Patent Application
20110256606
Clostridium difficile (C. difficile) Toxins A and B are responsible for C. difficile-associated disease (CDAD), which manifests itself as nosocomial diarrhea and pseudomembranous colitis (Kuijper et al., Clinical Microbiology and Infection 12(Suppl. 6):2-18, 2006; Drudy et al., International Journal of Infectious Diseases 11(1):5-10, 2007; Warny et al., Lancet 366(9491):1079-1084, 2005; Dove et al., Infection and Immunity 58(2):480-488, 1990; Barroso et al., Nucleic Acids Research 18(13):4004, 1990).
Toxins A and B are encoded by two separate but closely linked (and highly homologous) genes. Toxins A and B are produced simultaneously in C. difficile strain VPI 10463 (ATCC 43255), and the ratio of the produced toxins is usually 3:1, respectively (Karlsson et al., Microbiology 145:1683-1693, 1999). The toxins begin to be formed during the exponential growth phase, and are usually released from the bacteria between 36 and 72 hours of culture. Toxins present within the bacteria can be released earlier by sonication or by use of a French pressure cell.
Treatment of the toxins with formaldehyde results in the corresponding Toxoids A and B, which are completely inactivated and retain at least partial immunogenicity (Torres et al., Infection and Immunity 63(12):4619-4627, 1995). It has been shown that vaccination employing both toxoids is effective in hamsters, healthy adults, and patients with recurrent CDAD (Torres et al., Infection and Immunity 63(12):4619-4627, 1995; Kotloff et al., Infection and Immunity 69(2):988-995, 2001; Sougioultzis et al., Gastroenterology 128(3):764-770, 2005; Tones et al., Vaccine Research 5(3):149-162, 1996). Additionally, the administration of both free and aluminum salt (adjuvant) bound toxoids leads to appropriate immune responses (Torres et al., Vaccine Research 5(3):149-162, 1996; Giannasca et al., Infection and Immunity 67(2):527-538, 1999).
The administration of both toxoids simultaneously is more effective than administration of the individual proteins alone (Kim et al., Infection and Immunity 55(12):2984-2992, 1987). A toxoid composition found effective in inducing protective immune responses against toxin A and toxin B in patients with recurrent CDAD included both toxoids, at a ratio of 1.5:1, A:B (Sougioultzis et al., Gastroenterology 128(3):764-770, 2005).
Both the A and B toxoids are thus candidates for vaccine development. Greater production efficiency of Toxins A and B is desired to facilitate vaccine production.
In one aspect, the invention features a culture medium (e.g., for culturing a Clostridium difficile bacterium) at a pH of between 6.35 and 7.45 (e.g., 6.5, 7.28, or between 6.35 and 6.65) including peptone (e.g., soy peptone), a yeast extract (e.g., Difco Bacto Yeast extract), a buffering agent (e.g., NaHCO3), and a phosphate buffer (e.g., sodium phosphate, dibasic and potassium phosphate, monobasic). This culture medium can also include at least one additive (e.g., 2, 3, or more additives) selected from the group consisting of chromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose, D-sorbitol, sucrose, and a combination of azaserine, adenosine, and biotin.
In another aspect, the invention features a bacterial culture including Clostridium difficile and culture medium including at least one additive (e.g., 2, 3, or more additives) selected from the group consisting of chromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose, D-sorbitol, sucrose, and a combination of azaserine, adenosine, and biotin. This medium can also include peptone (e.g., soy peptone), yeast extract (e.g., Difco Bacto Yeast extract), sodium phosphate, dibasic, potassium phosphate, monobasic, and NaHCO3, and the culture medium can be at a pH of between 6.35 and 7.45 (e.g., 6.5, 7.28, or between 6.35 and 6.65).
In another aspect, the invention features a method of culturing Clostridium difficile including inoculating culture medium with Clostridium difficile, with the medium including at least one additive (e.g., 2, 3, or more additives) selected from the group consisting of chromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose, D-sorbitol, sucrose, and a combination of azaserine, adenosine, and biotin. This medium can also include peptone (e.g., soy peptone), yeast extract (e.g., Difco Bacto Yeast extract), sodium phosphate, dibasic, potassium phosphate, monobasic, and NaHCO3, and the culture medium can be at a pH of between 6.35 and 7.45 (7.28 or between 6.35 and 6.65). Preferably the culture medium is at a pH of 6.5.
In another aspect, the invention features a method for obtaining or preparing one or more C. difficile toxins including by preparing an aqueous growth medium including soy peptone, inoculating the medium with a C. difficile bacterium (e.g., using an aqueous C. difficile culture), culturing the inoculated medium (e.g., at a pH of 6.5, 7.28, between 6.35 and 7.45, or between 6.35 and 6.65)) under conditions which facilitate growth of bacterium and toxin production (e.g., at a temperature between 37° C. to 41° C.), and isolating the one or more C. difficile toxins from growth medium (e.g., by removing from the growth medium viable C. difficile organisms and spores, separating the one or more toxins from the growth media, and purifying the one or more toxins). This culture medium can also include yeast extract, NaHCO3, sodium phosphate, dibasic, potassium phosphate, monobasic, and D-sorbitol. This culturing can be carried out, e.g., under anaerobic conditions. The steps of inoculating the medium with a C. difficile bacterium (e.g., using an aqueous C. difficile culture) and culturing the inoculated medium can be repeated more than once, with inoculation into fresh growth medium with each repeat. This method can also include detoxifying the isolated one or more C. difficile toxins to prepare one or more toxoids (e.g., by reacting the one or more toxins by the addition of formaldehyde).
In another aspect, the invention features a method of enhancing the production of Toxin B from a C. difficile culture by preparing an aqueous growth medium including soy peptone, inoculating the medium with a C. difficile bacterium, culturing the inoculated medium at 37° C. to 41° C. and at a pH between pH 6.35 and pH 6.65 (e.g., at 37° C. and at a pH of 6.5). The pH and/or temperature can be held constant or vary during the culturing. The growth media can further include yeast extract, NaHCO3; sodium phosphate, dibasic, potassium phosphate, monobasic, and D-sorbitol. Toxin B production can be enhanced relative to Toxin A production, producing, e.g., ratios of Toxin A relative to Toxin B of less than 3:1, 2:1, 1.5:1, or less.
In any of the foregoing aspects, yeast extract can be between 10 and 30 g/L, the NaHCO3 can be between 2 and 5 g/L; the sodium phosphate, dibasic can be between 1 and 10 g/L, and the potassium phosphate, monobasic can be between 1 and 10 g/L. The adenosine can be present at a concentration of between 0.8 and 1.2 mM (e.g., 1 mM), the biotin at a concentration of between 40 and 60 nM (e.g., 50 nM), and the azaserine at a concentration between 15 and 50 μM (e.g., 50 μM). The concentration of D-sorbitol can be between 6 g/L and 20 g/L or between 8 g/L and 18 g/L (e.g., 12 g/L). The chromium trioxide can be present at a concentration of between 40 and 60 mg/L (e.g., 50 mg/L). The clindamycin can be present at a concentration between 0.4 and 0.6 mg/L (e.g., 0.5 mg/L). The ascorbic acid can be present at a concentration between 2.5 g/L and 10 g/L (e.g., 2.5 g/L and 10 g/L). The butyric acid can be present at a concentration between 30 mM and 60 mM (e.g., 30 mM and 60 mM). The D(+)xylose can be at a concentration between 6 g/L and 10 g/L (e.g., 6 g/L).
The invention provides several advantages. For example, the media and the methods of the invention allow increased production of Clostridium difficile toxins, which leads to increased efficiency and decreased costs in the production of toxin-based products such as vaccines. Other features and advantages of the invention will be apparent from the following Detailed Description, the Drawings, and the Claims.
In general, the invention features methods and compositions (such as for example, culture media) for culturing C. difficile and producing the C. difficile Toxins A and B. These two toxins can be used individually or in combination, in the preparation of toxoids.
As discussed further below, the culturing of C. difficile in the media of the invention leads to enhanced Toxin A and Toxin B production. Similarly, as discussed further below, enhanced toxin production is seen by culturing C. difficile in accordance to the methods of the invention.
The compositions and methods of the invention feature the use of a basal medium in conjunction with certain medium additives. In one example, the basal medium is comprised of peptone (e.g., 20-40 g/L), yeast extract (e.g., 10-30 g/L), a phosphate buffer (such as for example, potassium phosphate monobasic (e.g., 0.5-1.5 g/L) and sodium phosphate dibasic (e.g., 1-10 g/L)) and a buffering agent (such as for example, sodium bicarbonate (e.g., 1-10 g/L)). The peptone used may be soy-based or animal-derived (such as for example, tryptone).
In one example, the basal medium is SYS media. SYS medium contains the ingredients listed in Table 1A at the indicated concentrations. The basal medium may be titrated to a pH of between 6.35 and 7.45 (for example, 6.5, 7.28, or between 6.35 and 6.65). Exemplary ranges of concentrations for each of the indicated ingredients are also indicated.
Table 1B sets forth an alternative basal media useful in the compositions and methods of the invention, named TYS.
In substitution of the Soy peptone A3 and the Difco Bacto Tryptone any peptone (e.g., any soy peptone) can be utilized. Examples of soy peptones that can be used in the basal media (and their sources) include the following:
Kerry Biosciences: HyPer 1510,
IPS: Hy-Soy Kosher, and
Becton Dickinson: BD Select Phytone UF
In substitution of the Difco Bacto Yeast Extract, any yeast extract can also be used in the basal media. Examples of suitable yeast extracts (and their sources) are readily known to those skilled the art.
The suitability of a particular peptone or yeast extract for use in the invention can be determined using the experimental methods described herein. The invention also includes use of other bacterial growth media, in combination with the additives described below.
The invention also features the use of certain additives with a basal media (e.g., SYS media). Exemplary additives of the invention are set forth in Table 2, which also includes the exemplary concentration ranges for the indicated additives, as well as a single exemplary concentration. Additives include:
Chromium trioxide (Chromium(VI) oxide CrO3). Chromium trioxide is the acid anhydride of chromic acid. Chromium trioxide is a strong oxidant, highly toxic, corrosive, and carcinogenic compound.
Clindamycin (C18H33ClN2O5S). Clindamycin is a lincosamide antibiotic and is indicated for Clostridium difficile-associated diarrhea (the most frequent cause of pseudomembranous colitis). Clindamycin has a bacteriostatic effect. It interferes with bacterial protein synthesis by binding preferentially to the 50S subunit of the bacterial ribosome.
Azaserine (C5H7N3O4). Azaserine is a naturally occurring serine derivative diazo compound and is a known carcinogen. Azaserine is a glutamine analogue that irreversibly inhibits glutamine phosphoribosyl amidotransferase, which is involved in the biosynthesis of inosine monophosphate (IMP). IMP is an important precursor to the purine nucleotides which include adenosine monophosphate (AMP) and guanosine monophosphate (GMP).
Ascorbic acid (C6H8O6). Ascorbic acid is a sugar acid with antioxidant properties. L-Ascorbic acid is also known as vitamin C.
Butyric acid (C4H8O2). Butyric acid is a carboxylic acid and a short chain fatty acid. Butyric acid has been associated with the ability to inhibit the function of histone deacetylase enzymes, thereby favoring an acetylated state of histones in the cell.
Xylose (C5H10O5). Xylose (wood sugar) is a five-carbon monosaccharide. Xylose can be metabolized into useful products by a variety of organisms, e.g., Clostridium difficile.
Sorbitol (C6H14O6). Sorbitol, also known as glucitol, is a sugar alcohol. Sorbitol also is an osmotic stress agent (osmotic shock is induced by 0.5 M sorbitol).
Growth of C. difficile according to the methods of the invention proceeds in at least two phases: seed growth and fermentation. The seed growth phase, as described further below, may proceed in one or more seed culture stages (e.g, two stages or three stages).
A seed culture is first grown by inoculating seed medium with a sample from a stock culture (e.g., a working cell bank (WCB)). A sample of this seed culture is used either to inoculate a second seed culture or to inoculate a relatively large fermentation culture. Such seed cultures are typically carried out to allow the quantity of the microorganism from a stored culture (e.g., WCB) to be exponentially increased (scaled-up). Seed cultures can also be used to rejuvenate relatively dormant microbes in stored cultures. As is well understood in the art, more than one seed culture (e.g., two or three cultures or stages) can be used to scale-up the quantity of C. difficile for inoculation into the fermentation medium.
The number of seed cultures (or stages) used depends on, for example, the size and volume of the fermentation step. For example, the culture process may involve two seed cultures: a first seed culture is grown from an inoculation of a WCB (stage one seed culture), a sample of this seed culture is used to inoculate a second seed culture (stage two seed culture), and a sample from this second culture is used to inoculate a fermentation culture (fermentation stage). In a preferred embodiment of the present invention, the first and second seed cultures are grown in SYS media.
In stage one, a culture of C. difficile is suspended in seed medium and is incubated at a temperature between 30-40° C., preferably at 37±1° C., for 18 hours in an anaerobic environment. In stage two, a sample of the stage one seed medium is used to inoculate a stage two seed medium for further growth. After inoculation, the stage two medium is incubated at a temperature between 30-40° C., preferably at 37±1° C., for approximately 10 hours, also in an anaerobic environment. Preferably, growth in seed media at any stage does not result in cell lysis before inoculation of fermentation media. Additional growth in a third (fourth, etc.) stage seed culture can also be carried out.
In the fermentation stage, an appropriate concentration of seed culture, which can range from, e.g., 0.1-10%, is used to inoculate the fermentation media. Preferably, concentrations of 1.0% or 5.0% can be used. Most preferably, concentrations of 10% are used.
Fermentation is preferably carried out in an anaerobic chamber at approximately 35° C. to 45° C. and preferably at a temperature between 37° C. to 41° C. (e.g., 37° C.). The pH of the fermentation may be controlled at a pH between pH 6.35 to 7.45 (e.g., between 6.35 to 6.65, and preferably, at pH 6.5). Alternatively, the pH of the culture media is uncontrolled and is allowed to decrease naturally during the fermentation process.
C. difficile can be cultivated by fermentation with continuous exposure to a suitable gas or gas mixture (such as, for example, 80% nitrogen/10% CO2/10% hydrogen, 100% CO2, or 100% nitrogen). Such gases or gas mixtures may also be sparged (i.e., bubbled) through the medium during fermentation. As an alternative to sparging (or in addition to it), a gas mixture (e.g., 80% nitrogen/10% CO2/10% hydrogen) or a gas (e.g., CO2 or nitrogen) may be applied to the culture media as an overlay to degas the media throughout the fermentation process. The fermentation culture is preferably sparged prior to inoculation with either a mixture of 80% nitrogen/10% CO2/10% hydrogen, 100% CO2 or 100% nitrogen to remove any residual oxygen in the medium. During the fermentation process the culture may be sparged periodically. Alternatively, an overlay of a gas mixture or a gas (e.g., 100% nitrogen) may be applied to the culture.
Fermentation proceeds for approximately 16 to 24 hours (e.g., 18 to 21 hours). Preferably, agitation (e.g., 100 rpm) is applied to the culture medium during the fermentation process (and/or during stages one and two of seed cultures). Growth can be monitored by measuring the optical density (O.D.) of the medium.
C. difficile toxins can be isolated and purified from fermentation cultures using purification methods well known in the art such as for example, Kotloff et al., Infect. Immun 2001; 69:988-995, Coligan et al., “Current Protocols in Protein Science,” Wiley & Sons; Ozutsumi et al., Appl. Environ. Microbiol. 49:939-943, 1985; and Kim et al., Infection and Immunity 55:2984-2992, 1987; which are incorporated herein by reference. The purified toxins can then, for example, be inactivated by chemical treatments known in the art (e.g., formaldehyde treatment).
All references cited within this disclosure are hereby incorporated by reference in their entirety. Certain embodiments are further described in the following examples. These embodiments are provided as examples only and are not intended to limit the scope of the claims in any way.
The basal media and additives of the invention were used to culture Clostridium difficile and produce Clostridium difficile Toxins A and B. Tables 3 and 4 (and
This Example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media in the absence and presence of various metallic ions.
The following are the Example 1 test compounds, along with the compound formula and source.
AFC-Ammonium ferric citrate (C6H8O7.nFe.nH3N), USB Cat. 15751 Lot. 121753
FC-Ferric citrate (C6H5FeO7), FW 244.95, MB BIomedicals LLC, Cat. 195181, Lot R23927
FG-Ferrous gluconate Hydrade (C12 hrs.22FeO14)
FS-Ferric sulfate (FeSO4.7H2O), FW 278.02 CA-Calcium chloride Anhydrous (CaCl2), FW 110.98, J. T. Baker Cat. 1311-01, Lot. A13602
CC-Cobalt chloride 6 Hydrate Crystal (CoCl2.6H2O), FW 237.93, Mallinckrodt Chemicals Cat. 4535-02
CT-Chromium trioxide Crystal (CrO3), FW 99.99, J. T. Baker Cat. 1638-04, Lot.
MS-Magnesium sulfate (MgSO4.7H2O), FW 246.50
MC-Manganese chloride (MnCl24H2O), FW 197.90, J. T. Baker Cat. 2540-04, Lot E37335
The following table indicates the natural pH of the indicated compound in solution at the indicated concentration.
The following methods were used to test the production of Toxin A and B by Clostridium difficile when cultured in the presence of the above-listed additives.
1. The following table shows the amount of seed growth (OD600 nm) as measured by DU700.
2. The following table shows the amount of cell growth (OD600 nm) in cultures with the indicated compound.
3. The following table shows the amount of Toxin A produced (ng/mL) in cultures with the indicated compound (
4. The following table shows the amount of Toxin B produced (ng/mL) in cultures with the indicated compound.
5. The following table shows the amount of spore formation in 24 hour fermentation in cultures with the indicated compound. Broth was examined by microscope.
Chromium trioxide, when added to the SYS medium at 50 mg/L, caused increases in production of Toxin A (20%) and Toxin B (80%) after 24 hours in fermentation broth, but not after 12 hours in fermentation broth.
This example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media in the absence and presence of various antibiotics.
The following are antibiotics, along with the compound formula and source, which were tested in this example.
Cip-Ciprofloxacin (C17H18FN3O3) FW 331.35, BioChemika, Lot#WA19781, soluble with 0.2 mL of 5 N HCl.
Cli-Clindamycin hydrochloride (C18H33ClN3O5S.HCl) FW 461.44, Sigma C5269, Lot#37k1535, soluble in water.
Van-Vancomycin hydrochloride (C66H75Cl2N9O24.HCl) FW 1485, Sigma V20029, Lot#037K0686 soluble in water.
Pen G-Penicillin G Sodium salt (C16H17N2NaO4S) FW 356.4, Sigma P3032, Lot#057K04931, soluble in water.
Fe-EDTA was also tested (Ethylenediaminetetraacetic acid, Ferric Sodium Salt, (C10H12FeN2NaO8) FW 421.10, Acros Organics 304680050, Lot#A0245953).
Antibiotics were tested at the following concentrations:
Ciprofloxacin (2 mg/L and 10 mg/L), Clindamycin (0.5 mg/L and 2.5 mg/L), Vancomycin (0.1 mg/L and 0.5 mg/L), and Penicillin G (0.1 mg/L and 0.5 mg/L).
Ethylenediaminetetraacetic acid Ferric Sodium Salt was tested at a concentration of 100 mg/L.
The following materials were used to test the production of Toxin A and B by Clostridium difficile when cultured in the presence of the above antibiotics and compounds.
1. Make 100× concentration antibiotic solutions/10× Fe-EDTA solutions
2. Make 10× concentration of antibiotic solution:
Take 4 mL of 100× concentration solution+36 mL di water.
1. The following table shows the amount of seed growth (OD600 nm) as measured by DU700.
2. The following table shows the amount of cell growth (OD600 nm) in cultures with the indicated compound.
3. The following table shows the amount of Toxin A produced (ng/mL) in cultures with the indicated compound (
4. The following table shows the amount of Toxin B produced (ng/mL) in cultures with the indicated compound.
5. The following table indicates cell morphological characteristics in cultures with the indicated compound.
6. The following table shows the amount of spore formation in 24 h fermentation in cultures with the indicated compound. Broth was examined by microscope.
Clindamycin, when added to SYS medium at 0.5 mg/L, caused increases in Toxin A (28%) and Toxin B (94%) after 24 hours in fermentation broth, but not after 12 hours in fermentation broth.
This Example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media in the absence and presence of various vitamins and antibiotics
The following are the Example 3 test compounds, along with the compound formula and source.
Aza-Azaserine (C5H7N3O4) FW 173.10, (O-diaxoacetyl-L-serine) Fluka BioChemika, 11430 Lot#1301321, soluble in water.
Ade-Adenosine (C10H13N5O4) FW 267.25, Sigma A4036, Lot#046K06612, soluble with 5N HCl.
B12-Vitamin B12 (C63H88CoN14O14P) FW 1,355.37, Sigma, V6629Lot#124K17072, soluble in water.
Bio-d-Biotin (C10H16N2O3S) FW 244, Supelco 4-7868, Lot#LB5668 soluble with 5N HCl.
The following combinations of compounds were also tested at the indicated concentrations.
#1-50 μM Azaserine (8650 μg/L)+1 mM Adenosine (267 mg/L)+50 nM Biotin (12.2 μg/L)
50 μM Azaserine (432.5 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 nM Biotin (610 ng/50 mL)
#2-15 μM Azaserine (2595 μg/L)+1 mM Adenosine (267 mg/L)+50 nM Biotin (12.2 μg/L)
15 μM Azaserine (129.75 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 nM Biotin (610 ng/50 mL)
#3-15 μM Azaserine (2595 μg/L), (129.75 μg/50 m/l)
#4-5 μM Azaserine (865 μg/L)+1 mM Adenosine (267 mg/L)+50 nM Biotin (12.2 μg/L)
5 μM Azaserine (43.25 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 nM Biotin (610 ng/50 mL)
#5-5 μM Azaserine (865 μg/L)+1 mM Adenosine (267 mg/L)+50 μM Biotin (12.2 ng/L)
5 μM Azaserine (43.25 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 μM Biotin (0.61 ng/50 mL)
#6-0.05 nM d-Biotin (12.2 ng/L), (0.61 ng/50 mL)
#7-0.5 nM d-Biotin (122 ng/L), (6.1 ng/50 mL)
#8-5 nM d-Biotin (1.22 μg/L), (61 ng/50 mL)
#9-50 nM Vitamin B12 (67.77 μg/L), (3.39 μg/50 mL)
1. Make 50× concentration solutions.
2. Make d-Biotin (500 μM) solutions, then dilute to 2.5 μM, 50 nM, 5 nM, and 0.5 nM.
3. Make 50× concentration solutions then dilute to 10×.
1. The following table shows the amount of seed growth (OD600 nm) as measured by DU700.
2. The following table shows the amount of cell growth (OD600 nm) in cultures with the indicated compound.
3. The following table shows the amount of Toxin A produced (ng/mL) in cultures with the indicated compound.
4. The following table shows the amount of Toxin B produced (ng/mL) in cultures with the indicated compound.
50 μM Azaserine, 1 mM Adenosine, and 50 nM Biotin together, when added to the SYS medium, caused increases in production of Toxin A (80%) and Toxin B (238%) after 24 hours in fermentation broth.
This Example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media in the absence and presence of various amino acids and organic compounds.
The following are the Example 4 test compounds, along with the compound formula and source.
These compounds were tested using the following concentrations (10×):
L-Arginine Monohydrochloride (50 mM).
L-Tyrosine (50 mg/L).
L-Cysteine (0.33 mM, 10 mM, and 33 mM).
Ascorbic acid (2.5 g/L and 10 g/L).
Butyric acid (30 mM and 60 mM).
1. Make 10× Arginine solutions.
2. Make 10× Cysteine solutions at 33 mM then dilute to 1 mM and 0.33 mM.
3. Make 10× Tyrosine solutions at 50 mg/L.
4. Make 10× Ascorbic acid solutions at 2.5 g/L and 10 g/L.
5. Make 10× Butyric acid solutions at 30 mM.
1. The following table shows the amount of seed growth (OD600 nm) as measured by DU700.
2. The following table shows the amount of cell growth (OD600 nm) in cultures with the indicated compound.
3. The following table shows the amount of Toxin A produced (ng/mL) in cultures with the indicated compound (
4. The following table shows the amount of Toxin B produced (ng/mL) in cultures with the indicated compound.
5. The following table indicates cell morphological characteristics in cultures with the indicated compound.
6. The following table shows the amount of spore formation in 24 hour fermentation in cultures with the indicated compound. Broth was examined by microscope.
Ascorbic acid, when added to SYS medium at 10 g/L, caused increases in Toxin B of 19% after 12 hours and 49% after 24 hours of incubation in fermentation broth.
Butyric Acid, when added to SYS medium at 30 mM, caused increases in Toxin A of 14% and Toxin B of 34% after 12 hours of incubation in fermentation broth. It caused increases in Toxin A of 16% and Toxin B of 61% after 24 hours of incubation in fermentation broth. Butyric Acid, when added to SYS medium at 60 mM, caused increases in Toxin B of 89% after 24 hours of incubation in fermentation broth.
This Example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media in the absence and presence of various concentrations of carbohydrates.
The following table summarizes the data regarding toxin increases (%) following the addition of increasing concentrations of D-sorbitol.
The following are the Example 5 test compounds, along with the compound formula and source.
D(−)Fructose: (contained <0.05% glucose) C6H12O6, FW 180.2, Sigma F0127 Lot#60K0013
D(+)Galactose: C6H12O6, FW 180.2, Sigma G0625, Lot#102K0169 soluble in water (1 g/1.7 mL)
D(+)Mannose: C6H12O6, FW 180.16, Sigma M6020 Lot# soluble in water (50 mg/mL)
D(+)Maltose Monohydrate: (contained <0.3% glucose), C12 hrs.22O11.H2O, FW 360.3, Sigma M9171 Lot#80K10101 soluble in water
Sucrose: C12 hrs.22O11, FW 342.3, Sigma, 53929, Lot#127K0093 soluble in water
D(+)Xylose: C5H10O5, FW 150.132, Sigma X3877 Lot# soluble in water (1 g/0.8 mL)
D-Sorbitol: C6H14O6, FW 182.2, Sigma 53889, Lot#042K01355 soluble in water.
myo-Inositol: C6H12O6, FW 180.16, Sigma 17508, Lot# soluble in water (50 mg/mL)
10× solutions of the above carbohydrates were produced.
1. The following table shows the amount of seed growth (OD600 nm) as measured by DU700.
2. The following table shows the amount of cell growth (OD600 nm) in cultures with the indicated compound.
3. The following table shows the amount of Toxin A produced (ng/mL) in cultures with the indicated compound (
4. The following table shows the amount of Toxin B produced (ng/mL) in cultures with the indicated compound.
5. The following table indicates cell morphological characteristics in cultures with the indicated compound.
6. The following table shows the amount of spore formation in 24 h fermentation in cultures with the indicated compound. Broth was examined by microscope.
D(+)Xylose, when added to SYS medium at 6 g/L, slightly increased cell growth and Toxin A production. D(+)Xylose increased Toxin B production 9% after 12 hours of incubation in fermentation broth and 46% after 24 hours of incubation in fermentation broth.
D-Sorbitol, when added to SYS medium at 6 g/L, markedly increased cell growth and toxins production. Cell growth was increased 44% after 12 hours of incubation in fermentation broth. Toxin A production was increased 49% after 12 hours of incubation in fermentation broth and 86% after 24 hours of incubation in fermentation broth. Toxin B production was increased 68% after 12 hours of incubation in fermentation broth and 153% after 24 hours of incubation in fermentation broth.
This example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media supplemented with D-Sorbitol (12 g/L) under pH controlled conditions.
The following materials and equipment were used in this example.
A Biostat B Plus Fermentation System (Sartorius) was used for the first stage seed culture and second stage seed culture, using 2×2 L fermenters (Sartorius), one for each seed culture seed. A Biostat Q Plus Fermentation System (Sartorius) was used for the third stage seed culture using 4×1 L fermenters (Sartorius). A peristalitic pump (Masterflex) was used to transfer media for both systems. Media composition for the seed stages were as follows:
Media composition for the production stage was as follows:
The SYS media and SYS media+Sorbitol were each prepared using reverse osmosis deionized water (RODI-water).
Additional materials included:
The following methods were used to test the production of Toxin A and B when cultured under pH controlled conditions.
The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated pH control (
The highest yields of both Toxin A and Toxin B were produced by maintaining the pH of the culture at a low pH (i.e., 6.5). The control culture, which was subject to no pH control also showed significantly more toxin production than those cultures subjected to a controlled pH 7.2 or pH 8.0. Lacking pH control, the pH of the control culture declined naturally (typically, declining from a starting pH of approximately pH 7.3 to a final pH of approximately 6.3).
SDS-Page gels showed similar bands and intensities for the control and pH 6.5, with the only differences being in the intensity of a band in the 100 kDa range.
This example includes data on the amount of toxin produced when Clostridium difficile is cultured under pH controlled conditions in SYS basal media having a reduced sodium bicarbonate concentration of 2 g/L and supplemented with D-Sorbitol (12 g/L).
The materials and methods utilized in this experiment were as set out in Example 6, except as noted below.
SYS media+Sorbitol included 2 g/L NaHCO3 (reduced from 5 g/L used in Example 6)
1. 5×1 L vessels (3rd stage vessels: QB2, QB3, QA1, QA2, QA3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH control setpoints were implemented (see Table 6).
2. Prior to inoculation, bioreactors were sparged with an appropriate gas for 30 minutes at 300 mL/min (see Table 6).
3. 3rd stage cultures were incubated at 37° C. with no gassing for 21 hours.
1. The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated pH control (
2. Specific Toxin A productivity produced (ng/ml per OD unit) in the cultures subject to the indicated pH control is set out in
Lowering the sodium bicarbonate to 2 g/L in the SYS+Sorbitol medium allowed for a lower starting pH with less acid and/or CO2 sparged. It is possible to achieve a pH of 6.5 without the addition of acid, by sparging with CO2.
The uncontrolled pH condition in this experiment had at least equivalent total Toxin B production and slightly higher total Toxin A production than the pH 6.5 condition. Specific toxin production was similar for the uncontrolled and pH 6.5 conditions.
This example includes data on the effect of different concentrations of sodium bicarbonate (i.e., 0 g/L, 2 g/L, and 5 g/L) and carbon dioxide sparing on the pH of SYS basal media supplemented with D-Sorbitol (12 g/L).
The following materials and equipment were used in this example.
The Biostat Q Plus Fermentation System (Sartorius) was used for the first, second, and third stage cultures, using three 1 L fermenters (Sartorius). The composition of the SYS media supplemented with D-Sorbitol (SYS media+Sorbitol) was as described in Example 6, except that no NaHC03 was added to the initial 4 L batch prepared.
The following methods were used to test changes in pH.
1. 3 pH probes were calibrated on the Biostat Q Plus system
2. 4 L of SYS media with sorbitol was made without sodium bicarbonate and 1 L was added to a 1 L fermenter.
3. 6 g of sodium bicarb was added to the remaining 3 L of media for a bicarb concentration of 2 g/L. 1 L of the media was added to a 1 L fermenter.
4. 6 g of sodium bicarb was added to the remaining 2 L of media for a bicarb concentration of 5 g/L. 1 L of the media was added to a 1 L fermenter.
5. All fermenters were mixed at 100 rpm and the pH probes were installed
6. 100% CO2 was sparged at 500 ml/min and the data acquisition software was started to generate pH curves
7. After ˜3.5 hours, 5 ml of 5N HCl was added to each fermenter.
A lower final pH can be achieved in SYS medium with a lower sodium bicarbonate concentration when gassing with CO2. Using 2 g/L bicarb can lower the pH by 0.12 units with CO2 sparge alone compared to 5 g/L.
This example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media supplemented with D-Sorbitol (12 g/L) with different concentrations of sodium bicarbonate (i.e., 0 g/L, 2 g/L, and 5 g/L) and spared with carbon dioxide or an anaerobic gas mix (80% N2/10% CO2/10% H2).
The materials and methods utilized in this experiment were as set out in Example 6, except as noted below.
The composition of the SYS media for the first stage seed cultures was as described in Example 6. For the third stage cultures, an SYS media supplemented with Sorbitol was prepared having the following composition and using RODI-water:
Two separate batches of SYS media+Sorbitol culture media were also prepared having the same composition but with a different concentration of NaHCO3 (i.e., one with NaHCO3 2 g/L and one with 5 g/L NaHCO3).
1. 5×1 L vessels (3rd stage vessels: QB2, QB3, QA1, QA2, QA3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH control setpoints were implemented (see Table 7).
2. Prior to inoculation, bioreactors were sparged with an appropriate gas for 30 minutes at 300 mL/min (see Table 7).
3. 3rd stage cultures were incubated at 37° C. with no gassing for 21 hours.
1. The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated pH control (
2. The 1st stage cell growth in this experiment was lower than typically seen in this experiment. There was not a significant difference in the growth or toxin production of the fermentations controlled at pH 6.5 with either CO2 or anaerobic gas mix sparging. A sodium bicarbonate concentration of 2 g/L sodium bicarbonate provided a higher specific and total toxin A and B productivity compared to concentrations of 0 g/L and 5 g/L.
The use of CO2 for degassing the media is an option when controlling pH at 6.5 because of the comparable toxin yields to the anaerobic gas mix degassed fermentation.
This example includes data on the amount of toxin produced when Clostridium difficile is cultured under a range of temperatures (37-41° C. with a midpoint of 39° C.) and a range of pH (6.35 to 6.65 with a midpoint of 6.5) in SYS basal media supplemented with D-Sorbitol (12 g/L).
The materials and methods utilized in this experiment were as set out in Example 6, except as noted below.
The composition of the SYS media for the first stage seed cultures was as described in Example 6. For the third stage cultures, an SYS media supplemented with Sorbitol was prepared having the following composition and using RODI-water:
1. 6×1 L vessels (3rd stage vessels: QB1, QB2, QB3, QA1, QA2, QA3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH control setpoints were implemented (see Table 8).
2. Prior to inoculation, bioreactors were sparged with an appropriate gas for 30 minutes at 300 mL/min (see Table 8).
3. 3rd stage cultures were incubated at the applicable temperature with no gassing for 21 hours.
1. The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated temperature and the indicated pH control (
2. Cell growth was higher in lower temperature and higher pH conditions. Toxin A production was higher in the low temperature (37° C.) and low pH (6.35) conditions. Toxin B production was higher in the high temperature (41° C.) and high pH (6.65) conditions. Lower toxin A yields were seen in high temperature and low pH conditions and lower toxin B yields were seen in low temperature and high pH conditions.
Optimal conditions for production of Toxin A and B are different. Since Toxin B availability is a limiting factor for the production of a vaccine product comprising Toxoids A and B (e.g., in a ratio of 3:2), conditions which favor Toxin B production may be preferred. Higher temperature (41° C.) and higher controlled pH (6.65) are the best conditions for Toxin B production. While these conditions are not the most optimal for Toxin A yields, Toxin A is produced and at a level above other conditions.
This example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media supplemented with D-Sorbitol (12 g/L) under a controlled a pH 6.5, and at various temperatures (33, 35, 37, 39, 41, 43° C.) and 2 g/L sodium bicarbonate.
The materials and methods utilized in this experiment were as set out in Example 6, except as noted below.
The composition of the SYS media for the first stage seed cultures was as described in Example 6. For the third stage cultures, an SYS media supplemented with Sorbitol was prepared having the following composition and using RODI-water:
1. 6×1 L vessels (3rd stage vessels: QA1 to QA3, QB1 to QB3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH control setpoints were implemented (see Table 9).
2. Prior to inoculation, bioreactors were sparged with an appropriate gas for 30 minutes at 300 mL/min (see Table 9).
3. 3rd stage cultures were incubated at the applicable temperature with no gassing for 21 hours.
1. The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated temperature and the indicated pH control (
2. Cell growth decreases at temperatures higher than 37° C. Toxin A production is highest and similar within the range of 37-41° C. Toxin B yield increases almost linearly with increasing temperature from 37-41° C.
Culturing C. difficile at 37-41° C. is optimal for both Toxin A and B production. Culturing C. difficile at temperatures at the higher end of the 37-41° C. range favors increased Toxin B production.
This example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media supplemented with D-Sorbitol (12 g/L) using different inoculum concentrations (1%, 5%, and 10% of initial bioreactor volume) and under different pH conditions (controlled pH 6.5 and controlled at pH 6.5 with base-only).
The materials and methods utilized in this experiment were as set out in Example 6, except as noted below.
The composition of the SYS media for the first stage seed cultures was as described in Example 6. For the third stage cultures, an SYS media supplemented with Sorbitol was prepared having the following composition and using RODI-water:
1. 6×1 L vessels (3rd stage vessels: QA1 to QA3, QB1 to QB3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH setpoints were implemented (see Table 11). For vessels QA1, QA2, and QA3, pH was set at 6.5 for control with base-only (5N NaOH). Base-only control involves the addition of base to the culture to adjust the culture pH to pH 6.5 in the event the culture pH becomes lower than 6.5. Under such control, the pH of the culture naturally decreases from the initial media pH (approximately pH 7.4) to pH 6.5.
2. Prior to inoculation, bioreactors were sparged with the applicable gas for 30 minutes at 300 mL/min (see Table 10) and then an overlay of nitrogen gas was added to the applicable vessels.
3. 3rd stage cultures were incubated at the applicable temperature for 24 hours.
The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated temperature and the indicated pH control (
In this experiment, a 10 L vessel (Sartorius) was also utilized. The vessel was autoclaved and connected to the Biostat system and the following conditions were set: 37° C. and agitation (stirring) at 100 rpm. Culture pH was not controlled. The vessel was filled with 9 L of the SYS media also utilized in filling the 1 L fermenters (i.e., SYS media with 12 g/L sorbitol and 2 g/L Na2HCO3). The vessel was then de-gassed using Nitrogen gas and inoculated with 1 L of the Seed Bioreactor 2 culture. Toxin production and cell growth (OD) was measured following an 18 hour incubation: Toxin A (24533 ng/ml); Toxin B (14837 ng/ml); 2.94 OD(600 nm).
A 10 L vessel was also included in two of the experiments set out above (i.e., Examples 10 and 11) and was prepared, inoculated, and cultured similarly (except de-gassing was done with gas mix 80% N2/10% CO2/10% H2 and agitation was set at 75 rpm). The measured toxin production and cell growth following an 18 hour incubation was as follows: in Example 10, Toxin A (29605 ng/ml); Toxin B (10732 ng/ml); 2.95 OD(600 nm); in Example 11, Toxin A (25681 ng/ml); Toxin B (24898 ng/ml); 3.17 OD (600 nm). In a separate experiment, toxin production and cell growth in a 10 L culture with SYS media (with 12 g/L sorbitol and 2 g/L Na2HCO3) under similar conditions (i.e., a 10% inoculum concentration, culture temperature of 37° C. and 50 rpm agitation) was similar: Toxin A (21090 ng/ml); Toxin B (12228 ng/ml) and 3.02 OD (600 nm).
Similar toxin yields may be achieved by using inoculum rates lower than 10% although the culture duration may need to be increased. Inoculations of 1% and 5% achieved toxin yields >30 μg/ml for toxin A and >15 μg/ml of toxin B after 20 hours.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference in their entirety.
This application claims priority from U.S. Provisional Patent Application No. 61/099,759, filed on Sep. 24, 2008, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/58268 | 9/24/2009 | WO | 00 | 7/7/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61099759 | Sep 2008 | US |
