10 000 and 5000 μΜ of ascorbic acid.
AMINO ACID CONCENTRATIONS
The present inventors have further demonstrated that prior art media such as Staine-Scholte can be improved by adding high levels of aspartate, glycine, methionine and leucine. Therefore, in another embodiment, the invention relates to a chemically defined medium that comprises an amino acid sequence selected from the group consisting of aspartate at a concentration greater than 1000 μΜ, glycine at a higher concentration. at 1000 μΜ, methionine at a concentration greater than 500 μΜ and leucine at a concentration greater than 1500 μΜ.
In one embodiment, the chemically defined medium comprises aspartate at a concentration greater than 1,000 μΜ, greater than 2,000 μΜ, greater than 2450 μΜ, BE201 greater than 3,000 μΜ, greater than 3,500 μΜ, between 1 000 and 10,000 μΜ, between 1,000 and 5,000 μΜ, or about 4,000 μΜ. In another embodiment, the chemically defined medium comprises glycine at a concentration greater than 500 μΜ, greater than 1,000 μΜ, greater than 1,500 μΜ, greater than 1,750 μΜ, between 500 and 5,000 μΜ, between 500 and 5,000. 2500 μΜ or about 2000 μΜ. In another embodiment, the chemically defined medium comprises methionine at a concentration greater than 100 μΜ, greater than 300 μΜ, greater than 500 μΜ, greater than 600 μΜ, greater than 700 μΜ, and between 100 and 2,000 μΜ, between 100 and 1,000 μΜ or about 775 μΜ. In another embodiment, the chemically defined medium comprises leucine at a concentration greater than 500 μΜ, greater than 1,000 μΜ, greater than 1,500 μΜ, greater than 2,000 μΜ, greater than 2,500 μΜ, greater than 3 000 μΜ, between 500 and 10,000 μΜ, between 500 and 5,000 μΜ, between 3,000 and 4,000 μΜ, or about 3300 μΜ. In one embodiment, the chemically defined medium comprises at least 2, 3 or 4 amino acids among aspartate at a concentration greater than 100 μΜ, glycine at a concentration greater than 1,000 μΜ, methionine at a concentration greater than 500 μΜ and leucine at a concentration above 1500 μΜ. In a preferred embodiment, the chemically defined medium comprises aspartate at a concentration greater than 1000 μΜ, glycine at a concentration greater than 1000 μΜ, methionine at a concentration greater than 500 μΜ and the leucine at a concentration greater than 1500 μΜ.
In another preferred embodiment, the chemically defined medium comprises glutamate at a concentration greater than 50 μΜ, greater than 75 μΜ, greater than 90 μΜ, greater than 100 μΜ, greater than 110 μΜ, between 50 and 500 mM, 50 and 250 mM, between 100 and 150 mM or about 120 mM. In another embodiment, the chemically defined medium BE20 comprises alanine at a concentration greater than 1,000 μΜ, greater than 1,500 μΜ, greater than 2,000 μΜ, greater than 2,500 μΜ, greater than 3,000 μΜ, between 1,000 and 10,000 μΜ, between 1,000 and 5,000 μΜ, between 3,000 and 4,000 μΜ or about 3400 μΜ. In another embodiment, the chemically defined medium comprises phenylalanine at a concentration greater than 500 μΜ, greater than 750 μΜ, greater than 1,000 μΜ, greater than 1250 μΜ, greater than 1400 μΜ, between 500 and 10,000. μΜ, between 500 and 5000 μΜ, between 1000 and 2000 μΜ or about 1400 μΜ. In another embodiment, the chemically defined medium comprises histidine at a concentration greater than 50 μ, greater than 100 μ, greater than 150 μ, greater than 200 μ, between 50 and 1,000 μ, between 50 and 500. μΜ, between 150 and 250 μΜ or about 200 μΜ. In another embodiment, the chemically defined medium comprises isoleucine at a concentration greater than 500 μΜ, greater than 1,000 μΜ, greater than 1,500 μΜ, greater than 1,750 μΜ, between 500 and 5,000 μΜ, between 500 and 2500 μΜ, between 1000 and 2000 μΜ or about 1800 μΜ. In another embodiment, the chemically defined medium comprises lysine at a concentration greater than 500 μΜ, greater than 1,000 μΜ, greater than 1,500 μΜ, greater than 2,000 μΜ, between 500 and 10,000 μΜ, between 500 and 5,000 μΜ, between 1,500 and 2,500 μΜ or about 2,100 μΜ. In another embodiment, the chemically defined medium comprises proline at a concentration greater than 1,000 μΜ, greater than 3,000 μΜ, greater than 4,000 μΜ, greater than 5,000 μΜ, greater than 6,000 μΜ, greater than 7,000 μΜ, between 1,000 and 50,000 μΜ, between 1,000 and 10,000 μΜ, between 7,000 and 8,000 μΜ or about 7600 μΜ. In another embodiment, the chemically defined medium comprises serine at a concentration greater than 500 μΜ, greater than 1,000 μΜ, greater than 1,500 μΜ, greater than 1,700 μΜ, between 500 and 10,000 μΜ, between BE2014 500 and 5000 μΜ, between 1000 and 2000 μΜ or about 1700 μΜ. In another embodiment, the chemically defined medium comprises valine at a concentration greater than 1,000 μΜ, greater than 2,000 μΜ, greater than 2,500 μΜ, greater than 3,000 μΜ, between 1,000 and 10,000 μΜ, between 1,000 and 5,000 μΜ, between 3,000 and 4,000 μΜ or about 3400 μΜ. In another embodiment, the chemically defined medium comprises tyrosine at a concentration greater than 25 μΜ, greater than 50 μΜ, greater than 75 μΜ, greater than 100 μΜ, greater than 150 μΜ, greater than 175 μΜ, between 25 and 1,000 μΜ, between 25 and 500 μΜ, between 100 and 200 μΜ or about 180 μΜ. In another embodiment, the chemically defined medium comprises glutathione at a concentration greater than 100 μ, greater than 200 μ, greater than 400 μ, greater than 500 μ, greater than 600 μ, greater than 700 μ, between 100. and 5,000 μΜ, between 100 and 2,500 μΜ, between 100 and 1,000 μΜ or about 750 μΜ. In a preferred embodiment, the chemically defined medium comprises glutamate at a concentration greater than 50 mM, alanine at a concentration greater than 1000 μΜ, aspartate at a concentration greater than 1000 μΜ, phenylalanine at a concentration greater than 500 μΜ, glycine at a concentration greater than 500 μΜ, histidine at a concentration greater than 50 μΜ, isoleucine at a concentration greater than 500 μΜ, lysine at a concentration of greater than 500 μΜ, leucine at a concentration greater than 500 μΜ, methionine at a concentration greater than 100 μΜ, proline at a concentration greater than 1 000 μΜ, serine at a concentration greater than 500 μΜ, valine at a concentration above 1000 μΜ, tyrosine at a concentration greater than 25 μΜ and glutathione at a concentration greater than 700 μΜ . In another preferred embodiment, the chemically defined medium comprises BE201 glutamate at a concentration greater than 110 mM, alanine at a concentration greater than 3000 μΜ, aspartate at a concentration greater than 3500 μΜ, phenylalanine at a concentration higher than 1400 μΜ, glycine at a concentration higher than 1750 μΜ, histidine at a concentration greater than 200 μΜ, isoleucine at a concentration above 1750 μΜ, lysine at a concentration greater than 2,000 μΜ, leucine at a concentration greater than 3,000 μΜ, methionine at a concentration above 700 μΜ, proline at a concentration greater than 7,000 μΜ, serine at a higher concentration at 1700 μΜ, valine at a concentration greater than 3000 μΜ, tyrosine at a concentration greater than 175 μΜ and glutathione at a concentration of greater than 700 μΜ.
In a preferred embodiment, the chemically defined medium comprises aspartate at a concentration between 1,000 and 10,000 μΜ, glycine at a concentration between 500 and 5,000 μΜ, methionine at a concentration between 100 and 2 μg. 000 μΜ, leucine at a concentration between 500 and 10,000 μΜ, glutamate at a concentration between 50 and 500 mM, alanine at a concentration between 1000 and 10,000 μΜ, phenylalanine at a concentration between 500 and 10,000 μΜ, histidine at a concentration between 50 and 1,000 μΜ, isoleucine at a concentration between 500 and 5,000 μΜ, lysine at a concentration between 500 and 10,000 μΜ, proline at a concentration between 1,000 and 50,000 μΜ, serine at a concentration between 500 and 10,000 μΜ, valine at a concentration between 1,000 and 10,000 μΜ, tyrosine at a concentration between 25 and 1,000 μΜ and Glutathione at a concentration between 100 and 500 0 μΜ.
In a preferred embodiment, the chemically defined medium comprises aspartate at a
BE2C concentration between 1,000 and 5,000 μΜ, glycine at a concentration between 500 and 2,500 μΜ, methionine at a concentration between 100 and 1,000 μΜ, leucine at a concentration between 3,000 and 4,000 μΜ, glutamate at a concentration between 100 and 150 mM, alanine at a concentration between 3000 and 4000 μΜ, phenylalanine at a concentration between 1000 and 2000 μΜ, histidine at a concentration between 150 and 250 μΜ, isoleucine at a concentration between 1000 and 2000 μΜ, lysine at a concentration between 1500 and 2500 μΜ, proline at a concentration between 7000 and 8000 μΜ, serine at a concentration between 1,000 and 2,000 μΜ, valine at a concentration between 3,000 and 4,000 μΜ, tyrosine at a concentration between 100 and 200 μΜ and glutathione at a concentration between 100 and 1,000 μΜ.
COMPONENT REPORTS
The present inventors have surprisingly found that if certain ratios of compounds are used, the chemically defined medium will yield improved yields of virulence factors such as pertussis toxoid and FHA. This is why the invention relates to a chemically defined medium which comprises at least two components and wherein said at least two components are selected from the group consisting of: (a) carbon and phosphorus in a ratio greater than 100: 1, greater than 125: 1, greater than 150: 1, greater than 175: 1 or greater than 200: 1 (carbon.phosphorus) (mol / mol); (b) glutamate and phosphorus in a ratio greater than 20: 1, greater than 22: 1, greater than 24: 1 or greater than 25: 1 (glutamate: phosphorus) (mol / mol); BE2Q14 (c) carbon and magnesium in a ratio of less than 600: 1, less than 500: 1, less than 400: 1 or less than 300: 1 (carbon: magnesium) (mol / mol) ·; (d) glutamate and magnesium in a ratio of less than 115: 1, less than 110: 1, less than 105: 1 or less than 100: 1 (glutamate: magnesium) (mol / mol); (e) carbon and copper in a ratio greater than 3,000: 1, greater than 3,500: 1, or greater than 4,000: 1 (carbon: copper) (mol / mol); (f) glutamate and copper in a ratio greater than 170: 1, greater than 180: 1, greater than 200: 1 or greater than 250: 1 (glutamate: copper) (mol / mol); (g) carbon and iron greater than 9500: 1, greater than 1000: 1, greater than 1250: 1 or greater than 1500: 1 (carbon: iron) (mol / mol); (h) glutamate and iron in a ratio greater than 1600: 1, greater than 1800: 1, greater than 2000: 1 or greater than 2500: 1 (glutamate: iron) (mol / mol); (i) carbon and glycine in a ratio less than 500: 1, less than 400: 1, less than 300: 1 or less than 250: 1 (carbon: glycine) (mol / mol); (j) glutamate and glycine in a ratio less than 100: 1, less than 80: 1, less than 75: 1 or less than 60: 1 (glutamate: glycine) (mol / mol); (k) carbon and leucine in a ratio less than 440: 1, less than 400: 1, less than 350: 1 or less than 300: 1 (carbon: leucine) (mol / mol); (l) glutamate and leucine in a ratio of less than 75: 1, less than 70: 1, less than 60: 1 or less than 50: 1 (glutamate: leucine) (mol / mol); (m) carbon and methionine in a ratio of less than 1200: 1, less than 1000: 1, less than 800: 1 or less than 750: 1 (carbon: methionine) (mol / mol); , BE201 '(n) glutamate and methionine in a ratio of less than 200: 1, less than 175: 1, less than 150: 1 or less than 120: 1 (glutamate-methionine) (mol / mol); (o) carbon and calcium in a ratio greater than 3750: 1, greater than 4,000: 1, greater than 4,500: 1 or greater than 5,000: 1 (carbon: calcium) (mol / mol); (p) glutamate and calcium in a ratio greater than 620: 1, greater than 650: 1, greater than 675: 1 or greater than 750: 1 (glutamate: calcium) (mol / mol); (q) carbon and cobalt in a ratio greater than 3,000: 1, greater than 3,500: 1, greater than 4,750: 1 or greater than 5,000: 1 (carbon: cobalt) (mol / mol); (r) glutamate and cobalt in a ratio greater than 750: 1, greater than 1,000: 1, greater than 1,250: 1 or greater than 1,500: 1 (glutamate: cobalt) (mol / mol); (s) carbon and zinc in a ratio greater than 3,000: 1, greater than 3,500: 1, greater than 4,000: 1 or greater than 5,000: 1 (carbon: zinc) (mol / mol); (t) glutamate and zinc in a ratio greater than 750: 1, greater than 1,000: 1, greater than 1,250: 1 or greater than 1,500: 1 (glutamate: zinc) (mol / mol); (u) carbon and sulfate equivalents in a ratio greater than 750: 1, greater than 1,000: 1, greater than 1,250: 1 or greater than 1,500: 1 (carbon: sulfate equivalents) (mol / mol ); and (v) glutamate and sulfate equivalents in a ratio greater than 130: 1, greater than 150: 1, greater than 175: 1 or greater than 200: 1 (glutamate: sulfate equivalents) (mol / mol).
In one embodiment, the chemically defined medium comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21 or 22 components (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (1), (m), (n), (o), BE201 (p), (q), (r), (s), (t), (u) and (v). In one embodiment, the chemically defined medium comprises carbon and phosphorus in a ratio greater than 200: 1 (carbon: phosphorus) (mol / mol), glutamate and phosphorus in a ratio greater than 25: 1 (glutamate : phosphorus) (mol / mol), carbon and magnesium in a ratio of less than 300: 1 (carbon: magnesium) (mol / mol), glutamate and magnesium in a ratio of less than 100: 1 (glutamate: magnesium ) (mol / mol), carbon and copper in a ratio greater than 4,000: 1 (carbon: copper) (mol / mol), glutamate and copper in a ratio greater than 250: 1 (glutamate: copper) (mol / mol), carbon and iron in a ratio greater than 1500: 1 (carbon: iron) (mol / mol), glutamate and iron in a ratio greater than 2500: 1 (glutamate: iron) (mol / mol), carbon and glycine in a ratio of less than 250: 1 (carbon: glycine) (mol / mol), glutamate and glycine in a ratio of less than 250: 1 (carbon: glyci ne) (mol / mol), carbon and leucine in a ratio of less than 300: 1 (carbon: leucine) (mol / mol), glutamate and leucine in a ratio of less than 50: 1 (glutamate: leucine) (mol / mol), carbon and methionine in a ratio of less than 750: 1 (carbon: methionine) (mol / mol), glutamate and methionine in a ratio of less than 120: 1 (glutamate. methionine) (mol / mol), carbon and calcium in a ratio greater than 5,000: 1 (carbon: calcium) (mol / mol), glutamate and calcium in a ratio greater than 750: 1 (glutamate: calcium) (mol / mol), carbon and cobalt in a ratio greater than 5,000: 1 (carbon: cobalt) (mol / mol), glutamate and cobalt in a ratio greater than 1,500: 1 (glutamate: cobalt) (mol / mol), carbon and zinc in a ratio greater than 5000: 1 (carbon: zinc) (mol / mol), glutamate and zinc in a ratio greater than 1500: 1 (glutamate: zinc) (mol / mol), carbon and equivalents of sulphate in a report
BE2C greater than 1500: 1 (carbon: sulphate equivalents) and glutamate and sulphate equivalents in a ratio greater than 200: 1 (glutamate: sulphate equivalents).
The term "sulfate equivalents" refers to inorganic sulfate or inorganic compounds whose catabolism results in the production of sulphate (including, inter alia, cysteine, cystine and glutathione). MEDIUM COMPRISING THE FE (III)
Bordetella media tend to include iron in the form of Fe (II) ions such as Stainer-Scholte medium which comprises FeSO4 (Stainer and Schölte, Journal of General Microbiology (1971), 63: 211-220), however the present inventors have demonstrated that Fe (III) ions can also be used in Bordetella medium, and further that a medium comprising Fe (III) ions (such as Fe (III) citrate) production of virulence factors such as pertussis toxoid higher than a medium comprises Fe (II) ions (such as FeSO4).
Therefore, in one embodiment the chemically defined medium comprises Fe (III) ions. Similarly, in one embodiment, the chemically defined medium comprises Fe (II) or Fe (III) complexed with an organic compound, preferably the chemically defined medium comprises Fe (III) complexed with an organic compound. In one embodiment, the organic compound is an organic compound selected from the group consisting of heme, hemoglobin, myoglobin, transferrin, ferritin, lactoferrin, enterobactin, aerobactin, alcaligine a coprogenic substance, ferrichrome, desferrioxamine, ferroxamine, hydroxamate, citrate and dihydroxybenzoylserine. In one embodiment, the chemically defined medium comprises Fe (III) complexed with a citrate. In another embodiment, the chemically defined medium comprises more than 10 μΜ, more than 20 W1 more than 30 μΜ, more than 40 μΜ, more than 50 μΜ, between 10 and 500 μΜ, between 10 and 100 μΜ, between 25 and 75 μΜ, or approximately 60 μΜ of Fe (III) citrate.
OTHER COMPONENTS OF THE ENVIRONMENT
The medium according to the invention may comprise other components than those described above. For example, the chemically defined medium may include chloride. In one embodiment, the chemically defined medium comprises chloride at a concentration of less than 45 mM, less than 40 mM, less than 35 mM, less than 30 mM, less than 25 mM, less than 20 mM or less than 15 mM between 0.1 and 500 mM, between 10 and 20 mM or about 16 mM. The chemically defined medium may comprise acetate. In one embodiment, the chemically defined medium comprises acetate at a concentration greater than 1 mM, greater than 2 mM, greater than 3 mM, greater than 4 mM, between 1 and 100 mM, between 4 and 6 mM, or about 5 mM. The chemically defined medium may include potassium. In one embodiment, the chemically defined medium comprises potassium at a concentration greater than 1 mM, greater than 2 mM, greater than 3 mM, greater than 4 mM, greater than 5 mM, greater than 6 mM, between 1 and 100 mM, between 5.5 and 7 mM, or about 6.5 mM. The chemically defined medium may include a source of phosphorus. In one embodiment, the phosphorus source comprises phosphate at a concentration greater than 0.5 mM, at a concentration greater than 1 mM, at a concentration greater than 1.5 mM, at a concentration greater than 2 mM, at a concentration greater than 2.5 mM, between 0.5 and 100 mM, between 3 and 4 mM, or about 3.6 mM. The chemically defined medium may comprise a dimethyl-β-cyclodextrin. In one embodiment, the chemically defined medium comprises a BE201 dimethyl-β-cyclodextrin at a concentration greater than 0.1 mM, greater than 0.2 mM, greater than 0.3 mM, greater than 0.4 mM, greater than at 0.5 mM, greater than 0.6 mM, between 0.01 and 10 mM, between 0.7 and 0.8 mM, or about 0.75 mM.
In one embodiment, the chemically defined medium does not comprise sulphate, cysteine or cystine and comprises more than 0.008 mM thiosulfate, more than 11 mM MOPS, more than 6 μΜ copper, more than 400 μΜ magnesium, more than 700 μΜ of zinc, more than 0.15 μΜ of cobalt, more than 29 μΜ of thiamine, more than 0.8 μΜ of riboflavin, more than 8.0 μΜ of pantothenate, more than 0.8 μΜ biotin, more than 140 μΜ of calcium, more than 35 μΜ of niacin, more than 3,000 μΜ of ascorbic acid, glutamate at a concentration greater than 110 mM, and alanine at a concentration greater than 3,000 μΜ, aspartate at a concentration greater than 3,500 μΜ, phenylalanine at a concentration greater than 1400 μΜ, glycine at a concentration above 1750 μΜ, histidine at a concentration greater than 200 μΜ, isoleucine at a concentration greater than 1750 μΜ, lysine at a higher concentration at 2000 μΜ, leucine at a concentration greater than 3 000 μΜ, methionine at a concentration greater than 700 μΜ, proline at a concentration greater than 7 000 μΜ, serine at a concentration greater than 1 700 μΜ μΜ, valine at a concentration greater than 3 000 μΜ, tyrosine at a concentration greater than 175 μΜ, glutathione at a concentration above 700 μΜ, less than 15 mM chloride, more than 4 mM acetate more than 6 mM potassium, more than 0.6 mM dimethyl-β-cyclodextrin and more than 2.5 mM phosphate; optionally the chemically defined medium further comprises sodium and more than 50 μΜ Fe (III) citrate.
In one embodiment, the chemically defined medium does not comprise sulphate, cysteine or cystine and BE2 comprises between 0.005 and 0.100 mM thiosulfate, between 2 and 100 mM MOPS, between 2 and 200 μM copper, between 2 and 100 mM and 6,000 μM magnesium, between 10 and 150 μM zinc, between 0.10 and 0.30 μM cobalt, between 25 and 200 μM thiamine, between 0.1 and 10 μM riboflavin, between 0.5 and 100 μM pantothenate, between 0.5 and 100 μM biotin, between 50 and 1000 μM calcium, between 1 and 500 μM niacin, between 25 and 10,000 μM ascorbic acid, 11 aspartate at a concentration between 1 and And 10,000 μM, glycine at a concentration between 500 and 5000 μM, methionine at a concentration between 100 and 2000 μM, leucine at a concentration between 500 and 10,000 μM, glutamate at a concentration of between 50 and 500 mM, alanine at a concentration between 1000 and 10 000 μM, phenylalanine at a concentration between 500 and 10 000 μM, histidine at a concentration between 50 and 1000 μM, isoleucine at a concentration between 500 and 5000 μM, lysine at a concentration between 500 and 10,000 μM, proline at a concentration between 1000 and 50,000 μM , serine at a concentration between 500 and 10,000 μM, valine at a concentration between 1000 and 10,000 μM, tyrosine at a concentration between 25 and 1000 μM, glutathione at a concentration between 100 and 5000 μM, between 0.1 and 500 mM chloride, between 1 and 100 mM acetate, between 1 and 100 mM potassium, between 0.01 and 10 mM dimethyl-β-cyclodextrin and between 0.5 and 100 mM phosphate; optionally the chemically defined medium further comprises sodium and between 10 and 500 μM of Fe (III) citrate.
In one embodiment, the chemically defined medium does not include sulfate, cysteine or cystine and comprises between 0.005 and 0.025 mM thiosulfate, between 5 and 20 mM MOPS, between 4 and 10 pM copper, between 1000 and and 6,000 μM magnesium, between 30 and 80 μM zinc, between 0.10 and 0.20 μM cobalt, between 25 and 50 μM thiamine, between 0.5 BE20 and 1.0 μΜ riboflavin, between 5 , 0 and 10.0 μΜ of pantothenate, between 5.0 and 10.0 μΜ of biotin, between 100 and 200 μΜ of calcium, between 25 and 75 μΜ of niacin, between 1000 and 5000 μΜ of ascorbic acid, aspartate at a concentration between 1000 and 5000 μΜ, glycine at a concentration between 500 and 2500 μΜ, methionine at a concentration between 100 and 1000 μΜ, leucine at a concentration between 3000 and and 4000 μΜ, glutamate at a concentration between 100 and 150 mM, alanine at a concentration between 3000 and 4000 μΜ, phenylalanine at a concentration between 100 0 and 2000 μΜ, histidine at a concentration between 150 and 250 μΜ, isoleucine at a concentration between 1000 and 2000 μΜ, lysine at a concentration between 1500 and 2500 μΜ, proline at a concentration between 7000 and 8000 μΜ, serine at a concentration between 1000 and 2000 μΜ, valine at a concentration between 3000 and 4000 μΜ, tyrosine at a concentration between 100 and 200 μΜ μΜ, glutathione at a concentration between 100 and 1000 μΜ, between 10 and 20 mM chloride, between 4 and 6 mM acetate, between 5.5 and 7 mM potassium, ente 0.7 and 0.8 mM dimethyl-O-cyclodextrin and between 3 and 4 mM phosphate; optionally the chemically defined medium further comprises sodium and between 25 and 75 μΜ of Fe (III) citrate.
FERMENTATION PROCESS
This invention further relates to a fermentation method for growing a species of the genus Bordetella in a chemically defined medium (CDM) comprising (a) inoculating the chemically defined medium according to the invention with the species of the genus Bordetella; (b) maintenance of the Bordetella species in the chemically defined medium for a period of time sufficient to allow accumulation of biomass. BE20
The term "fermentation process" refers to a process on an industrial scale for growing cells and / or expressing a virulence factor from these cells. The term "industrial scale" refers to a process in a fermenter. In one embodiment, the "process on an industrial scale" refers to a process in a fermenter having a working volume of between 5 and 10,000 liters, between 10 and 5,000 liters, between 20 and 2,000 liters, between 50 and 1,000 liters, greater than or equal to 5 liters, greater than or equal to 10 liters, greater than or equal to 15 liters, greater than or equal to 20 liters, greater than or equal to 25 liters, greater than or equal to 50 liters, greater than or equal to 100 liters, less than or equal to 10 000 liters, less than or equal to 5 000 liters or less than or equal to 2 500 liters. In another embodiment, the "industrial scale process" is a process suitable for the production of more than 10 mg / L, more than 15 mg / L or more than 20 mg / L of pertussis toxoid.
In one embodiment, the fermentation process has an average generation time less than 15h, less than 12h, less than 10h or less than 9h. The method of determining the average generation time is described above.
In another embodiment, the method of
Fermentation produces more than 10 mg / L, 15 mg / L or 20 mg / L of pertussis toxoid. The method for determining pertussis toxoid yields is described above.
In one embodiment, the fermentation process is carried out at a temperature greater than or equal to 32 ° C, greater than or equal to 33 ° C, greater than or equal to 34 ° C, lower than or equal to 45 ° C, lower or equal to 42 ° C, less than or equal to 40 ° C, less than or equal to 38 ° C, between 32 and 45 ° C, between 33 and 42 ° C, between 33 and 40 ° C or between 33 and 38 ° C .
In one embodiment, an antifoaming agent is used during the fermentation process. In another embodiment, the antifoam agent is a polydimethyl-siloxane.
In one embodiment, the dissolved oxygen level is between 1 and 160 μΜ, between 15 and 140 μΜ, between 30 and 120 μΜ, between 45 and 110 μΜ, between 60 and 100 μΜ, or about 80 μΜ.
In one embodiment, the pH of the fermentation process is between pH 6.0 and 7.5, between pH 6.5 and 7.0 or about pH 7.2.
EXPRESSION AND PURIFICATION OF VIRULENCE FACTORS
In one embodiment, the species of the genus Bordetella expresses at least one virulence factor including pertussis toxoid (PT), filamentous haemagglutinin (FHA), pertactin (PRN), agglutinogen 2 or agglutinogen 3. In one embodiment, the species of the genus Bordetella expresses PT, in one embodiment the species of the genus Bordetella expresses FHA, in one embodiment the species of the genus Bordetella expresses PRN, in a In one embodiment, the species of the genus Bordetella expresses PT and FHA, in one embodiment the species of the genus Bordetella expresses PT and PRN, in one embodiment the species of the genus Bordetella expresses PRN and FHA, in one embodiment. embodiment, the species of the genus Bordetella expresses PT, PRN and FHA. PT, FHA and PRN are well known in the art.
In one embodiment, the method further comprises a step c) of purifying the virulence factor to obtain a purified virulence factor. The purified virulence factor may be pertussis toxoid (PT), filamentous haemagglutinin (FHA), pertactin (PRN), agglutinogen 2 or purified agglutinogen 3. The purified virulence factor may be altered after purification, for example the pertussis toxoid may be chemically detoxified after purification. See also EP 427462 and WO 91/12020 for the preparation of pertussis antigens. In one embodiment, step c) involves cell purification by chromatography. In one embodiment, the chromatographic technique is affinity chromatography, gel filtration, high pressure liquid chromatography (HPLC) or ion exchange chromatography. Optionally, the affinity chromatography uses a label affinity purification column, an antibody purification column, a lectin affinity column, a prostaglandin purification column, or a strepavidin column. HPLC optionally uses an ion exchange column, a reverse phase column or an exclusion column. Optionally, the ion exchange column is an anion exchange column or a cation exchange column.
The method may further comprise a step d) of formulating an immunogenic composition comprising the purified virulence factor.
The method may further comprise a step e) of adding at least one additional antigen to the immunogenic composition. In one embodiment, the at least one additional antigen is selected from the group consisting of pertussis toxoid, filamentous haemagglutinin, pertactin, fimbrial agglutinogen, diphtheria toxoid, tetanus toxoid, at least one derived conjugated saccharide antigen. N. meningitidis, a Hepatitis B surface antigen, inactivated polio virus (IPV) and a conjugated saccharide antigen derived from Haemophilus influenzae b. Said at least conjugated saccharide antigen derived from N. meningitidis may be MenC, MenY, MenA and MenW (eg A + C, A + Y, A + W, C + Y, C + W, Y + W, A +). C + Y, A + C + W, A + Y + W, C + Y + W, A + C + Y + W); possibly MenC and / or MenY are / is included, eventually all four are included.
Alternatively or in addition to the above meningococcal antigens, the immunogenic composition may comprise one or more pneumococcal capsular oligosaccharide or polysaccharide conjugated to a carrier protein.
The oligosaccharides or pneumococcal capsular polysaccharides (preferably the latter) typically represented in the compositions according to the invention include antigens derived from at least four pneumococcal serotypes. Preferably, the four serotypes comprise 6B, 14, 19F and 23F. More preferably, at least 7 serotypes are included in the composition, for example those derived from serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. More preferably, at least 11 serotypes are included in the composition (11-valent), for example those derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. In a preferred embodiment according to the invention, at least 13 of these conjugated pneumococcal antigens are included, although additional antigens, for example 23-valents (such as serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F) are also contemplated by the invention.
In one embodiment, the immunogenic composition comprises a pharmaceutically acceptable excipient. In one embodiment, the fermentation process comprises a step f) of adding a pharmaceutically acceptable excipient to the immunogenic composition.
In one embodiment, the immunogenic composition comprises an adjuvant such as aluminum phosphate or aluminum hydroxide. In one embodiment, the fermentation process comprises a step g) of adding an adjuvant to the immunogenic composition. Methods for adsorbing DTPa and DTPw antigens on aluminum adjuvants are known in the art. See for example WO 93/24148 and WO 97/00697. Generally, the adsorbed components on an adjuvant are left there for at least 10 minutes at room temperature at a suitable pH to adsorb the majority and preferably all the antigen before incorporation of the antigens by mixing in the combined immunogenic compositions according to the present invention. Other components are preferably non-adsorbed (as IPV) or adsorbed specifically on other adjuvants. Hepatitis B surface antigen (HBsAg) is preferably adsorbed on aluminum sulfate (as described in WO 93/24148) before mixing with other components.
In another embodiment, the invention relates to a virulence factor obtainable by the method. In another embodiment, the invention relates to a virulence factor obtained by the method.
In another embodiment, the invention relates to an immunogenic composition comprising the virulence factor and a pharmaceutically acceptable excipient. In one embodiment, the immunogenic composition comprises at least one additional antigen. In one embodiment, the at least one additional antigen is selected from the group consisting of pertussis toxoid, filamentous haemagglutinin, pertactin, fimbrial agglutinogen, diphtheria toxoid, tetanus toxoid, at least one derived conjugated saccharide antigen. N. meningitidis, a Hepatitis B surface antigen, inactivated polio virus (IPV) and a conjugated saccharide antigen derived from Haemophilus influenzae b (possibly conjugated to tetanus toxoid). Said at least conjugated saccharide antigen derived from N. meningitidis may be MenC, MenY, MenA and MenW (eg A + C, A + Y, A + W, C + Y, C + W, Y + W, A +). C + Y, A + C + W, A + Y + W, C + Y + W, A + C + Y + W); possibly MenC and / or MenY are / is included, eventually all four are included. In one embodiment, the vaccine comprises diphtheria toxoid, tetanus toxoid, and at least one virulence factor from PT, FHA, and PRN (a DTPa vaccine).
In one embodiment, the invention relates to a vaccine comprising the immunogenic composition.
Vaccine preparation is generally described in Vaccine Design - The Subunit and Adjuvant Approach Ed Powell and Newman; Pellum Press. Advantageously, the combined vaccine according to the invention is a pediatric vaccine.
The amount of polysaccharide or oligosaccharide antigen conjugated in each vaccine is chosen as an amount that induces an immunoprotective response without significant side effects in typical vaccinated individuals. This amount will vary depending on the specific immunogens used. Generally each dose should comprise from 1 to 1000 μg of conjugated polysaccharide or oligosaccharide (expressed as saccharide amount), preferably from 2 to 100 μg, more preferably from 4 to 40, 2 to 15, or 3 to 10 μg, of most preferably about or exactly 5 μg.
The protein antigen content in the vaccine will typically be in the range of 1 to 100 μg, preferably 5 to 50 μg, more typically in the range of 5 to 25 μg.
A suitable amount of antigen for a particular vaccine can be determined by standard studies involving observation of antibody titers and other responses in subjects. After an initial vaccination, subjects may receive one or two booster shots at approximately 4 week intervals or more.
The vaccine preparations according to the present invention may be used to protect or treat a mammal (preferably human) susceptible to infection, by administering said vaccine systemically or mucosally. Such administrations may include intramuscular, intraperitoneal, intradermal or subcutaneous injection.
According to another aspect, the invention relates to the immunogenic composition or the vaccine as described previously used in the prevention or treatment of the disease.
According to another aspect, the invention relates to the immunogenic composition or the vaccine as described previously used for the prevention or treatment of whooping cough.
According to another aspect, the invention relates to a use of the immunogenic composition or the vaccine as described above that can be used in the prevention or treatment of the disease.
In another aspect, the invention relates to a use of the immunogenic composition or vaccine as described above in the preparation of a medicament for the treatment or prevention of a bacterial disease.
In another aspect, the invention relates to a method for preventing or treating a disease comprising administering the immunogenic composition or vaccine as described above to a patient.
In one embodiment, the disease is pertussis.
The term "pertussis toxoid" refers to pertussis toxoid or alternatively a genetic toxoid form of pertussis toxoid. In one embodiment, the pertussis toxoid is not a genetic toxoid of pertussis toxoid.
The term "comprising", "understand / understand" and "understands" may be replaced in any case by the terms "consisting of / consisting of", "consisting of / consisting of" and "consisting of / consists of". The term "includes" means "includes". Thus, unless otherwise indicated, it will be understood that the term "includes", and its variants such as "understand / understand", and "include" imply the inclusion of the indicated compound or composition (eg, nucleic acid, polypeptide, antigen) or a step, or a group of compounds or
No steps, but not the exclusion of any other compound, composition, step, or group. The term "constituted by / consists of" means "contains" excluding any other compound, composition, stage, or group, etc.
Unless otherwise indicated, the singular terms "one", "one", and "the", "the" include plural referents. Similarly, and unless otherwise indicated, the term "or" is intended to include "and". The term "plurality" refers to two or more. It will be further understood that all base sizes or amino acid sizes, and all molecular weight or molecular weight values, indicated for the nucleic acids or polypeptides are approximate, and are indicated for descriptive purposes. In addition, the numerical limits indicated with respect to the concentrations or levels of a substance, such as an antigen, are intended to be approximate. Thus, when it is indicated that a concentration is at least (for example) 200 μg, it will be understood that the concentration is at least approximately (or "about" or "~") 200 μg.
Example 1 - Fermentation on a 20L scale of Bordetella pertussis in chemically defined basic media
A chemically defined medium (CDM), based on the composition of the medium of Stainer & Schölte (SS, Stainer and Schölte, J. Gen. Microbiol 63: 211-220 (1971)) was designed, containing amino acid supplements as well as dimethyl-β-cyclodextrin. Table 1 compares the composition of the Stainer & Schölte (SS) original, a modified version of SS medium containing dimethyl-β-cyclodextrin-growth promoter of B. pertussis (Imaizumi et al., J. Clin Microbiol., 17: 781-786 (1983)) - and other minor alterations (SS-cyclo), and the chemically defined basic medium (B-CDM).
SS-cyclo and B-CDM media were evaluated in COQ467 and COQ365 fermentations, respectively. For both fermentations, a first shake flask pre-culture containing 7.5 ml of fresh medium (B-CDM) was inoculated with 109 CFU of B. pertussis and incubated at 35 ° C (+/- 1 ° C). ) and 150 rpm for 24 hours (+/- 1 hour). The first preculture was used to inoculate a second shake flask pre-culture containing 100 ml of fresh medium (B-CDM). The second preculture was incubated at 35 ° C (+/- 1 ° C) and 150 rpm for 24 h (+/- 1 h), and used to inoculate two shake flasks each containing fresh medium ( SS-cyclo for COQ467 and B-CDM for COQ365, see composition in Table 1). After growth at 35 ° C. (+/- 1 ° C.) and 150 rpm for 40 h (+/- 4 h), the two shake flasks of the third preculture were pooled. The pooled preculture was used to inoculate a fermenter as soon as the third preculture was stopped.
A 20L fermentor (Biolafitte) was used. 10L of medium ("SS cyclo" for COQ467 and "B-CDM" for COQ365) were transferred under aseptic conditions into the fermenter. The following conditions were used to establish the 100% dissolved oxygen (DO) level: temperature (35 ° C) and head pressure (0.4 bar). Inoculation was performed by adding 1.5L of pooled preculture.
During the fermentation, the temperature (35 ° C), the head pressure (0.4 bar), and the air flow (20 L min-1) were kept constant. The foaming was controlled by automatically adding a polydimethylsiloxane emulsion via a foam controller. The dissolved oxygen level was set at 25% and regulated by acceleration of agitation when the OD dropped below 25%. The minimum stirring speed was set at 50 rpm and the maximum stirring speed at 1000 rpm. The pH was regulated at 7.2 by addition of 50% (w / v) phosphoric acid in COQ467 (SS-cyclo) and addition of 50% (w / v) acetic acid in COQ365 (B-). CDM).
During fermentation, growth was monitored via optical density at 650 nm (OD 650 nm). At the end of the fermentation (defined by the time when the oxygen consumption decreases - following the depletion of glutamate, resulting in a reduction of the stirring speed), the production of pertussis toxoid (PT) in the supernatant of culture was determined by Elisa. Table 2 compares biomass yield, PT yield, and mean generation time of fermentation COQ365 (B-CDM) and COQ467 (SS-cyclo). Determination of PT concentration. The concentration of PT in the culture supernatants was determined by an enzyme-linked immunosorbent assay (ELISA). The wells of polystyrene microdilution plates (4-39454; Nunc) were coated overnight at 4 ° C with 100 μΐ of purified guinea pig anti-PT polyclonal antiserum (1: 16,000 dilution in carbonate buffer 50). mM, pH 9.6). The plate was washed three times with DPBST (Dubelcco phosphate free saline solution without Ca or Mg, containing 0.1% (v / v)
Tween 20). Successive dilutions of purified PT references and culture supernatants (in DPBST) were then added to each well (100 μΐ per well). After incubation for 30 minutes at room temperature, the plate was washed three times with DPBST. Goat anti-PT antiserum (1: 500 dilution in DPBST) and guinea pig serum without anti-PT (1: 1000 dilution in DPBST) were then added to each well (100 μΐ per well). . After incubation for 30 minutes at room temperature, the plate was washed three times with DPBST. Rabbit anti-goat immunoglobulin G conjugated to alkaline phosphatase (Zymed, 1: 1000 dilution in DPBST) was then added to each well (100 μΐ per well). After incubation for 30 minutes at room temperature, the plate was washed three times with DPBST. The plate was revealed by addition of a 10 g / L solution of p-nitrophenyl phosphate (Calbiochem) in a diethanolamine buffer (diethanolamine 9.7% (v / v), sodium azide 0.2 g / L 0.214 g / L MgCl2.6H2O, pH 9.8) to each well (100 μΐ per well). The color development was carried out at room temperature, and stopped by adding 50 μl of 3M NaOH to each well. Absorbance of the wells was read at 405 nm within 1 hour of adding NaOH using a Versamax microplate reader (Molecular Devices).
B-CDM conditions resulted in higher yields and growth rates than SS-cyclo. PT production also increased significantly. (See Table 2)
SS = Stainer & Schölte B-CDM = chemically defined basic medium
Tris = tris (hydroxymethyl) aminomethane * Initial calculated biomass concentration based on OD650nm
The yield was calculated as the difference between DOssonm at the end of the fermentation and DOesonm at the beginning of the fermentation. *** The total fermentation time is defined as the time when the oxygen consumption decreases (following glutamate depletion), resulting in a reduction of the stirring speed **** Average generation time calculated as follows . First, the number of generations is calculated as a ratio between DOesonm at the end of the fermentation and DOgsonm at the beginning of fermentation, converted to log2. The average generation time is then calculated by dividing the total fermentation time by the number of generations.
Example 2 Effect of the iron source on the 20L scale fermentation of Bordetelle pertussis in a chemically defined medium
Ferric citrate has been evaluated as an alternative to ferrous sulfate in fermentation COQ348.
A first shake flask pre-culture containing 7.5 ml of fresh medium (B-CDM;
Table 1) was inoculated with 109 CFU of B. pertussis and incubated at 35 ° C (+/- 1 ° C) and 150 rpm for 24 h (+/- 1 h). The first preculture was used to inoculate a second shake flask pre-culture containing 100 ml of fresh medium (B-CDM). The second preculture was incubated at 35 ° C (+/- 1 ° C) and 150 rpm for 24 h (+/- 1 h), and used to inoculate two shake flasks each containing fresh medium ( B-CDM modified to contain 10 mg / L of Fe (III) citrate trihydrate, without FeSCh). After growth at 35 ° C. (+/- 1 ° C.) and 150 rpm for 40 h (+/- 4 h), the two shake flasks of the third preculture were combined. The pooled preculture was used to inoculate a fermenter as soon as the third preculture was stopped.
A 20L fermentor (Biolafitte) was used. 10L of medium (B-CDM modified to contain 10mg / L of Fe (III) citrate trihydrate, without FeSCu) was transferred under aseptic conditions into the fermenter. The following conditions were used to establish the 100% dissolved oxygen (DO) level: temperature (35 ° C) and head pressure (0.4 bar). Inoculation was performed by adding 1.5L of the pooled preculture.
During the fermentation, the temperature (35 ° C), the head pressure (0.4 bar), and the air flow (20 L min-1) were kept constant. The foaming was controlled by automatically adding a polydimethylsiloxane emulsion via a foam controller. The dissolved oxygen level was set at 25% and regulated by acceleration of agitation when the OD dropped below 25%. The minimum stirring speed was set at 50 rpm and the maximum stirring speed at 1000 rpm. The pH was regulated at 7.2 by addition of 50% (w / v) phosphoric acid.
During fermentation, growth was monitored via the optical density at 650 nm (ODesonm). At the end of the fermentation (defined by the time when oxygen consumption decreases - following the depletion of glutamate, resulting in a reduction of the stirring speed), the production of pertussis toxoid (PT) in the supernatant culture was determined by Elisa.
Table 3 compares the biomass yield, the PT yield, and the average generation time of COQ352 fermentation (modified B-CDM to contain 10 mg / L of Fe (III) citrate trihydrate, without FeSCL) and fermentation COQ365 ( B-CDM containing FeSCu, see Example 1).
The yield and growth rate were similar between the two conditions, in terms of maximum biomass concentration, indicating that the inorganic sulfate can be omitted from the composition of the medium, and that the iron can be supplied as Fe (II) or Fe (III) without affecting the growth of B. pertussis. PT production also increased significantly when ferric citrate was used as a source of iron instead of ferrous sulphate.
* Initial calculated biomass concentration based on the measured OD6sonm of pre-culture, ie 1.5 * pre-cooked / H, 5. ** The yield was calculated as the difference between DO650nm at the end of the fermentation and DO650nm at the beginning of the fermentation. *** The total fermentation time is defined as the time when the oxygen consumption decreases (following glutamate depletion), resulting in a reduction of the stirring speed **** Average generation time calculated as follows . First, the number of generations is calculated as a ratio between DC> 650nm at the end of the fermentation and DO650nm at the beginning of the fermentation, converted to log2. The average generation time is then calculated by dividing the total fermentation time by the number of generations. BE2 (
Example 3 Thiosulfate as Sulfur Source for the Growth of Bordetella Pertussis
On the basis of the literature, the growth of B. pertussis is only possible in the presence of an organic source of sulfur, which can be provided in the form of cystine, cysteine, and / or glutathione (Jebb and Tomlinson (1957 J. Gen. Microbiol 17:59).
Assays were performed to determine whether inorganic sources of sulfur were able to support the growth of B. pertussis. A shake flask containing 7.5 ml of fresh medium (B-CDM modified to contain 0.604 g / L of niacin) was inoculated with 109 CFU of B. pertussis and incubated at 35 ° C (+/- 1 ° C). and 150 rpm for 24h (+/- 5h). The cells were harvested by centrifugation, washed twice with 0.9% (w / v) NaCl, and resuspended in fresh medium containing no S source (see composition in Table 4) at a theoretical OD 6 50 nm of 0.5, calculated from the DC> 65onm of the crop before harvest. 20 μl of this cell suspension were used to inoculate each well of a 96-well microtiter plate filled with 180 μΐ of fresh media containing no S source (see composition in Table 4). To each of the wells, 20 μl of a complement solution containing one of the compounds shown in Table 5 were added. Only the inner wells of the plate were used for the cultures, in order to reduce evaporation to a minimum, and a control was included, in which the supplemental solution was replaced by water. The plate was then incubated for 53 hours at 35 ° C in a Biotek Synergy H1 reader with constant shaking, and growth was monitored automatically every 10 minutes via DOösonm. The results of the growth assay are shown in Table 5.
Inorganic sulphate and sulphite have not been able to support the growth of B. pertussis. However, growth was observed in the presence of thiosulfate as the sole source of sulfur in the medium. These results demonstrate that i) sulfate can be omitted from the medium, and ii) that an organic source of sulfur such as cystine, cysteine, or glutathione does not necessarily need to be present, as long as a growth-supporting compound such as thiosulfate is present.
* calculated as the difference between the OD650nm after 53h and the initial ODgsonm ** +, biomass yield greater than or equal to the negative control; -, biomass yield less than or equal to the negative control
Example 4 Screening for identifying alternative buffers that can be used in the chemically defined medium
Screening was performed to identify alternatives to Tris buffer in B-CDM medium. A first shake flask pre-culture containing 7.5 ml of fresh medium (B-CDM) was inoculated with 109 CFU of B. pertussis and incubated at 35 ° C (+/- 1 ° C) and 150 rpm. mn for 24h (+ / -lh). The first preculture was used to inoculate a second shake flask pre-culture containing 100 ml of fresh medium (B-CDM). The second preculture was incubated at 35 ° C (+/- 1 ° C) and 150 rpm for 24h (+/- 1h). The cells were then harvested by centrifugation, washed in 0.9% NaCl and resuspended in 0.9% NaCl. This cell suspension was used to inoculate a set of 9 flasks under agitation, each containing 50 ml of fresh medium (B-CDM) or a medium in which the Tris buffer in the CDM was replaced with another medium among those indicated in Table 6. The flasks were incubated at 35 ° C and 150 rpm for 48h. Growth was monitored via the DOssonm after 24h and 48h. The results are shown in Table 6.
The β-glycerophosphate and MOPS buffers were both able to support the growth of B. pertussis in the B-CDM. Overall, MOPS was superior to β-glycerophosphate in terms of growth rate (biomass yield after 24h) and yield (biomass yield after 48h). With both buffers, lower concentrations resulted in faster growth and higher final biomass yield. At the lowest concentration tested (2.5 g / L), MOPS demonstrated a beneficial effect on growth rate (biomass yield after 24h), compared to control conditions using Tris as a buffer.
* The biomass yield is expressed relative to the control conditions (Tris buffer) at the same time of incubation
Example 5 - Impact of the addition of Cu2 + on the. 20L scale fermentation of Bordetella pertussis in a chemically defined medium The effect of a Cu2 + complement was evaluated in COQ348 fermentation.
A first shake flask pre-culture containing 7.5 ml of fresh medium (B-CDM, see composition in Table 1) was inoculated with 109 CFU of B. pertussis and incubated at 35 ° C (+/- 1). ° C) and 150 rpm for 24h (+/- lh). The first preculture was used to inoculate a second shake flask pre-culture containing 100 ml of fresh medium (B-CDM). The second preculture was incubated at 35 ° C. (+/- 1 ° C.) and 150 rpm for 24 h (+/- 1 h) and used to inoculate two flasks under agitation each containing 1 L of fresh medium (B). -CMD supplemented with 1.28 mg / L (7.5 μl) of CUCI2.2H2O). After growth at 35 ° C. (+/- 1 ° C.) and 150 rpm for 40 h (+/- 4 h), the two shake flasks of the third preculture were pooled. The pooled preculture was used to inoculate a fermenter as soon as the third preculture was stopped. A 20L fermentor (Biolafitte) was used. 10L of medium (B-CMD supplemented with 1.28mg / L (7.5mL) of CUCI2.2H2O) was transferred under aseptic conditions into the fermenter. The following conditions were used to establish the 100% dissolved oxygen (DO) level: temperature (35 ° C) and head pressure (0.4 bar). Inoculation was performed by adding 1.5L of the pooled preculture. During the fermentation, the temperature (35 ° C), the head pressure (0.4 bar), and the air flow (20 L min-1) were kept constant. The foaming was controlled by automatically adding a polydimethylsiloxane emulsion via a foam controller. The dissolved oxygen level was set at 25% and regulated by acceleration of agitation when the OD dropped below 25%. The minimum stirring speed was set at 50 rpm and the maximum stirring speed at 1000 rpm. The pH was controlled at 7.2 by addition of 50% (w / v) acetic acid.
During fermentation, growth was monitored via optical density at 650 nm (OD650nm) · At the end of fermentation (defined as the time when oxygen consumption decreases - following glutamate depletion , resulting in a reduction of the stirring rate), the production of pertussis toxoid (PT) in the culture supernatant was determined by Elisa. Table 7 compares the biomass yield, the PT yield, and the average generation time of fermentation COQ348 (Cu-complemented B-CDM) and COQ365 fermentation (Cu-complement-free B-CDM, see Example 1). The addition of CuCl2 to the chemically defined medium resulted in a significant increase in biomass yield. Growth rate and PT yield were also positively affected.
* Initial calculated biomass concentration based on the measured pre-culture DOesonm, ie 1.5 * pre-cooked / 11.5. ** The yield was calculated as the difference between DC> 650nm at the end of the fermentation and DOesonm at the beginning of the fermentation. *** The total fermentation time is defined as the time when oxygen consumption decreases (following glutamate depletion), resulting in a reduction of the average generation TertlpS stirring rate calculated as follows . First, the number of generations is calculated as a ratio between DOesonm at the end of the fermentation and DOssonm at the beginning of the fermentation, converted into a logo. The average generation time is then calculated by dividing the total fermentation time by the number of generations.
Example 6 - 20L Fermentation of Bordetella. pertussis in an improved chemically defined medium
An improved formulation of basic CDM (B-CDM) was evaluated in COQ426 fermentation.
A first shake flask pre-culture containing 7.5 ml of fresh medium (B-CDM, see composition in Table 1) was inoculated with 109 CFU of B. pertussis and incubated at 35 ° C (+ / -1). ° C) and 150 rpm for 24h (+/- lh). The first preculture was used to inoculate a second shake flask pre-culture containing 100 ml of fresh medium (B-CDM). The second preculture was incubated at 35 ° C. (+/- 1 ° C.) and 150 rpm for 24 h (+/- 1 h) and used to inoculate two flasks under agitation each containing 1 L of fresh medium (CMD). improved, see composition in Table 8). After growth at 35 ° C. (+/- 1 ° C.) and 150 rpm for 40 h (+/- 4 h), the two shake flasks of the third preculture were pooled. The pooled preculture was used to inoculate a fermenter as soon as the third preculture was stopped. A 20L fermentor (Biolafitte) was used. 10L of medium was transferred under aseptic conditions into the fermenter. The following conditions were used to establish the 100% dissolved oxygen (DO) level: temperature (35 ° C) and head pressure (0.4 bar). Inoculation was performed by adding 1.5L of pooled preculture. During the fermentation, the temperature (35 ° C), the head pressure (0.4 bar), and the air flow (20 L min-1) were kept constant. The foaming was controlled by automatically adding a polydimethylsiloxane emulsion via a foam controller. The dissolved oxygen level was set at 25% and regulated by acceleration of agitation when the OD dropped below 25%. The minimum stirring speed was set at 50 rpm and the maximum stirring speed at 1000 rpm. The pH was regulated at 7.2 by addition of 50% (w / v) phosphoric acid.
During fermentation, growth was monitored via optical density at 650 nm (OD 650 nm). At the end of the fermentation (defined by the time when the oxygen consumption decreases - following the depletion of glutamate, resulting in a reduction of the stirring speed), the production of pertussis toxoid (PT) in the supernatant of culture was determined by Elisa.
Table 9 compares the biomass yield, the PT yield, and the average generation time of COQ426 fermentation (enhanced CDM) and COQ365 fermentation (B-CDM, see Example 1).
Improved CDM conditions resulted in a slightly lower growth yield compared to basic CDM. The growth rate was also slightly lower. However, PT production was significantly increased (+170%).
* Initial calculated biomass concentration based on the measured pre-culture DOesonm, i.e. 1.5 * pre-cooked / H, 5.
** The yield was calculated as the difference between DOesonm at the end of the fermentation and DOesonm at the beginning of the fermentation. *** The total fermentation time is defined as the time when the oxygen consumption decreases (following glutamate depletion), resulting in a reduction of the stirring speed **** Average generation time calculated as follows . First, the number of generations is calculated as a ratio between DOsonm at the end of the fermentation and DOesonm at the beginning of fermentation, converted to log2. The average generation time is then calculated by dividing the total fermentation time by the number of generations.
Example 7 - 20L Scale Fermentation of Bordetella pertussis in an Improved Chemically Defined Medium Containing Thiosulfate as a Source of Sulfur
A modified improved CDM formulation (Example 6) was evaluated in fermentation COQ454. In this medium, cysteine was replaced by thiosulfate as a source of sulfur.
A first shake flask pre-culture containing 7.5 ml of fresh medium (B-CDM;
Table 1) was inoculated with 109 CFU of B. pertussis and incubated at 35 ° C (+/- 1 ° C) and 150 rpm for 24h (+/- 1 h). The first preculture was used to inoculate a second shake flask pre-culture containing 100 ml of fresh medium (B-CDM). The second preculture was incubated at 35 ° C. (+/- 1 ° C.) and 150 rpm for 24h (+/- 1 h) and used to inoculate two flasks under agitation each containing 1 L of fresh medium (CMD). improved with thiosulfate, see composition in Table 10). After growth at 35 ° C. (+/- 1 ° C.) and 150 rpm for 40 h (+/- 4 h), the two shake flasks of the third preculture were combined. The pooled preculture was used to inoculate a fermenter as soon as the third preculture was stopped. A 20L fermentor (Biolafitte) was used. 10L of medium was transferred under aseptic conditions into the fermenter. The following conditions were used to establish the 100% dissolved oxygen (DO) level: temperature (35 ° C) and head pressure (0.4 bar). Inoculation was performed by adding 1.5L of the pooled preculture.
During the fermentation, the temperature (35 ° C), the head pressure (0.4 bar), and the air flow (20 L min-1) were kept constant. The foaming was controlled by automatically adding a polydimethylsiloxane emulsion via a foam controller. The dissolved oxygen level was set at 25% and regulated by acceleration of agitation when the OD dropped below 25%. The minimum stirring speed was set at 50 rpm and the maximum stirring speed at 1000 rpm. The pH was regulated at 7.2 by addition of 50% (w / v) phosphoric acid.
During the fermentation, growth was monitored via the optical density at 650 nm (DOδsonm). At the end of the fermentation (defined by the time when the oxygen consumption decreases - following the depletion of glutamate, resulting in a reduction of the stirring speed), the production of pertussis toxoid (PT) in the supernatant of culture was determined by Elisa. Table 11 compares biomass yield, PT yield, and mean generation time of COQ454 fermentation (enhanced CDM with thiosulfate), COQ426 fermentation (enhanced CDM, see Example 6), and fermentation COQ365 (B-CDM). see Example 1).
The biomass yield in the "thiosulfate enhanced CDM" was slightly lower compared to the baseline CDM, but resulted in a higher growth rate and higher TP production (+310%). Compared to the "enhanced CDM", the "Thiosulfate Enhanced CDM" medium resulted in a similar biomass yield, higher growth rate, and higher PT production (+ 52%).
* Initial calculated biomass concentration based on measured pre-cultured DOgsonm, ie 1.5 * DOPre-bake / H # 5. ** Yield was calculated as the difference between DO650nm at the end of fermentation and DOgsonm at the beginning of the fermentation. *** The total fermentation time is defined as the time when the oxygen consumption decreases (following glutamate depletion), resulting in a reduction of the stirring speed **** Average generation time calculated as follows . First, the number of generations is calculated as a ratio between DOesonm at the end of fermentation and DOgsonm at the beginning of fermentation, converted to log2- The average generation time is then calculated by dividing the total fermentation time by the number of generations.
Example 8 - Growth of B. pertussis in a minimal medium containing a single amino acid
Assays were performed to determine whether growth of B. pertussis is possible in a minimal medium containing a single amino acid as the sole source of carbon and nitrogen. A shake flask containing 7.5 ml of fresh medium (B-CDM containing 0.604 g / L of niacin) was inoculated with 109 CFU of B. pertussis and incubated at 35 ° C (+/- 1 ° C) and 150 rpm for 24h (+/- lh). The cells were harvested by centrifugation, washed twice with 0.9% (w / v) NaCl, and resuspended in fresh medium (see composition in Table 12) at a theoretical OD 50 of from the DOôsonm of the crop before harvest. 1 ml of this cell suspension was used to inoculate shake flasks containing 30 ml of the medium described in Table 12, supplemented with a single amino acid (125 mM L-cysteine, 125 mM L-proline, 125 mM L-glutamate 125 mM L-glutamine, 30 mM L-aspartate, 125 mM L-asparagine, 125 mM L-serine, or 125 mM L-alanine) as a source of C and N, and 0.25 mM thiosulfate as a source of S (except for medium supplemented with L-Cys, where no thiosulfate was added). The same medium with ammonium chloride (25 mM) and thiosulfate (0.25 mM), but without amino acid, was used as a negative control. The shake flasks were then incubated for about 10 days at 35 ° C with constant shaking (150 rpm). Growth was monitored via DOssonm. The results of the growth assay are illustrated in Figure 1.
All the amino acids tested were able to support the growth of B. pertussis as the only source of C and N, provided that a source of S (thiosulfate) is present. When L-Cys was used as the amino acid, no additional source of sulfur was required.
Example 9 - Growth of B. pertussis in minimal medium containing no amino acid
Assays were performed to determine whether growth of B. pertussis is possible in a minimal medium in which nitrogen was provided only as inorganic ammonia, sulfur as thiosulfate, and carbon in the form of organic acid. A stirred flask containing 7.5 ml of fresh medium (B-CDM containing 0.604 g / L niacin) was inoculated with 109 CFU of B. pertussis and incubated at 35 ° C (+/- 1 ° C). and 150 rpm for 24h (+/- 5h). The cells were harvested by centrifugation, washed twice with 0.9% (w / v) NaCl, and resuspended in fresh medium (see composition in Table 13) at a theoretical OD 50 of from the DC> 650nm of the crop before harvest. 1 ml of this cell suspension was used to inoculate shake flasks containing 30 ml of the medium described in Table 13, supplemented with a single organic acid (100 mM citrate, 100 mM L-lactate, 100 mM acetate, 100 mM pyruvate 100 mM fumarate, or 100 mM succinate). The same medium without additional organic acid was used as a negative control. The shake flasks were then incubated for about 10 days at 35 ° C with constant shaking (150 rpm). Growth was monitored via the DO650nm. The results of the growth assay are illustrated in Figure 2.
All organic acids tested were able to support the growth of B. pertussis as the sole source of C.

权利要求:
Claims (18)
[1]
A chemically defined medium for culturing on an industrial scale a species of the genus Bordetella comprising: (i) an iron component selected from the group consisting of Fe (II) complexed with an organic compound and Fe (III) ) complexed with an organic compound, wherein the organic compound is selected from heme, hemoglobin, myoglobin, transferrin, ferritin, lactoferrin, enterobactin, aerobactin, alkaligine, a coprogenic substance, ferrichrome, desferrioxamine, ferroxamine, hydroxamate, citrate and dihydroxybenzoylserine; (ii) 3- (N-morpholino) propanesulfonic acid (MOPS); (iii) dimethyl-β-cyclodextrin; and (ii) an amino acid selected from the group consisting of aspartate at a concentration of 1000 μΜ or greater, glycine at a concentration of 1000 μΜ or greater, methionine at a concentration of 500 μΜ or greater, and leucine at a concentration of 1500 μΜ or more, wherein said chemically defined medium does not include FeSO4 or tris (hydroxymethyl) aminomethane.
[2]
2. Chemically defined medium according to claim 1, further comprising (vi) 2 μΜ or more, 3 μΜ or more, 4 μΜ or more, 5 μΜ or more, or 6 μΜ or more of copper; (vii) 2 μΜ or more, 5 μΜ or more, 10 μΜ or more, 50 μΜ or more, 100 μΜ or more, or 400 μΜ or more of magnesium; (x) an additive selected from the group consisting of zinc, cobalt, thiamine, riboflavin and pantothenate; (xi) an additive selected from the group consisting of 0.4 μΜ or more of biotin, 50 μl or more of calcium, 15 μl or more of niacin, and 25 μl or more of ascorbic acid.
[3]
A chemically defined medium according to claim 1 or 2, comprising an inorganic source of sulfur selected from the group consisting of thiosulfate, trithionate, tetrathionate, peroxodisulfate, sulfide and sulfite, and wherein said medium does not comprise organic source of sulfur.
[4]
4. A chemically defined medium according to any one of the preceding claims comprising more than 0.005 mM, more than 0.006 mM, more than 0.007 mM, more than 0.008 mM, more than 0.010 mM, more than 0.050 mM, more than 0.100 mM, approximately 0.120 mM or about 0.011 mM thiosulfate.
[5]
A chemically defined medium according to any one of the preceding claims comprising more than 0.003 mM, more than 0.004 mM, more than 0.005 mM, more than 0.008 mM, more than 0.010 mM, more than 0.020 mM, more than 0.050 mM, approximately 0.007 mM or about 0.080 mM trithionate.
[6]
The chemically defined medium of any preceding claim comprising greater than 0.002 mM, greater than 0.003 mM, greater than 0.004 mM, greater than 0.005 mM, greater than 0.025 mM, greater than 0.050 mM, approximately 0.060 mM or approximately 0 , 0006 mM tetrathionate.
[7]
The chemically defined medium of any preceding claim comprising greater than 0.005 mM, greater than 0.006 mM, greater than 0.007 mM, greater than 0.008 mM, greater than 0.010 mM, greater than 0.050 mM, greater than 0.100 mM, about 0.120 mM or about 0.011 mM peroxodisulfate.
[8]
8. A chemically defined medium according to any one of the preceding claims comprising more than 0.010 mM, more than 0.012 mM, more than 0.014 mM, more than 0.016 mM, more than 0.020 mM, more than 0.100 mM, more than 0.200 mM, approximately 0.240 mM or about 0.022 mM sulfide.
[9]
9. A chemically defined medium according to any one of the preceding claims comprising more than 0.010 mM, more than 0.012 mM, more than 0.014 mM, more than 0.016 mM, more than 0.020 mM, more than 0.100 mM, more than 0.200 mM, approximately 0.240 mM or about 0.022 mM sulfite.
[10]
The chemically defined medium of claim 1 comprising an MOPS buffer. at a concentration greater than 2 mM, greater than 5 mM, greater than 7 mM, greater than 9 mM, greater than 10 mM or greater than 11 mM.
[11]
11. The chemically defined medium of claim 2 comprising copper in the form of copper chloride.
[12]
12. Chemically defined medium according to any one of the preceding claims comprising an inorganic nitrogen source selected from an ammonium salt and an ammonium chloride.
[13]
13. A chemically defined medium according to any one of the preceding claims comprising a carbon source selected from the group consisting of glutamate, proline, citrate, lactate, acetate, pyruvate, fumarate and succinate.
[14]
14. Chemically defined medium according to any preceding claim further comprising a component selected from the group consisting of: (i) more than 0.1 μΜ, more than 1 μΜ, more than 50 μΜ, more than 100 μΜ, more than 200 μΜ, more than 300 μΜ, more than 400 μΜ, more than 500 μΜ, more than 600 μΜ or more than 700 μΜ of zinc; (ii) more than 0.05 μΜ, more than 0.10 μΜ, or more than 0.15 μΜ of cobalt; (iii) more than 100 μΜ, more than 120 μΜ or more than 140 μΜ calcium; (iv) more than 20 μΜ, more than 30 μΜ or more than 35 μΜ niacin; (v) more than 50 μΜ, more than 75 μΜ, more than 100 μΜ, more than 1,000 μΜ, more than 2,000 μΜ or more than 3,000 μΜ of ascorbic acid; (vi) more than 0.1 μΜ, more than 1 μΜ, more than 5 μΜ, more than 10 μΜ or more than 25 μΜ of thiamine; (vii) more than 0.4 μΜ, more than 0.5 μΜ, more than 0.6 μΜ or more than 0.8 μΜ of biotin; (viii) more than 0.1 μΜ, more than 0.2 μΜ, more than 0.3 μΜ, more than 0.4 μΜ, more than 0.5 μΜ, more than 0.6 μΜ or more than 0.8 μΜ of riboflavin; and (ix) more than 0.1 μΜ, more than 0.5 μΜ, more than 1.0 μΜ, more than 2.0 μΜ, more than 5.0 μΜ, or more than 7.0 μΜ of pantothenate.
[15]
15. A chemically defined medium according to any one of the preceding claims comprising an amino acid selected from the group consisting of: (i) glutamate at a concentration greater than 50 mM, greater than 75 mM, greater than 90 mM, greater than 100 mM or greater than 110 mM; (ii) alanine at a concentration greater than 1,000 μΜ, greater than 1,500 μΜ, greater than 2,000 μΜ, greater than 2,500 μΜ or greater than 3,000 μΜ; (iii) phenylalanine at a concentration greater than 500 μΜ, greater than 750 μΜ, greater than 1,000 μΜ, greater than 1,250 μΜ or greater than 1,400 μΜ; (iv) histidine at a concentration greater than 50 μΜ, greater than 100 μΜ, greater than 150 μΜ or greater than 200 μΜ; (v) isoleucine at a concentration greater than 500 μΜ, greater than 1,000 μΜ, greater than 1,500 μΜ or greater than 1,750 μΜ; (vi) lysine at a concentration greater than 500 μΜ, greater than 1,000 μΜ, greater than 1,500 μΜ or greater than 2,000 μΜ; (vii) proline at a concentration greater than 1,000 μΜ, greater than 3,000 μΜ, greater than 4,000 μΜ, greater than 5,000 μΜ, greater than 6,000 μΜ or greater than 7,000 μΜ; (viii) serine at a concentration greater than 500 μπιΜ, greater than 1,000 μΜ, greater than 1,500 μΜ or greater than 1,700 μΜ; (ix) valine at a concentration greater than 1,000 μΜ, greater than 2,000 μΜ, greater than 2,500 μΜ or greater than 3,000 μΜ; and (x) tyrosine at a concentration greater than 25 μΜ, greater than 50 μΜ, greater than 75 μΜ, greater than 100 μΜ, greater than 150 μΜ or greater than 175 μΜ.
[16]
16. Chemically defined medium according to any one of the preceding claims further comprising a glutathione at a concentration greater than 100 μΜ, greater than 200 μΜ, greater than 400 μΜ, greater than 500 μΜ, greater than 600 μΜ or greater than 700 pm .
[17]
17. A chemically defined medium according to any one of the preceding claims further comprising a component selected from the group consisting of: (i) chloride at a concentration of less than 45 mM, less than 40 mM, less than 35 mM, less than 30 mM, less than 25 mM, less than 20 mM, between 10 and 20 mM or about 16 mM; (ii) acetate at a concentration greater than 1 mM, greater than 2 mM, greater than 3 mM, greater than 4 mM, between 4 and 6 mM or approximately 5 mM; and (iii) potassium at a concentration greater than 1 mM, greater than 2 mM, greater than 3 mM, greater than 4 mM, greater than 5 mM, greater than 6 mM, between 5.5 and 7 mM or greater 6.5 mM.
[18]
18. A fermentation method for growing a Bordetella species in a chemically defined medium (CDM) comprising (a) inoculating a chemically defined medium according to any one of the preceding claims with a species of the genus Bordetella; and (b) maintaining the Bordetella species in the chemically defined medium for a period of time sufficient to allow accumulation of biomass.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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GBGB1316351.4A|GB201316351D0|2013-09-13|2013-09-13|

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GB13163514|2013-09-13|

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