WO2021001540A1

WO2021001540A1 - Detoxification of proteins

Info

Publication number: WO2021001540A1
Application number: PCT/EP2020/068835
Authority: WO
Inventors: Aria AMINZADEH; René Jørgensen
Original assignee: Proxi Biotech Aps
Priority date: 2019-07-04
Filing date: 2020-07-03
Publication date: 2021-01-07

Abstract

A method for producing an immunogenic composition comprising inactivated TcdA and/or TcdB is described. The method comprises contacting TcdA and/or TcdB with a solution comprising at least one destabilizing agent; at least one oxidizing agent; at least one transition metal ion, and at least one antioxidant.

Description

DETOXIFICATION OF PROTEINS

TECHNICAL FIELD

The present invention relates to a method for producing an inactivated protein, which inactivated protein may be used in an immunogenic composition. The present invention in particular relates to a method for producing inactivated large clostridial toxins (LCTs) from Clostridioides difficile (C. difficile )\ Toxin A (TcdA) and Toxin B (TcdB). Such inactivated TcdA and/or TcdB can be used in an immunogenic composition. The inactivation step can be contacting TcdA and/or TcdB with a solution comprising at least one destabilizing agent, at least one oxidizing agent.

BACKGROUND

Inactivated proteins, such as inactivated bacterial toxins, are currently used in vaccine production. Pertussis toxin is e.g. used for vaccine production against whooping cough. In order to be able to use the proteins as safe vaccine components, the proteins must be detoxified while preserving the immunogenicity.

C. difficile is a gram-positive anaerobic bacterium that is the leading cause of healthcare- associated diarrhea in the western world, with a mortality rate as high as 30%. Colonization of C. difficile usually occurs in the colon of elderly people or immunocompromised patients, when the natural gut microbiota is altered by treatment with antibiotics. An infection with the bacteria can give rise to a spectrum of diseases, ranging from mild diarrhea to pseudomembranous colitis, toxic megacolon and death.

The primary cause of pathogenicity by C. difficile is due to the two large clostridial toxins (LCTs): Toxin A (TcdA) and Toxin B (TcdB), which are large proteins with molecular weight of 308 kDa and 270 kDa, respectively. TcdA and TcdB are both potent cytotoxins which cause colonic tissue damage during an infection, with TcdB having about 100- to 1000-fold higher cytotoxic potency than TcdA.

TcdA and TcdB share about 66% sequence homology and both function as glucosyltransferases that inactivate Rho/Rac/Ras family of GTPases. The inactivation results in loss of epithelial cell-cell junctions, dysregulation of the actin cytoskeleton and/or necrotic lesions in the colonic epithelium, leading to the disease symptoms mentioned supra.

The standard of care treatment for C. difficile infection starts with the discontinuance of the triggering antibiotic treatment followed by use of the narrow spectrum antibiotics vancomycin or metronidazole, or in some cases the recently approved Fidaxomicin. In recent years, nonresponders to metronidazole and vancomycin have been increasing, likely due to hypervirulent strains with higher antibiotic resistance. Treatment with either metronidazole or vancomycin results in recurrence of disease in 20-30% of patients, and up to 40-60% of those patients experience additional recurrences. However, studies in both mice and humans have shown that toxinneutralizing antibodies against TcdA and TcdB can completely protect against C. difficile disease symptoms and recurrence, and therefore a vaccine against C. difficile infections containing immunogenic TcdA and TcdB toxoids is highly needed.

Methods for detoxification of proteins, such as bacterial toxins, that are currently available involve the use of chemical agents such as formaldehyde, trinitrobenzenesulfonic acid, beta-propiolactone, glutaraldehyde, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) or combinations, to chemically modify the material of the toxins.

Toxoids may be produced by addition of formaldehyde to the toxins for a significant period of time, usually several days to weeks. Formaldehyde, however, has several disadvantages. Besides the time-consuming detoxification process, which is necessary for lowering the residual toxicity to acceptable levels, there is the risk of toxic reversibility over time, which has been reported in several studies. To prevent this reversibility, low amounts of formaldehyde are often left in the final formulated vaccine, released for injection into humans. While the amount of formaldehyde in each dose of vaccine given to a patient is low, the total amount of formaldehyde injected into infants and small children with the schemes of multiple routine vaccinations might become substantial. There is also considerable evidence that e.g., formaldehyde and beta-propiolactone are carcinogens and therefore hazardous to work with, and the removal thereof before human administration is essential.

Formaldehyde inactivation also results in a highly cross-linked toxoid with significant chemical modifications, making it immunogenically less toxin-like and lacking critical neutralising epitopes. For this reason, formaldehyde may not be suitable for detoxification of a number of toxins for use as antigens in a vaccine, including botulinum toxin, TcdA and TcdB from C. difficile.

EP 0338566 discloses a method for producing an immunogenic composition comprising pertussis toxoid, the method comprising contacting pertussis toxin with 1 % cholic acid and 6%

tetranitromethane (TNM), which is a nitrating agent, and not an oxidizing agent.

EP 0834322 discloses a method for producing an immunogenic composition comprising inactivation by adding thiomersal.

US 4762710 discloses a method for chemical inactivation of a toxin with an oxidant and a trace amount of a metal ion and preparation of an acellular, detoxified vaccine therefrom.

There is thus a need to develop new alternative methods for preparing toxoids, in particular of TcdA and/or TcdB, which methods result in safer, more stable and more immunogenic toxoids, which are substantially free from undesirable components. SUMMARY OF THE INVENTION

It is an object of certain aspects of the present invention to provide an improvement over the above described techniques and known art; particularly to provide a novel method for producing an immunogenic composition comprising an irreversibly inactivated protein.

Accordingly, a first aspect of the present invention provides a method for producing an immunogenic composition comprising an inactivated protein, the method comprising contacting the protein with a solution comprising at least one destabilizing agent and at least one oxidizing agent.

A presently preferred embodiment of the present invention provides a method for producing an immunogenic composition comprising inactivated Ted A and/or TcdB as the protein of choice, the method comprising contacting the toxins with a solution comprising at least one destabilizing agent and at least one oxidizing agent.

In one or more exemplary embodiments, the method further comprises contacting the protein with a solution comprising at least one transition metal ion.

In one or more exemplary embodiments, the method further comprises contacting the protein with a solution comprising at least one antioxidant.

A second aspect of the present invention relates to an immunogenic composition produced according to the any of the methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects, features and advantages of which embodiments of the invention are capable of, will be apparent and elucidated from the following description of embodiments and aspects of the present invention, reference being made to the accompanying drawings, in which:

Fig. 1 shows an SDS-PAGE analysis of purified native TcdA and TcdB, where 8 mI_ of each sample consisting of 1 mM toxin was mixed with 8 mI_ of Laemmli Sample Buffer and left at room temperature for 30 min. Both samples along with molecular weight marker were electrophoresed using 4-20% Mini-PROTEAN TGX Stain-Free gels. M: Molecular weight marker, 1 : TcdA, 2: TcdB.

Fig. 2 shows farllV (200-260 nm) circular dichroism analysis of native and inactivated TcdA, depicting the change in secondary structural features. A Jasco J-1500 CD spectrometer was used with a scanning speed of 50 nm/min and a bandwith of 2 nm. Protein concentrations between 0.2- 0.3 mg/ml in a volume of 200 pL was used in a 1 mm cuvette (Hellma, art no. 1 10-1-40). All CD spectra are an average of 3-5 runs. Molar ellipticity ([0]) in units of mdeg cm² dmol ¹ was calculated as

mdeg M_w

10 - L c where /0/ is calculated molar ellipticity, mdeg is experimentally measured ellipticity in mdeg, M_w is protein molecular weight (g/mol), L is the optical path length (cm), c is the protein concentration (mg/ml). Solid: native TcdA, dashed: inactivated TcdA (example 4A), dotted: inactivated TcdA (example 5A), dashed/dotted: inactivated TcdA (example 2A).

Fig. 3 shows nearllV (250-320 nm) circular dichroism analysis of native and inactivated TcdA, depicting the change in tertiary structural features. A Jasco J-1500 CD spectrometer was used with a scanning speed of 50 nm/min and a bandwith of 2 nm. Protein concentrations between 0.3-0.5 mg/ml in a volume of 100 pL was used in a 10 mm cuvette (Hellma, art no. 105-201-15-40). All CD spectra are an average of 3-5 runs. Solid: native TcdA, dashed: inactivated TcdA (example 4A), dotted: inactivated TcdA (example 5A), dashed/dotted: inactivated TcdA (example 2A).

Fig. 4 shows farllV (200-260 nm) circular dichroism analysis of native and inactivated TcdB, depicting the change in secondary structural features. A Jasco J-1500 CD spectrometer was used with a scanning speed of 50 nm/min and a bandwith of 2 nm. Protein concentrations between 0.2- 0.3 mg/ml in a volume of 200 pL was used in a 1 mm cuvette (Hellma, art no. 1 10-1-40). All CD spectra are an average of 3-5 runs. Solid: native TcdB, dashed: inactivated TcdB (example 10).

Fig. 5 shows nearllV (250-320 nm) circular dichroism analysis of native and inactivated TcdB, depicting the change in tertiary structural features. A Jasco J-1500 CD spectrometer was used with a scanning speed of 50 nm/min and a bandwith of 2 nm. Protein concentrations between 0.3-0.5 mg/ml in a volume of 100 pL was used in a 10 mm cuvette (Hellma, art no. 105-201-15-40). All CD spectra are an average of 3-5 runs. Solid: native TcdB, dashed: inactivated TcdB (example 10).

Fig. 6 shows a Kaplan-Meier survival curve for mice that were immunised intramuscularly two times with toxoids produced according to the present invention, comprising 5 pg inactivated TcdA and 5 pg inactivated TcdB adjuvanted with aluminium hydroxide (Alhydrogel). TcdA and TcdB toxoids were assessed and compared to adjuvant alone (Group 1 ). Mice were challenged orally with a lethal dose of Clostridioides difficile Ribotype 027 (NCTC 13366) on day 56 after the first immunisation and monitored for 4 days following the infection (7.5 c 10⁵ CFU, n = 8 per group).

Fig. 7 shows a relative weight graph of mice immunised intramuscularly two times with toxoids produced according to the present invention (Group 2) or adjuvant alone (Group 1 ). Mice in Group 2 received TcdA and TcdB toxoids comprising 5 pg inactivated TcdA and 5 pg inactivated TcdB adjuvanted with aluminium hydroxide (Alhydrogel). Mice were challenged orally with a lethal dose of Clostridioides difficile Ribotype 027 (NCTC 13366) on day 56 after the first immunisation and the weight of each mouse were monitored for 3 days following the infection (7.5 c 10⁵ CFU, n = 8 per group). Relative weight is based on the weight at day 0.

Fig. 8 shows serum IgG responses against native purified TcdA from the serum of immunised mice measured by indirect ELISA. Individual serum samples from day 49 were tested for the level of antitoxin produced after two doses of vaccine comprising 5 pg inactivated TcdA and 5 pg inactivated TcdB adjuvanted with aluminium hydroxide (Alhydrogel). Mice in Group 2 received TcdA and TcdB toxoids, and Group 1 received adjuvant alone. A four-parameter dose-response curve was outlined for each serum sample by plotting the absorbance at 450 nm as a function of the serum dilution. Antitoxin IgG titers are shown as IC50 values, representing the dilution of serum where the antitoxin response is reduced by 50%. n = 8 per group.

Fig. 9 shows serum IgG responses against native purified TcdB from the serum of immunised mice measured by indirect ELISA. Individual serum samples from day 49 were tested for the level of antitoxin produced after two doses of vaccine comprising 5 pg inactivated TcdA and 5 pg inactivated TcdB adjuvanted with aluminium hydroxide (Alhydrogel). Mice in Group 2 received TcdA and TcdB toxoids, and Group 1 received adjuvant alone. A four-parameter dose-response curve was outlined for each serum sample by plotting the absorbance at 450 nm as a function of the serum dilution. Antitoxin IgG titers are shown as IC50 values, representing the dilution of serum where the antitoxin response is reduced by 50%. n = 8 per group.

DEFINITIONS

Immunogenic composition

The term“immunogenic composition” refers to a composition that elicits an immune response in a human and/or animal subject to which the composition is administered.

Immune response

The term“immune response” refers to the development of a humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a protein in a recipient human and/or animal subject. The immune response may be humoral, cellular, or both. The presence of a humoral (antibody-mediated) immune response can be determined by for example ELISA or cell-based assay known in the art such as neutralizing antibody assay etc.

Vaccine composition

In one or more exemplary embodiments, an immunogenic composition is a vaccine composition. The term“vaccine composition” refers to a composition that elicits an immune response in a human and/or animal subject to which the composition is administered. The vaccine composition may protect the human and/or animal subject against subsequent challenge by an immunizing agent or an immunologically cross-reactive agent. Protection can either be partial or complete with regard to decrease in symptoms or infection as compared to a human and/or animal subject under the same conditions to which the vaccine composition has not been administered. The vaccine composition may further contain one or more adjuvants and/or a pharmaceutically acceptable diluent or carrier. Animal subject

The term“animal subject” refers to all animals, such as for example, mice, hamsters, rabbits, primates, pigs, horses, dogs, cats, alpacas, sheep, goats, donkeys, camels etc.

Inactivated protein

The term“inactivated protein” refers to a protein, such as but not limited to TcdA and/or TcdB, that has been chemically modified and thereby has a reduced biological activity relative to the native protein, also called detoxified. The desired reduction in biological activity can vary among different proteins, but in general, sufficiently reduced to not cause toxic effects in human and/or animal subjects upon injection of an immunogenic composition comprising the inactivated protein. The reduction in biological activity can be 100-, 200-, 300-, 400-, 500-, 1000-, 2000-, 3000-, 4000-, 5000-fold, 10000-fold, 20000-fold, 50000-fold, 100000-fold, or more, relative to the corresponding native protein. Biological activity can be quantified in vitro for example as the exerted cytotoxicity in mammalian cells such as Vero kidney cells from Cercopithecus aethiops or IMR90 cells or other cell-based cytotoxicity assay known in the art.

Native

The term“native” as used herein, refers to the form found in nature. For example, a native protein is a protein present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

Destabilizing agent

The term“destabilizing agent” refers to any agent that can unfold and/or denature and/or distort the quaternary and/or tertiary and/or secondary structure and/or disrupt non-covalent interactions such as ionic bonds and/or van der Waals forces and/or hydrogen bonds, and/or dipole-dipole interactions and/or hydrophobic effects and/or disulfide bonds of a protein. Destabilizing agent herein refers to agents selected from the group consisting of ionic detergents, zwitterionic detergents, chaotropic agents and reducing agents.

Oxidizing agent

The term“oxidizing agent” refers to a reactant that removes electrons from other reactants.

Transition metal ion

The term“transition metal ion” herein refers to ions of a transition metal or transition metal salt that has been dissolved in a solution. Transition metal ions are in solution either as monoatomic ions or complex ions. Usually the transition metal ion is obtained by dissolving a transition metal salt such as ferrous sulfate, ferric sulfate, ferrous chloride, ferric chloride, copper dichloride, copper chloride, copper sulfate, silver nitrate, cobalt chloride, and chromium chloride or others in a solution.

Antioxidant

The term“antioxidant” herein refers to a specific type of reactant that is capable of reducing transition metal ions. Reducing agent

The term“reducing agent” herein refers to a reactant that reduces disulfide bonds.

Ionic detergent

The term“ionic detergent” refers to an amphipathic molecule, containing a polar hydrophilic head group with either a negative (anionic) or a positive (cationic) charge, that is attached to a long- chain hydrophobic carbon tail.

Zwitterionic detergent

The term“zwitterionic detergent” refers to an amphipathic molecule, containing a polar hydrophilic head group with both a negative (anionic) and a positive (cationic) charge, that is attached to a long-chain hydrophobic carbon tail. As the polar hydrophilic head group contains both negatively and positively charged atomic groups, the overall charge is neutral.

Non-ionic detergent

The term“non-ionic detergent” refers to an amphipathic molecule, containing a polar hydrophilic head group that is uncharged, attached to a long-chain hydrophobic carbon tail.

Chaotropic agent

The term“chaotropic agent” refers to a molecule that can disrupt non-covalent interactions such as ionic bonds and/or hydrogen bonds and/or van der Waals forces, and/or dipole-dipole interactions and/or hydrophobic effects, and thereby reduce the stability of the protein.

DETAILED DESCRIPTION OF THE INVENTION

Specific embodiments of the invention will now be described with reference to the accompanying drawings.

The different aspects, alternatives and embodiments of the invention disclosed herein can be combined with one or more of the other aspects, alternatives and embodiments described herein. Two or more aspects can be combined.

In describing the embodiments of the invention specific terminology will be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.

When describing the embodiments of the present invention, the combinations and permutations of all possible embodiments have not been explicitly described. Nevertheless, the mere fact that certain measures are recited in mutually different dependent claims or described in different embodiments does not indicate that a combination of these measures cannot be used to advantage. The present invention envisages all possible combinations and permutations of the described embodiments. The terms“comprising”,“comprise” and“comprises” herein are intended by the inventors to be optionally substitutable with the terms“consisting of”,“consist of and“consists of”, respectively, in every instance.

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Destabilizing agent

In one or more exemplary embodiments, the destabilizing agent is a detergent.

Ionic detergent

In one or more exemplary embodiments, the detergent is an ionic detergent.

In one or more exemplary embodiments, the ionic detergent is selected from the group consisting of Sodium Dodecyl Sulfate (SDS), sodium deoxycholate, sodium cholate, sodium lauroyl sarcosinate, dioctyl suifosuccinate and cetyltrimethylammonium bromide.

In one or more exemplary embodiments, the ionic detergent is Sodium Dodecyl Sulphate (SDS).

In one or more exemplary embodiments, the ionic detergent is SDS at a concentration of 0.01 to 30 mM, preferably 0.1 to 10 mM, more preferably 0.1 to 5 mM, even more preferably, 0.1 to 3.5 mM, most preferably 0.17 mM.

In one or more examplary embodiments, the ionic detergent is SDS at a concentration of 0.1 to 20 mM, preferably 0.2 to 10 mM, more preferably 0.35 to 3.5 mM, even more preferably, 0.5 to 1 mM, most preferably 0.5 mM.

In one or more exemplary embodiments, the ionic detergent is SDS at a concentration of 0.01 mM, or 0.05 mM, 0.01 mM, or 0.15 mM, or 0.17 mM or 0.2 mM, or 0.3 mM, or 0.4 mM, or 0.5 mM, or 0.75 mM, or 1 mM, or 1.2 mM, or 1.5 mM, or 1.75 mM, or 2 mM, or 2.5 mM, or 3 mM. or 3.5 mM, or 4 mM, or 5 mM, or 6 mM, or 7 mM, or 8 mM, or 9 mM, or 10 mM.

In one or more exemplary embodiments, the ionic detergent is SDS at a concentration of 1 1 mM, or 12 mM, or 13 mM, or 14 mM, or 15 mM, or 16 mM, or 17 mM, or 18mM, or 19 mM, or 20, or 25 mM, or 30 mM.

In one or more exemplary embodiments, Ted A and/or TcdB is contacted with SDS at a

concentration of 0.1 mM, or 0.15 mM, or 0.17 mM, or 0.2 mM, or 0.25 mM, or 0.3 mM, or 0.35 mM, or 0.4 mM, or 0.5 mM, or 0.70 mM, or 1 mM, or 1.2 mM, or 1.5 mM, or 1 .75 mM, or 2 mM, or 2.5 mM, or 3 mM, or 3.5 mM, or 4 mM, or 5 mM, or 6 mM, or 7 mM, or 8 mM, or 9 mM, or 10 mM.

Zwitterionic detergent

In one or more exemplary embodiments, the detergent is a zwitterionic detergent. In one or more exemplary embodiments, the zwitterionic detergent is selected from the group consisting of n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate

(sulfobetaines/zwittergent), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

(CHAPS), (3-(3 Cholamidopropyl)dimethylammonio)-2-hydroxy-1-propanesulfonate) (CHAPSO), amidosulfobetaines, non-detergent sulfobetaines (NDSB) and Lauryldimethylamine N-oxide.

In one or more exemplary embodiments, the zwitterionic detergent is 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).

In one or more exemplary embodiments, the zwitterionic detergent is CHAPS at a concentration of 0.1 to 3 mM, preferably 0.2 to 2 mM, more preferably 0.3 to 1.5 mM, most preferably 0.5 mM.

In one or more exemplary embodiments, the zwitterionic detergent is CHAPS at a concentration of 0.1 mM, or 0.3 mM, or 0.5 mM, or 1 mM, or 1.5 mM.

In one or more exemplary embodiments, the zwitterionic detergent is n-T etrad ecyl-N , N-d i methyl-3- ammonio-1-propanesulfonate (sulfobetaines/zwittergent).

In one or more exemplary embodiments, the zwitterionic detergent is zwittergent at a concentration of 0.1 to 3 mM, preferably 0.2 to 2 mM, more preferably 0.3 to 1.5 mM, most preferably 0.5 mM.

In one or more exemplary embodiments, the zwitterionic detergent is zwittergent at a concentration of 0.1 mM, or 0.3 mM, or 0.5 mM, or 1 mM, or 1.5 mM.

Chaotropic agent

In one or more exemplary embodiments, the destabilizing agent is a chaotropic agent.

In one or more exemplary embodiments, the chaotropic agent is selected from the group consisting of urea, thiourea, guanidinium chloride, ethanol, n-butanol, lithium perchlorate, lithium acetate, phenol and 2-propanol.

In one or more exemplary embodiments, the chaotropic agent is urea.

In one or more exemplary embodiments, the chaotropic agent is urea at a concentration of 0.1 to 8

M, preferably 0.5 to 4 M, more preferably 1 to 2.5 M, most preferably 2 M.

In one or more exemplary embodiments, the chaotropic agent is urea at a concentration of 0.1 M, or 0.3 M, or 0.5 M, or 0.75 M, or 1 M, or 1.25 M, or 1.5 M, or 1.75 M, or 2M, or 2.5 M, or 3 M, or 4

M, or 5 M, or 6 M, or 7 M, or 8 M.

In one or more exemplary embodiments, Ted A and/or TcdB is contacted with urea at a

concentration of 0.1 M, or 0.3 M, or 0.5 M, or 0.75 M, or 1 M, or 1.25 M, or 1.5 M, or 1.75 M, or 2M.

Reducing agent

In one or more exemplary embodiments, the destabilizing agent is a reducing agent. In one or more exemplary embodiments, the reducing agent is selected from the group consisting of b-mercaptoethanol (BME), dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) and 2- mercaptoethylamine-HCL.

In one or more exemplary embodiments, the reducing agent is b-mercaptoethanol (BME).

In one or more exemplary embodiments, the reducing agent is BME at a concentration of 1 to 200 mM, preferably 10 to 100 mM, more preferably 20 to 40 mM, most preferably 40 mM.

In one or more exemplary embodiments, the reducing agent is b-mercaptoethanol (BME) at a concentration of 10 mM, or 15 mM, 20 mM, or 25 mM, or 30 mM, or 32.5 mM, or 35 mM or 40 mM.

In one or more exemplary embodiments, the reducing agent is dithiothreitol (DTT).

In one or more exemplary embodiments, the reducing agent is DTT at a concentration of 1 to 100 mM, preferably 10 to 50 mM, more preferably 20 to 40 mM, most preferably 40 mM.

In one or more exemplary embodiments, the reducing agent is DTT at a concentration of 10 mM, or 15 mM, 20 mM, or 25 mM, or 30 mM, or 32.5 mM, or 35 mM, or 40 mM.

Destabilization by acidification

In one or more exemplary embodiments, the destabilization can be achieved solely by changing the pH of the solution, preferably to pH 4 to 6, more preferably pH 4 to 5 most preferably pH 4.5.

In one or more examplary embodiments, the destabilization of Ted A and/or TcdB can be achieved by changing the pH of the solution, preferably to pH 4 to 5.5, more preferably pH 4 to 5, most preferably pH 4.5.

Oxidizing agent

In one or more exemplary embodiments, the oxidizing agent is selected from the group consisting of hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium

permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4- methylbenzenesulfonamide sodium salt, and dioxaneperoxide.

In one or more exemplary embodiments, the oxidizing agent is hydrogen peroxide (H2O2).

In one or more exemplary embodiments, the oxidizing agent is H2O2 at a concentration of 0.001 to 1000 mM, preferably 0.01 to 100 mM, more preferably 0.1 to 80 mM, even more preferably 1 to 50 mM, and most preferably 10 mM.

In one or more exemplary embodiments, Ted A and/or TcdB is contacted with hydrogen peroxide (H2O2) at a concentration of 0.001 to 1000 mM, preferably 0.01 to 100 mM, more preferably 0.1 to 30 mM, even more preferably 0.5 to 10 mM, and most preferably 3 mM. In a presently preferred embodiment, when producing an immunogenic composition comprising inactivated Ted A and/or TcdB, then hydrogen peroxide (H2O2) at the above mentioned concentrations is preferable. In one or more exemplary embodiments, the oxidizing agent is H2O2 at a concentration of 0.001 mM, or 0.005 mM, or 0.01 mM, or 0.03 mM, or 0.05 mM, or 0.1 mM, or 0.5 mM, or 1 mM, 2 mM, 3 mM, or 4 mM, or 5 mM, or 6 mM, or 7 mM, or 8 mM, or 9 mM, or 10 mM.

In one or more exemplary embodiments, the oxidizing agent is H2O2 at a concentration of 15 mM, or 20 mM, or 30 mM, or 40 mM, or 50 mM, or 75 mM, or 100 mM.

In one or more exemplary embodiments, the oxidizing agent is H2O2 at a concentration of 200 mM, or 500 mM, or 1000 mM.

In one or more exemplary embodiments, Ted A and/or TcdB is contacted with H2O2 at a concentration of 0.1 mM, or 0.3 mM or 0.5 mM, or 0.75 mM, or 1 mM, or 1.5 mM, or 2 mM, or 3 mM, or 4 mM, or 5 mM.

Transition metal ion

In one or more exemplary embodiments, the transition metal ion is selected from the group consisting of ferrous, ferric, cuprous, cupric, silver, cobalt, chromium metals or metal salt thereof.

In one or more exemplary embodiments, the transition metal ion is from ferrous sulfate (FeSC ), ferric sulfate (Fe₂(SC>₄)3), ferrous chloride, ferric chloride, copper dichloride, copper chloride, copper sulfate, silver nitrate, cobalt chloride, and chromium chloride.

In one or more exemplary embodiments, the transition metal ion is from ferrous sulfate (FeSC ).

In one or more exemplary embodiments, the transition metal ion is from FeSC at a concentration of 0.001 to 10 mM, preferably 0.001 to 1 mM, more preferably 0.01 to 0.5 mM, even more preferably 0.01 to 0.1 mM, and most preferably 0.1 mM.

In one or more exemplary embodiments, the transition metal ion is from FeSC at a concentration of 0.001 mM, or 0.003 mM, or 0.005 mM, or 0.01 mM, 0.015 mM, or 0.02 mM, or 0.025 mM, or 0.03 mM, or 0.04 mM, 0.05 mM, or 0.06 mM, or 0.07 mM, or 0.08 mM, or 0.09 mM, or 0.1 mM, or 0.2 mM, or 0.3 mM, or 0.4 mM, or 0.5 mM.

In one or more exemplary embodiments, the transition metal ion is from ferric sulfate (Fe₂(SC>₄)3).

In one or more exemplary embodiments, the transition metal ion is from Fe₂(SC>₄)3 at a concentration of 0.001 to 10 mM, preferably 0.001 to 1 mM, more preferably 0.01 to 0.5 mM, even more preferably 0.05 to 0.2 mM, and most preferably 0.1 mM.

In one or more exemplary embodiments, the transition metal ion is from Fe₂(SC>₄)3 at a concentration of 0.001 mM, or 0.003 mM, or 0.005 mM, or 0.01 mM, 0.015 mM, or 0.02 mM, or 0.025 mM, or 0.03 mM, or 0.04 mM, 0.05 mM, or 0.06 mM, or 0.07 mM, or 0.08 mM, or 0.09 mM, or 0.1 mM, or 0.2 mM, or 0.3 mM, or 0.4 mM, or 0.5 mM.

In one or more exemplary embodiments, the transition metal ion is from copper sulfate (CUSO4). In one or more exemplary embodiments, the transition metal ion is from CuSC at a concentration of 0.001 to 10 mM, preferably 0.001 to 1 mM, more preferably 0.01 to 0.5 mM, even more preferably 0.02 to 0.1 mM, and most preferably 0.01 mM.

In one or more exemplary embodiments, the transition metal ion is from CuSC at a concentration of 0.001 mM, or 0.003 mM, or 0.005 mM, or 0.01 mM, 0.015 mM, or 0.02 mM, or 0.025 mM, or 0.03 mM, or 0.04 mM, 0.05 mM, or 0.06 mM, or 0.07 mM, or 0.08 mM, or 0.09 mM, or 0.1 mM, or 0.2 mM, or 0.3 mM, or 0.4 mM, or 0.5 mM.

In one or more exemplary embodiments, the transition metal ion is from CuCh at a concentration of 0.001 to 10 mM, preferably 0.001 to 1 mM, more preferably 0.01 to 0.5 mM, even more preferably 0.02 to 0.1 mM, and most preferably 0.05 mM.

In one or more exemplary embodiments, the transition metal ion is from CuCh at a concentration of 0.001 mM, or 0.003 mM, or 0.005 mM, or 0.01 mM, 0.015 mM, or 0.02 mM, or 0.025 mM, or 0.03 mM, or 0.04 mM, 0.05 mM, or 0.06 mM, or 0.07 mM, or 0.08 mM, or 0.09 mM, or 0.1 mM, or 0.2 mM, or 0.3 mM, or 0.4 mM, or 0.5 mM.

In one or more exemplary embodiments, Ted A and/or TcdB is contacted with FeSC at a concentration of 0.01 mM, 0.015 mM, or 0.02 mM, or 0.025 mM, or 0.03 mM, or 0.04 mM, 0.05 mM, or 0.06 mM, or 0.07 mM, or 0.08 mM, or 0.09 mM, or 0.1 mM, or 0.2 mM, or 0.3 mM, or 0.4 mM, or 0.5 mM.

In one or more exemplary embodiments, Ted A and/or TcdB is contacted with CuSC at a concentration of 0.01 mM, 0.015 mM, or 0.02 mM, or 0.025 mM, or 0.03 mM, or 0.04 mM, 0.05 mM, or 0.06 mM, or 0.07 mM, or 0.08 mM, or 0.09 mM, or 0.1 mM, or 0.2 mM, or 0.3 mM, or 0.4 mM, or 0.5 mM.

Antioxidant

When antioxidants are added to the methods of the present disclosure, it can enhance the extent of inactivation of proteins. The mechanism is not exactly known, and without being bound to any theory, it is suggested that it works by reducing the oxidized transition metal ions in the reaction solution. Antioxidants e.g. sodium ascorbate when added up to 1 mM enhances the extent of inactivation of T cd A and/or TcdB, but above this concentration e.g. at 3 mM it has the opposite effect. Therefore, the optimal concentration of antioxidants can vary depending on the protein and inactivation conditions.

In one or more exemplary embodiments, the antioxidant is sodium ascorbate (ascorbic acid) or mineral salts thereof. In one or more exemplary embodiments, the antioxidant is sodium ascorbate (ascorbic acid) at a concentration of 0.1 mM to 5 mM, preferably 0.3 mM to 3 mM, more preferably 0.5 mM to 2 mM, most preferably 1 mM.

In one or more exemplary embodiments, the antioxidant is sodium ascorbate at a concentration of 0.1 mM or 0.2 mM, or 0.3 mM, or 0.4 mM, or 0.5 mM, or 0.6 mM, or 0.7 mM, or 0.8 mM, or 0.9 mM, or 1 mM.

In one or more exemplary embodiments, the antioxidant is sodium ascorbate at a concentration of 1 mM or 1 .5 mM, or 2 mM, or 3 mM, or 4 mM, or 5 mM.

In one or more exemplary embodiments, Ted A and/or TcdB is contacted with sodium ascorbate at a concentration of 0.1 mM or 0.2 mM, or 0.3 mM, or 0.4 mM, or 0.5 mM, or 0.6 mM, or 0.7 mM, or 0.8 mM, or 0.9 mM, or 1 mM, or 1 .5 mM.

In one or more exemplary embodiments, the antioxidant is uric acid.

In one or more exemplary embodiments, the antioxidant is uric acid at a concentration of 0.1 mM to 5 mM, preferably 0.3 mM to 3 mM, more preferably 0.5 mM to 2 mM, most preferably 1 mM.

In one or more exemplary embodiments, the antioxidant is uric acid at a concentration of 0.1 mM or 0.2 mM, or 0.3 mM, or 0.4 mM, or 0.5 mM, or 0.6 mM, or 0.7 mM, or 0.8 mM, or 0.9 mM, or 1 mM.

In one or more exemplary embodiments, the antioxidant is uric acid at a concentration of 1 mM or 1.5 mM, or 2 mM, or 3 mM, or 4 mM, or 5 mM.

In one or more exemplary embodiments, the antioxidant is homocysteine.

In one or more exemplary embodiments, the antioxidant is homocysteine at a concentration of 0.1 mM to 5 mM, preferably 0.3 mM to 3 mM, more preferably 0.5 mM to 2 mM, most preferably 1 mM.

In one or more exemplary embodiments, the antioxidant is homocysteine at a concentration of 0.1 mM or 0.2 mM, or 0.3 mM, or 0.4 mM, or 0.5 mM, or 0.6 mM, or 0.7 mM, or 0.8 mM, or 0.9 mM, or 1 mM.

In one or more exemplary embodiments, the antioxidant is homocysteine at a concentration of 1 mM or 1.5 mM, or 2 mM, or 3 mM, or 4 mM, or 5 mM.

Time-period

The methods of the present disclosure are all very fast to produce immunogenic compositions. The time-period needed for a sufficient inactivation is dependent on the temperature of the reaction solution. A lower temperature requires a longer time-period, whereas at a higher temperature a faster time-period is sufficient.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 37°C. In one or more exemplary embodiments, the immunogenic composition can be produced in less than 5 hours at a temperature of 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 3 hours at a temperature of 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 1 hours at a temperature of 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 30 min at a temperature of 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 15 min at a temperature of 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 10 min at a temperature of 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 5 min at a temperature of 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 1 min at a temperature of 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 22°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 18 hours at a temperature of 22°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 12 hours at a temperature of 22°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 10 hours at a temperature of 22°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 8 hours at a temperature of 22°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 22°C. In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 22°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 22°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 1 hour at a temperature of 22°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 7 days at a temperature of 4°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 5 days at a temperature of 4°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 3 days at a temperature of 4°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 48 hours at a temperature of 4°C.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 4°C.

In one or more exemplary embodiments, the immunogenic composition can be produced when the protein is contacted with a solution comprising at least one destabilizing agent, at least one oxidizing agent, and at least one transition metal ion during a time-period of 1 minute to 24 hours, preferably 30 to 360 minutes, more preferably 60 to 180 minutes, most preferably 120 minutes.

In one or more exemplary embodiments, the immunogenic composition can be produced when the protein is contacted with a solution comprising at least one destabilizing agent, and at least one oxidizing agent during a time-period of 1 minute to 24 hours, preferably 30 to 360 minutes, more preferably 60 to 180 minutes, most preferably 120 minutes.

In one or more exemplary embodiments, the immunogenic composition can be produced when the protein is contacted with a solution comprising at least one destabilizing agent, and at least one oxidizing agent, at least one transition metal ion and at least one antioxidant during a time-period of 1 minute to 24 hours, preferably 30 to 360 minutes, more preferably 60 to 180 minutes, most preferably 120 minutes.

In one or more exemplary embodiments, the immunogenic composition can be produced when the protein is contacted with a solution comprising at least one destabilizing agent, and at least one oxidizing agent, at least one transition metal ion and at least one antioxidant during a time-period of 1 minute to 7 days, preferably 30 minutes to 48 hours, more preferably 60 minutes to 24 hours, most preferably 24 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced when TcdA and/or TcdB is contacted with a solution comprising at least one destabilizing agent, at least one oxidizing agent, at least one transition metal ion and at least one antioxidant during a time- period of 1 minute to 7 days, preferably 30 minutes to 48 hours, more preferably 60 minutes to 24 hours, most preferably 24 hours.

Temperature

The temperature of the reaction solution can be varied between 0 to 55°C. Lower temperatures e.g. 4°C are in many cases preferred as proteins tend to be more stable, which in return will extend the time-period for a sufficient inactivation of said protein. If a faster inactivation time-period is preferred, the temperature of the reaction solution can be raised to e.g. 22°C to 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced when the step of contacting the protein with a solution comprising at least one destabilizing agent, at least one oxidizing agent, and at least one transition metal ion is conducted at a temperature between 0 to 55°C, preferably 20 to 40°C, more preferably 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced when the step of contacting the protein with a solution comprising at least one destabilizing agent, and at least one oxidizing agent is conducted at a temperature between 0 to 55°C, preferably 20 to 40°C, more preferably 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced when the step of contacting the protein with a solution comprising at least one destabilizing agent, at least one oxidizing agent, at least one transition metal ion, and at least one antioxidant is conducted at a temperature between 0 to 55°C, preferably 20 to 40°C, more preferably 37°C.

In one or more exemplary embodiments, the immunogenic composition can be produced when the step of contacting the protein with a solution comprising at least one destabilizing agent, at least one oxidizing agent, at least one transition metal ion, and at least one antioxidant is conducted at a temperature between 0 to 50°C, preferably 4 to 37°C, more preferably 4 to 22°C, most preferably 4°C.

In one or more exemplary embodiments, the immunogenic composition can be produced when the step of contacting TcdA and/or TcdB with a solution comprising at least one destabilizing agent, at least one oxidizing agent, at least one transition metal ion and at least one antioxidant is conducted at a temperature between 0 to 50°C, preferably 4 to 37°C, more preferably 4 to 22°C, most preferably 4°C. pH of buffers

When pH is not intended to be used as a means of achieving destabilization, but solely for stabilizing the protein in a buffer during the inactivation reaction, a pH between 6 to 8.5 is preferred.

In one or more exemplary embodiments, the step of contacting the protein with a solution comprising at least one destabilizing agent, at least one oxidizing agent, at least one transition metal ion and at least one antioxidant is conducted between pH 6 to 8.5, preferably pH 6.5 to 8, more preferably pH 7 to 8, and most preferably pH 7.5.

Specific embodiments for producing an immunogenic composition

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 7 days at a temperature of 0 to 10°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 7 days at a temperature of 0 to 10°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 7 days at a temperature of 0 to 10°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 7 days at a temperature of 0 to 10°C and at pH 7 to 8 using urea with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 7 days at a temperature of 0 to 10°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 5 days at a temperature of 0 to 10°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 5 days at a temperature of 0 to 10°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid. In one or more exemplary embodiments, the immunogenic composition can be produced in less than 5 days at a temperature of 0 to 10°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 5 days at a temperature of 0 to 10°C and at pH 7 to 8 using urea with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 5 days at a temperature of 0 to 10°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 3 days at a temperature of 0 to 10°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 3 days at a temperature of 0 to 10°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 3 days at a temperature of 0 to 10°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 3 days at a temperature of 0 to 10°C and at pH 7 to 8 using urea with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 3 days at a temperature of 0 to 10°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 48 hours at a temperature of 0 to 10°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant. In one or more exemplary embodiments, the immunogenic composition can be produced in less than 48 hours at a temperature of 0 to 10°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 48 hours at a temperature of 0 to 10°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 48 hours at a temperature of 0 to 10°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 48 hours at a temperature of 0 to 10°C and at pH 7 to 8 using urea with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 48 hours at a temperature of 0 to 10°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 0 to 10°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 0 to 10°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 0 to 10°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 0 to 10°C and at pH 7 to 8 using urea with H2O2 with FeSC and with ascorbic acid. In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 0 to 10°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 0 to 10°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 0 to 10°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSCU.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 10 to 25°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 10 to 25°C and at pH 7 to 8 using SDS with H2O2 with FeSCU and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 10 to 25°C and at pH 7 to 8 using urea with H2O2 with FeSCU and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 24 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 18 hours at a temperature of 10 to 25°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant. In one or more exemplary embodiments, the immunogenic composition can be produced in less than 18 hours at a temperature of 10 to 25°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 18 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 18 hours at a temperature of 10 to 25°C and at pH 7 to 8 using urea with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 18 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 12 hours at a temperature of 10 to 25°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 12 hours at a temperature of 10 to 25°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 12 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 12 hours at a temperature of 10 to 25°C and at pH 7 to 8 using urea with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 12 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 12 hours at a temperature of 10 to 25°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC>₄ and with 0.5 to 2 mM ascorbic acid. In one or more exemplary embodiments, the immunogenic composition can be produced in less than 12 hours at a temperature of 10 to 25°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC .

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 8 hours at a temperature of 10 to 25°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 8 hours at a temperature of 10 to 25°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 8 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 8 hours at a temperature of 10 to 25°C and at pH 7 to 8 using urea with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 8 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 10 to 25°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 10 to 25°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 10 to 25°C and at pH 7 to 8 using urea with H2O2 with FeSC and with ascorbic acid. In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 10 to 25°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC>₄ and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 10 to 25°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC .

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 10 to 25°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 10 to 25°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 10 to 25°C and at pH 7 to 8 using urea with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 10 to 25°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 10 to 25°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid. In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 10 to 25°C and at pH 7 to 8 using urea with H2O2 with FeSCU and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 10 to 25°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 10 to 25°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 10 to 25°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSCU.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 25 to 37°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 25 to 37°C and at pH 7 to 8 using SDS with H2O2 with FeSCU and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 25 to 37°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 25 to 37°C and at pH 7 to 8 using urea with H2O2 with FeSCU and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 6 hours at a temperature of 25 to 37°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 25 to 37°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 25 to 37°C and at pH 7 to 8 using SDS with H2O2 with FeSCU and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 25 to 37°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 25 to 37°C and at pH 7 to 8 using urea with H2O2 with FeSCU and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 25 to 37°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 25 to 37°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 4 hours at a temperature of 25 to 37°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSCU.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 25 to 37°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 25 to 37°C and at pH 7 to 8 using SDS with H2O2 with FeSCU and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 25 to 37°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 25 to 37°C and at pH 7 to 8 using urea with H2O2 with FeSCU and with ascorbic acid. In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 25 to 37°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 25 to 37°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC>₄ and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 2 hours at a temperature of 25 to 37°C and at pH 4 to 5 using 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC .

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 1 hour at a temperature of 25 to 37°C and at pH 7 to 8 using at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 1 hour at a temperature of 25 to 37°C and at pH 7 to 8 using SDS with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 1 hour at a temperature of 25 to 37°C and at pH 7 to 8 using 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 1 hour at a temperature of 25 to 37°C and at pH 7 to 8 using urea with H2O2 with FeSC and with ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced in less than 1 hour at a temperature of 25 to 37°C and at pH 7 to 8 using 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 4°C for a time-period of 24 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 24 hours at pH 7.5. In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 24 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSCU and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 24 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC at a temperature of 4°C for a time-period of 24 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 4°C for a time-period of 48 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 48 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 48 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 48 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC at a temperature of 4°C for a time-period of 48 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 4°C for a time-period of 3 days.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 3 days at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 3 days at pH 7.5. In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 4°C for a time-period of 5 days.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 5 days at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 5 days at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 4°C for a time-period of 7 days.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 7 days at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 4°C for a time-period of 7 days at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 22°C for a time-period of 1 hour.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 1 hour at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 1 hour at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 22°C for a time-period of 2 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 2 hours at pH 7.5. In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 2 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSCU and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 2 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC at a temperature of 22°C for a time-period of 2 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 22°C for a time-period of 4 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 4 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 4 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 4 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC at a temperature of 22°C for a time-period of 4 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 22°C for a time-period of 6 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 6 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 6 hours at pH 7.5. In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 6 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC at a temperature of 22°C for a time-period of 6 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 22°C for a time-period of 8 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 8 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 8 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 22°C for a time-period of 12 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 12 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 12 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 22°C for a time-period of 18 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 18 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 18 hours at pH 7.5. In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 22°C for a time-period of 24 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 24 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 22°C for a time-period of 24 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 37°C for a time-period of 1 to 5 min.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 1 to 5 min at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 1 to 5 min at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 37°C for a time-period of 5 to 15 min.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 5 to 15 min at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 5 to 15 min at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 37°C for a time-period of 15 to 30 min.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 15 to 30 min at pH 7.5. In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 15 to 30 min at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 37°C for a time-period of 30 to 60 min.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 30 to 60 min at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 30 to 60 min at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 30 to 60 min at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC at a temperature of 37°C for a time-period of 30 to 60 min at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 37°C for a time-period of 90 min.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 90 min at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 90 min at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 90 min at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC at a temperature of 37°C for a time-period of 90 min at pH 4.5. In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 37°C for a time-period of 120 min.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 120 min at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 120 min at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 120 min at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC at a temperature of 37°C for a time-period of 120 min at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 37°C for a time-period of 3 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 3 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 3 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 3 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM CuSC at a temperature of 37°C for a time-period of 3 hours at pH 4.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 37°C for a time-period of 4 hours. In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 4 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSCU and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 4 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 37°C for a time-period of 5 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 5 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 5 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with at least one destabilizing agent with at least one oxidizing agent with at least one transition metal ion and with at least one antioxidant at a temperature of 37°C for a time-period of 6 hours.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.1 to 0.5 mM SDS with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 6 hours at pH 7.5.

In one or more exemplary embodiments, the immunogenic composition can be produced with 0.5 to 2 M urea with 0.1 to 10 mM H2O2 with 0.01 to 0.1 mM FeSC and with 0.5 to 2 mM ascorbic acid at a temperature of 37°C for a time-period of 6 hours at pH 7.5.

Ionic detergent / oxidizing agent / transition metal ion

In one embodiment one or more destabilizing agents such as Sodium Dodecyl Sulfate (SDS), sodium cholate, sodium deoxycholate, sodium lauroyl sarcosinate, dioctyl sulfosuccinate, cetyltrimethylammonium bromide or other well known ionic detergents could be used in combination with one or more oxidizing agents such as hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4-methylbenzenesulfonamide sodium salt, dioxaneperoxide or other well known oxidizing agents and in combination with one or more transition metal ions such as those from ferrous, ferric, cuprous, cupric, silver, cobalt, chromium or other known transition metal ions. SDS / oxidizing agent / transition metal ion

In one preferred embodiment SDS was used as the destabilizing agent in combination with one or more oxidizing agents such as hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4-methylbenzenesulfonamide sodium salt, dioxaneperoxide or other well known oxidizing agents and in combination with one or more transition metal ions such as those from ferrous, ferric, cuprous, cupric, silver, cobalt, chromium or other known transition metal ions.

In yet another preferred embodiment SDS at a concentration of 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.40 mM, 0.6 mM, 0.75 mM, 1 mM, 1 .5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM was used as the destabilizing agent in combination with one or more oxidizing agents such as hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4-methylbenzenesulfonamide sodium salt, dioxaneperoxide or other well known oxidizing agents and in combination with one or more transition metal ions such as those from ferrous, ferric, cuprous, cupric, silver, cobalt, chromium or other known transition metal ions.

In one more preferred embodiment SDS was used as the destabilizing agent in combination with hydrogen peroxide (H2O2) as the oxidizing agent and in combination with one or more transition metal ions such as those from ferrous, ferric, cuprous, cupric, silver, cobalt, chromium or other known transition metal ions.

SDS / hydrogen peroxide / transition metal ion

In yet another more preferred embodiment SDS at a concentration of 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.40 mM, 0.6 mM, 0.75 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM,

3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM was used as the destabilizing agent in combination with (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1 .5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM as the oxidizing agent and in combination with one or more transition metal ions such as those from ferrous, ferric, cuprous, cupric, silver, cobalt, chromium or other known transition metal ions.

SDS / hydrogen peroxide / ferrous sulfate

In one most preferred embodiment SDS at a concentration of 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.40 mM, 0.6 mM, 0.75 mM, 1 mM, 1 .5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM was used as the destabilizing agent in combination with hydrogen peroxide (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM as the oxidizing agent and in combination with a ferrous transition metal ion such as from ferrous sulfate at a concentration of 0.001 mM, 0.005 mM, 0.01 mM, 0.015 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.5 mM, 0.06 mM, 0.07 mM, 0 08 mM, 0 9 mM, 0 1 mM, 0.15 mM, 0 175 mM, 0.2 mM, 0 3 mM, 0 4 mM, or 0 5 mM

SDS / hydrogen peroxide / ferric sulfate

In one most preferred embodiment SDS at a concentration of 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.40 mM, 0.6 mM, 0.75 mM, 1 mM, 1 .5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM was used as the destabilizing agent in combination with hydrogen peroxide (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM,

2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM as the oxidizing agent and in combination with a ferric transition metal ion such as from ferric sulfate at a concentration of 0.001 mM, 0.005 mM, 0.01 mM, 0.015 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.5 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.9 mM, 0.1 mM, 0.15 mM, 0.175 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM.

SDS / hydrogen peroxide / copper(ll)sulfate

2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM as the oxidizing agent and in combination with a copper transition metal ion such as from copper(ll)sulfate at a concentration of 0.001 mM, 0.005 mM, 0.01 mM, 0.015 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.5 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.9 mM, 0.1 mM, 0.15 mM, 0.175 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM.

SDS / oxidizing agent

According to another embodiment TcdA and/or TcdB were chemically inactivated for a period of time sufficient to reduce the cytotoxicity more than 3 log-io relative to native toxin. Protein concentrations of around 0.1 to 10 mg/ml, preferably 0.2 to 2 mg/ml, more preferably 0.3 to 1 mg/ml, most preferably 0.3 mg/ml were used in a suitable buffer such as TRIS, PBS, HEPES or others well known in the art. The reaction pH could be varied from about pH 4 to 10, preferably pH 4 to 8, more preferably pH 4.5 to 7.5, most preferably pH 7.5 at a temperature between 0 to 50°C, preferably 4 to 37°C, more preferably 4 to 22°C, most preferably 4°C for 1 minute to 24 hours, preferably 1 minute to 7 days, preferably 30 minutes to 48 hours, more preferably 60 minutes to 24 hours, most preferably 24 hours. Chemical inactivation of the toxins was achieved by using one or more destablizing agents in combination with an oxidizing agent.

In one preferred embodiment SDS was used as the destabilizing agent in combination with one or more oxidizing agents such as hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4-methylbenzenesulfonamide sodium salt, dioxaneperoxide or other well known oxidizing agents.

3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM was used as the destabilizing agent in combination with one or more oxidizing agents such as hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4-methylbenzenesulfonamide sodium salt, dioxaneperoxide or other well known oxidizing agents.

SDS / hydrogen peroxide

3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM was used as the destabilizing agent in combination with hydrogen peroxide (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM,

1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM as the oxidizing agent.

Chaotropic agent / oxidizing agent / transition metal ion

In one embodiment one or more destabilizing agents such as urea, thiourea, guanidinium chloride, ethanol, n-butanol, lithium perchlorate, lithium acetate, phenol, 2-propanol and other well known chaotropic agents could be used in combination with one or more oxidizing agents such as hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen , ozone, N-chloro-4- methylbenzenesulfonamide sodium salt, dioxaneperoxide or other well known oxidizing agents and in combination with one or more transition metal ions such as those from ferrous, ferric, cuprous, cupric, silver, cobalt, chromium or other known transition metal ions.

Protein concentrations of around 0.1 to 10 mg/ml, preferably 0.2 to 2 mg/ml, more preferably 0.3 to 1 mg/ml were used in a suitable buffer such as TRIS, PBS, HEPES or others well known in the art. The reaction pH could be varied from about pH 4 to 10, preferably pH 4 to 8, more preferably pH 4.5 to 7.5, most preferably pH 7.5 at a temperature between 0 to 50°C, preferably 4 to 37°C, more preferably 4 to 22°C, most preferably 4°C for 1 minute to 7 days, preferably 30 minutes to 48 hours, more preferably 60 minutes to 24 hours, most preferably 24 hours. Chemical inactivation of the proteins was achieved by using one or more chaotropic agents in combination with an oxidizing agent and a transition metal ion.

Urea / oxidizing agent / transition metal ion

In one preferred embodiment urea was used as the destabilizing agent in combination with one or more oxidizing agents such as hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4-methylbenzenesulfonamide sodium salt, dioxaneperoxide or other well known oxidizing agents and in combination with one or more transition metal ions such as those from ferrous, ferric, cuprous, cupric, silver, cobalt, chromium or other known transition metal ions.

In yet another preferred embodiment urea at a concentration of 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.75 M, 1 M, 1.25 M, 1.5 M, 1.75 M, 2M, 2.5 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M was used as the destabilizing agent in combination with one or more oxidizing agents such as hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4-methylbenzenesulfonamide sodium salt, dioxaneperoxide or other well known oxidizing agents and in combination with one or more transition metal ions such as those from ferrous, ferric, cuprous, cupric, silver, cobalt, chromium or other known transition metal ions.

Urea / hydrogen peroxide / transition metal ion

In yet another more preferred embodiment urea at a concentration of 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.75 M, 1 M, 1.25 M, 1.5 M, 1.75 M, 2M, 2.5 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M was used as the destabilizing agent in combination with hydrogen peroxide (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM,

7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM as the oxidizing agent and in combination with one or more transition metal ions such as those from ferrous, ferric, cuprous, cupric, silver, cobalt, chromium or other known transition metal ions.

Urea / hydrogen peroxide / ferrous sulfate

In one preferred embodiment urea at a concentration of 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.75 M,

1 M, 1.25 M, 1.5 M, 1.75 M, 2M, 2.5 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M was used as the destabilizing agent in combination with hydrogen peroxide (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or

8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM as the oxidizing agent and in combination with a ferrous transition metal ion such as from ferrous sulfate at a concentration of 0.001 mM, 0.005 mM, 0.01 mM, 0.015 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.5 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.9 mM, 0.1 mM, 0.15 mM, 0.175 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM.

Urea / hydrogen peroxide / ferric sulfate

1 M, 1.25 M, 1.5 M, 1.75 M, 2M, 2.5 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M was used as the destabilizing agent in combination with hydrogen peroxide (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM as the oxidizing agent and in combination with a ferric transition metal ion such as from ferric sulfate at a concentration of 0.001 mM, 0.005 mM, 0.01 mM, 0.015 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.5 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.9 mM, 0.1 mM, 0.15 mM, 0.175 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM.

Urea / hydrogen peroxide / copper(ll)sulfate

1 M, 1.25 M, 1.5 M, 1.75 M, 2M, 2.5 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M was used as the destabilizing agent in combination with hydrogen peroxide (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM as the oxidizing agent and in combination with a copper transition metal ion such as from copper(ll)sulfate at a concentration of 0.001 mM, 0.005 mM, 0.01 mM, 0.015 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.5 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.9 mM, 0.1 mM, 0.15 mM, 0.175 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM.

Urea / hydrogen peroxide

In one preferred embodiment urea was used as the destabilizing agent in combination with one or more oxidizing agents such as hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4-methylbenzenesulfonamide sodium salt, dioxaneperoxide or other well known oxidizing agents.

In yet another more preferred embodiment urea at a concentration of 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.75 M, 1 M, 1.25 M, 1.5 M, 1.75 M, 2M, 2.5 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M was used as the destabilizing agent in combination with hydrogen peroxide (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM as the oxidizing agent.

Ionic detergent / chaotropic agent / oxidizing agent / transition metal ion

In one embodiment one or more destabilizing agents from the group consisting of ionic detergents such as Sodium Dodecyl Sulfate (SDS), sodium cholate, sodium deoxycholate, sodium lauroyl sarcosinate, di octyl sulfosuccinate, cetyltrimethylammonium bromide or other well known ionic detergents could be used in combination with one or more chaotropic agents such as urea, thiourea, guanidinium chloride, ethanol, n-butanol, lithium perchlorate, lithium acetate, phenol, 2- propanol and other well known chaotropic agents in combination with one or more oxidizing agents such as hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4- methylbenzenesulfonamide sodium salt, dioxaneperoxide or other well known oxidizing agents and in combination with one or more transition metal ions such as those from ferrous, ferric, cuprous, cupric, silver, cobalt, chromium or other known transition metal ions.

SDS / urea / hydrogen peroxide / ferrous sulfate

In one preferred embodiment SDS at a concentration of 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.40 mM, 0.6 mM, 0.75 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM was used in combination with urea at a concentration of 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.75 M, 1 M, 1.25 M, 1.5 M, 1.75 M, 2M, 2.5 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M in combination with hydrogen peroxide (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM in combination with ferrous sulfate at a concentration of 0.001 mM, 0.005 mM, 0.01 mM, 0.015 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.5 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.9 mM, 0.1 mM, 0.15 mM, 0.175 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM.

SDS / urea / hydrogen peroxide / ferric sulfate

In one preferred embodiment SDS at a concentration of 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.40 mM, 0.6 mM, 0.75 mM, 1 mM, 1 .5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM was used in combination with urea at a concentration of 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.75 M, 1 M, 1.25 M, 1.5 M, 1.75 M, 2M, 2.5 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M in combination with hydrogen peroxide (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM in combination with ferric sulfate at a concentration of 0.001 mM, 0.005 mM, 0.01 mM, 0.015 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.5 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.9 mM, 0.1 mM, 0.15 mM, 0.175 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM.

SDS / urea / hydrogen peroxide / copper(ll)sulfate

In one preferred embodiment SDS at a concentration of 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.40 mM, 0.6 mM, 0.75 mM, 1 mM, 1 .5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM was used in combination with urea at a concentration of 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.75 M, 1 M, 1.25 M, 1.5 M, 1.75 M, 2M, 2.5 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M in combination with hydrogen peroxide (H2O2) at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM or 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM in combination with copper(ll)sulfate at a concentration of 0.001 mM,

0.005 mM, 0.01 mM, 0.015 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.5 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.9 mM, 0.1 mM, 0.15 mM, 0.175 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM.

In one or more exemplary embodiments, the protein is contacted with a solution comprising at least one destabilizing agent; at least one oxidizing agent; and at least one transition metal ion during a time-period of 1 min, or 5 min, or 10, min, or 20 min, or 30 min, or 45 min, or 60 min, or 75 min, or 90 min, or 105 min, or 120 min, or 135 min, or 150 min, or 165 min, or 180 min. The time-period could also be 200 min, 240 min, 300 min, or 360 min. It could also be 10 hours or up to 24 hours.

In one or more exemplary embodiments, Ted A and/or TcdB is contacted with a solution comprising at least one destabilizing agent; at least one oxidizing agent; at least one transition metal ion; and at least one antioxidant during a time-period of 1 min, or 5 min, or 10, min, or 20 min, or 30 min, or 45 min, or 60 min, or 75 min, or 90 min, or 105 min, or 120 min, or 135 min, or 150 min, or 165 min, or 180 min. The time-period could also be 200 min, 240 min, 300 min, or 360 min. It could also be 24 hours, 36 hours, 48 hours, 60 hours, 72 hours or up to 7 days.

In one or more exemplary embodiments, the protein is contacted with a solution comprising at least one destabilizing agent; and at least one oxidizing agent during a time-period of 1 min, or 5 min, or 10, min, or 20 min, or 30 min, or 45 min, or 60 min, or 75 min, or 90 min, or 105 min, or 120 min, or 135 min, or 150 min, or 165 min, or 180 min. The time-period could also be 200 min, 240 min, 300 min, or 360 min. It could also be 10 hours or up to 24 hours.

In one or more exemplary embodiments, the step of contacting the protein with a solution comprising at least one destabilizing agent; at least one oxidizing agent; and at least one transition metal ion is conducted at pH 6, or pH 6.5, or pH 7, or pH 7.5, or pH 8, or pH 8.5.

In one or more exemplary embodiments, the step of contacting the protein with a solution comprising at least one destabilizing agent; at least one oxidizing agent is conducted at pH 6, or pH 6.5, or pH 7, or pH 7.5, or pH 8, or pH 8.5. In one or more exemplary embodiments, the step of contacting TcdA and/or TcdB with a solution comprising at least one destabilizing agent; at least one oxidizing agent; at least one transition metal ion; and at least one antioxidant is conducted at pH 6, or pH 6.5, or pH 7, or pH 7.5, or pH 8, or pH 8.5.

According to one embodiment the step of contacting the protein with a solution comprising at least one destabilizing agent; at least one oxidizing agent; and at least one transition metal ion is conducted with a protein concentration of 0.1 mg/ml, or 0.2 mg/ml, or 0.3 mg/ml, or 0.4 mg/ml, or 0.5 mg/ml, or 0.6 mg/ml, or 0.7 mg/ml, or 0.8 mg/ml, or 0.9 mg/ml, or 1 mg/ml, or 1.25 mg/ml, or

1.5 mg/ml, or 1.75 mg/ml, or 2 mg/ml, or 2.25 mg/ml, or 2.5 mg/ml, or 3 mg/ml, or 4 mg/ml, or 5 mg/ml, or 6 mg/ml, or 7 mg/mi, or 8 mg/ml, or 9 mg/ml, or 10 mg/ml.

In one or more exemplary embodiments, the step of contacting TcdA and/or TcdB with a solution comprising at least one destabilizing agent; at least one oxidizing agent; at least one transition metal ion; and at least one antioxidant is conducted with a protein concentration of 0.1 mg/ml, or 0.2 mg/ml, or 0.3 mg/ml, or 0.4 mg/ml, or 0.5 mg/ml, or 0.6 mg/ml, or 0.7 mg/ml, or 0.8 mg/ml, or 0.9 mg/ml, or 1 mg/ml, or 1.25 mg/ml, or 1.5 mg/ml, or 1 .75 mg/ml, or 2 mg/ml, or 2.25 mg/ml, or

2.5 mg/ml, or 3 mg/ml, or 4 mg/ml, or 5 mg/ml, or 6 mg/ml, or 7 mg/ml, or 8 mg/ml, or 9 mg/ml, or 10 mg/ml.

Protein

In one or more exemplary embodiments, the protein is a toxic protein.

In one or more exemplary embodiments, the protein is a recombinant protein.

In one or more exemplary embodiments, the protein is a bacterial toxin.

In one or more exemplary embodiments, the bacterial toxin is from a gram-positive bacterium.

In one or more exemplary embodiments, the bacterial toxin is from a gram-negative bacterium.

In one or more exemplary embodiments, the bacterial toxin is from a bacteria of genera

Clostridioides, Clostridium, Bacillus, Haemophilus, Corynebacterium, Bordetella, Escherichia, Staphylococcus, Vibrio, Pseudomonas, Neisseria, Mycobacterium, Acinetobacter, Campylobacter, Enterococcus, Salmonella, Streptococcus, or the family of Enterobactehaceae, or from Shigella or Yersinia.

In one or more exemplary embodiments, the bacteria of genera Clostridioides is Clostridioides difficile, the bacteria of genera Clostridium is Clostridium tetani, Clostridium perfringens,

Clostridium sordellii, Clostridium novyi, or Clostridium botulinum, the bacteria of genera Bacillus is Bacillus anthracis or Bacillus cereus, the bacteria of genera Haemophilus is Haemophilus influenzae, the bacteria of genera Corynebacterium is Corynebacterium diphtheria, the bacteria of genera Bordetella is Bordetella pertussis, the bacteria of genera Escherichia is Escherichia coli, the bacteria of genera Staphylococcus is Staphylococcus aureus or Staphylococcus epidermidis, the bacteria of genera Vibrio is Vibrio cholerae, the bacteria of genera Pseudomonas is

Pseudomonas aeruginosa, the bacteria of genera Neisseria is Neisseria meningitidis or Neisseria gonorrhoeae, the bacteria of genera Mycobacterium is Mycobacterium tuberculosis, the bacteria of genera Acinetobacter is Acinetobacter baumannii, the bacteria of genera Campylobacter is Campylobacter jejuni, the bacteria of genera Enterococcus is Enterococcus faecium or

Enterococcus faecalis, the bacteria of genera Salmonella is Salmonella enterica Typhi, the bacteria of genera Streptococcus is Streptococcus pneumoniae, Group A Streptococcus, Group B Streptococcus, the bacteria of genera Shigella is Shigella dysenteriae, Shigella flexneri, Shigella boydii or Shigella sonnei, the bacteria of genera Yersnia is Yersnia pestis or Yersinia

enterocolitica.

In one or more exemplary embodiments, the bacterial toxin is Toxin A (Ted A) or Toxin B (TcdB) from Clostridioides difficile, tetanus toxin from C. tetani, alpha toxin, beta toxin, epsilon toxin or iota toxin from C. perfringens, lethal toxin (TcsL) and/or haemorrhagic toxin (TcsH) from C. sordellii, alpha toxin, beta toxin, gamma toxin, epsilon toxin and zeta toxin from C. novyi, botulinum toxin A (BoNT A), botulinum toxin B (BoNT B), botulinum toxin C (BoNT C), botulinum toxin D (BoNT D), botulinum toxin E (BoNT E), botulinum toxin F (BoNT F), botulinum toxin G (BoNT G) from C. botulinum, anthrax toxin from B. anthracis, emetic toxin (ETE), enterotoxins Nhe, HBL or EntK from B. cereus, diphtheria toxin from C. diphtheria, pertussis toxin from B. pertussis, shiga toxin, shiga- like verotoxin, heat-stable enterotoxin, heat-labile enterotoxin from E. coli, alpha-toxin, beta-toxin, delta-toxin and toxic shock syndrome toxin (TSST) from S. aureus, cholera toxin, cholix toxin from V. cholerae, exotoxin A, exoenzyme S, exoenzyme U from P. aeruginosa, MafB toxin from N. meningitidis, tuberculosis necrotizing toxin (TNT) from M. tuberculosis, cytolethal distending toxin from C. jejuni, typhoid toxin from S. Typhi, pneumolysin toxin from S. pneumoniae, ShET1 toxin, ShET2 toxin from S. flexneri, shiga toxin from S. dysenteriae, YitA, YitB, YitC, YipA, YipB from Y. pestis or yersinia stable toxin from Y. enterocolitica, SLO, SLS, SpyCEP, SpeB and SpeA from Group A Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is Toxin (Ted A) and Toxin B (TcdB) from Clostridioides difficile.

In one or more exemplary embodiments, the bacterial toxin is Toxin (Ted A) from Clostridioides difficile.

In one or more exemplary embodiments, the bacterial toxin is Toxin B (TcdB) from Clostridioides difficile.

In one or more exemplary embodiments, the bacterial toxin is tetanus toxin from C. tetani. In one or more exemplary embodiments, the bacterial toxin is alpha toxin, beta toxin, epsilon toxin and/or iota toxin from C. perfringens.

In one or more exemplary embodiments, the bacterial toxin is alpha toxin from C. perfringens.

In one or more exemplary embodiments, the bacterial toxin is beta toxin from C. perfringens.

In one or more exemplary embodiments, the bacterial toxin is epsilon from C. perfringens.

In one or more exemplary embodiments, the bacterial toxin is iota toxin from C. perfringens.

In one or more exemplary embodiments, the bacterial toxin is lethal toxin (TcsL) and haemorrhagic toxin (TcsH) from C. sordellii.

In one or more exemplary embodiments, the bacterial toxin is lethal toxin (TcsL) from C. sordellii.

In one or more exemplary embodiments, the bacterial toxin is haemorrhagic toxin (TcsH) from C. sordellii.

In one or more exemplary embodiments, the bacterial toxin is alpha toxin, beta toxin, gamma toxin, epsilon toxin and zeta toxin from C. novyi.

In one or more exemplary embodiments, the bacterial toxin is alpha toxin from C. novyi.

In one or more exemplary embodiments, the bacterial toxin is beta toxin from C. novyi.

In one or more exemplary embodiments, the bacterial toxin is gamma toxin from C. novyi.

In one or more exemplary embodiments, the bacterial toxin is epsilon toxin from C. novyi.

In one or more exemplary embodiments, the bacterial toxin is zeta toxin from C. novyi.

In one or more exemplary embodiments, the bacterial toxin is tetanus toxin from C. tetani.

In one or more exemplary embodiments, the bacterial toxin is botulinum toxin A (BoNT A), botulinum toxin B (BoNT B), botulinum toxin C (BoNT C), botulinum toxin D (BoNT D), botulinum toxin E (BoNT E), botulinum toxin F (BoNT F) and botulinum toxin G (BoNT G) from C. botulinum.

In one or more exemplary embodiments, the bacterial toxin is botulinum toxin A (BoNT A) and botulinum toxin B (BoNT B) from C. botulinum.

In one or more exemplary embodiments, the bacterial toxin is botulinum toxin A (BoNT A) from C. botulinum.

In one or more exemplary embodiments, the bacterial toxin is botulinum toxin B (BoNT B) from C. botulinum.

In one or more exemplary embodiments, the bacterial toxin is botulinum toxin C (BoNT C), botulinum toxin D (BoNT D) and botulinum toxin G (BoNT G) from C. botulinum. In one or more exemplary embodiments, the bacterial toxin is botulinum toxin C (BoNT C) from C. botulinum.

In one or more exemplary embodiments, the bacterial toxin is botulinum toxin D (BoNT D) from C. botulinum.

In one or more exemplary embodiments, the bacterial toxin is botulinum toxin G (BoNT G) from C. botulinum.

In one or more exemplary embodiments, the bacterial toxin is botulinum toxin E (BoNT E) and botulinum toxin F (BoNT F) from C. botulinum.

In one or more exemplary embodiments, the bacterial toxin is botulinum toxin E (BoNT E) from C. botulinum.

In one or more exemplary embodiments, the bacterial toxin is botulinum toxin F (BoNT F) from C. botulinum.

In one or more exemplary embodiments, the bacterial toxin is anthrax toxin from B. anthracis.

In one or more exemplary embodiments, the bacterial toxin is emetic toxin (ETE), enterotoxins

Nhe, HBL and EntK from B. cereus.

In one or more exemplary embodiments, the bacterial toxin is emetic toxin (ETE) from B. cereus.

In one or more exemplary embodiments, the bacterial toxin is enterotoxin Nhe from B. cereus.

In one or more exemplary embodiments, the bacterial toxin is enterotoxin HBL from B. cereus.

In one or more exemplary embodiments, the bacterial toxin is enterotoxin EntK from B. cereus.

In one or more exemplary embodiments, the bacterial toxin is diphtheria toxin from C. diphtheria.

In one or more exemplary embodiments, the bacterial toxin is pertussis toxin from B. pertussis.

In one or more exemplary embodiments, the bacterial toxin is shiga toxin, shiga-like verotoxin, heat-stable enterotoxin, heat-labile enterotoxin from E. coli.

In one or more exemplary embodiments, the bacterial toxin is shiga toxin and shiga-like verotoxin from E. coli.

In one or more exemplary embodiments, the bacterial toxin is shiga toxin from E. coli.

In one or more exemplary embodiments, the bacterial toxin is shiga-like verotoxin from E. coli.

In one or more exemplary embodiments, the bacterial toxin is heat-stable enterotoxin and heat- labile enterotoxin from E. coli.

In one or more exemplary embodiments, the bacterial toxin is heat-stable enterotoxin from E. coli. In one or more exemplary embodiments, the bacterial toxin is heat-labile enterotoxin from E. coli.

In one or more exemplary embodiments, the bacterial toxin is alpha-toxin, beta-toxin and delta- toxin and toxic shock syndrome toxin (TSST) from S. aureus.

In one or more exemplary embodiments, the bacterial toxin is alpha-toxin from S. aureus.

In one or more exemplary embodiments, the bacterial toxin is beta-toxin from S. aureus.

In one or more exemplary embodiments, the bacterial toxin is delta-toxin from S. aureus.

In one or more exemplary embodiments, the bacterial toxin is toxic shock syndrome toxin (TSST) from S. aureus.

In one or more exemplary embodiments, the bacterial toxin is cholera toxin and cholix toxin from V. cholerae.

In one or more exemplary embodiments, the bacterial toxin is cholera toxin from V. cholerae.

In one or more exemplary embodiments, the bacterial toxin is cholix toxin from V. cholerae.

In one or more exemplary embodiments, the bacterial toxin is exotoxin A, exoenzyme S and exoenzyme U from P. aeruginosa.

In one or more exemplary embodiments, the bacterial toxin is exotoxin A from P. aeruginosa.

In one or more exemplary embodiments, the bacterial toxin is exoenzyme S from P. aeruginosa.

In one or more exemplary embodiments, the bacterial toxin is exoenzyme U from P. aeruginosa.

In one or more exemplary embodiments, the bacterial toxin is MafB toxin from N. meningitidis.

In one or more exemplary embodiments, the bacterial toxin is tuberculosis necrotizing toxin (TNT) from M. tuberculosis.

In one or more exemplary embodiments, the bacterial toxin is cytolethal distending toxin from C. jejuni.

In one or more exemplary embodiments, the bacterial toxin is typhoid toxin from S. Typhi.

In one or more exemplary embodiments, the bacterial toxin is pneumolysin toxin from S.

pneumoniae.

In one or more exemplary embodiments, the bacterial toxin is ShET1 toxin and ShET2 toxin from S. flexneri.

In one or more exemplary embodiments, the bacterial toxin is ShET1 toxin from S. flexneri.

In one or more exemplary embodiments, the bacterial toxin is ShET2 toxin from S. flexneri.

In one or more exemplary embodiments, the bacterial toxin is shiga toxin from S. dysenteriae. In one or more exemplary embodiments, the bacterial toxin is YitA, YitB, YitC, YipA and YipB from Y. pestis.

In one or more exemplary embodiments, the bacterial toxin is YitA, YitB and YitC from Y. pestis.

In one or more exemplary embodiments, the bacterial toxin is YitA from Y. pestis.

In one or more exemplary embodiments, the bacterial toxin is YitB from Y. pestis.

In one or more exemplary embodiments, the bacterial toxin is YitC from Y. pestis.

In one or more exemplary embodiments, the bacterial toxin is YipA and YipB from Y. pestis.

In one or more exemplary embodiments, the bacterial toxin is YipA from Y. pestis.

In one or more exemplary embodiments, the bacterial toxin is YipB from Y. pestis.

In one or more exemplary embodiments, the bacterial toxin is yersinia stable toxin from Y.

enterocolitica.

In one or more exemplary embodiments, the bacterial toxin is SLO, SLS, SpyCEP, SpeB and SpeA from Group A Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is SLO from Group A Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is SLS from Group A Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is SpyCEP from Group A

Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is SpeB from Group A Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is SpeA from Group A Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is beta-hemolysin, C5a peptidase, CAMP factor, Oligopeptidase, Hyaluronate lyase and carbohydrate exotoxin CM101 from Group B Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is beta-hemolysin from Group B Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is C5a peptidase from Group B Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is CAMP factor from Group B

Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is Oligopeptidase from Group B Streptococcus. In one or more exemplary embodiments, the bacterial toxin is Hyaluronate from Group B Streptococcus.

In one or more exemplary embodiments, the bacterial toxin is carbohydrate exotoxin CM101 from Group B Streptococcus.

In one or more exemplary embodiments, the protein is from a fungus.

In one or more exemplary embodiments, the protein is from a virus.

In one or more exemplary embodiments, the virus is from the family of Adenoviridae_^

In one or more exemplary embodiments, the virus is from the family of Papovaviridae_^

In one or more exemplary embodiments, the virus is from the family of Parvoviridae_^

In one or more exemplary embodiments, the virus is from the family of Herpesviridae_^

In one or more exemplary embodiments, the virus is from the family of Poxviridae_^

In one or more exemplary embodiments, the virus is from the family of Anelloviridae_^

In one or more exemplary embodiments, the virus is from the family of Pleolipoviridae_^

In one or more exemplary embodiments, the virus is from the family of Reoviridae_L

In one or more exemplary embodiments, the virus is from the family of Picornaviridae_^

In one or more exemplary embodiments, the virus is from the family of Caliciviridae_^

In one or more exemplary embodiments, the virus is from the family of Togaviridae_^

In one or more exemplary embodiments, the virus is from the family of Arenaviridae_^

In one or more exemplary embodiments, the virus is from the family of Flaviviridae_^

In one or more exemplary embodiments, the virus is from the family of Orthomyxoviridae_^

In one or more exemplary embodiments, the virus is from the family of Paramyxoviridae_^

In one or more exemplary embodiments, the virus is from the family of Bunyaviridae_^

In one or more exemplary embodiments, the virus is from the family of Rhabdoviridae_^

In one or more exemplary embodiments, the virus is from the family of Filoviridae_^

In one or more exemplary embodiments, the virus is from the family of Coronaviridae_L

In one or more exemplary embodiments, the virus is from the family of Astroviridae_^

In one or more exemplary embodiments, the virus is from the family of Bornaviridae_^

In one or more exemplary embodiments, the virus is from the family of Arteriviridae_^ In one or more exemplary embodiments, the virus is from the family of Hepeviridae_^

In one or more exemplary embodiments, the virus is from the family of Retroviridae_^

In one or more exemplary embodiments, the virus is from the family of Caulimoviridae_^

In one or more exemplary embodiments, the virus is from the family of Hepadnaviridae_^

In one or more exemplary embodiments, the virus is Influenzavirus A.

In one or more exemplary embodiments, the virus is Influenzavirus B.

In one or more exemplary embodiments, the virus is Influenzavirus C.

In one or more exemplary embodiments, the virus is Ebola virus.

In one or more exemplary embodiments, the virus is Zika virus.

In one or more exemplary embodiments, the virus is Marburg virus.

In one or more exemplary embodiments, the virus is MERS-CoV.

In one or more exemplary embodiments, the virus is SARS-CoV.

In one or more exemplary embodiments, the virus is SARS-CoV-2.

In one or more exemplary embodiments, the virus is HIV.

In one or more exemplary embodiments, the protein is from a venom.

In one or more exemplary embodiments, the venom protein is a snake toxin such as ot- bungarotoxin, b-bungarotoxin, cobratoxin, crotoxin, erabutoxin, taicatoxin and textilotoxin, or a spider toxin such as agatoxin, atracotoxin, grammotoxin, latrotoxin, phoneutriatoxin, phrixotoxin and versutoxin or a scorpion toxin such as margatoxin, iberiotoxin and noxiustoxin.

In one or more exemplary embodiments, the protein is from a plant.

In one or more exemplary embodiments, the plant protein is ricin or abrin.

In one or more exemplary embodiments, the present invention provides a method for inactivation of proteins that is generally applicable to protein toxins to produce protein toxoids. In one or more exemplary embodiments, the protein is the bacterial toxins - Toxin A (TcdA) or Toxin B (TcdB) - from Clostridioides difficile.

In one or more exemplary embodiments, the protein concentration is from 0.1 to 10 mg/ml, preferably 0.2 to 2 mg/ml, more preferably 0.3 to 1 mg/ml, most preferably 0.5 mg/ml.

In one or more exemplary embodiments, the concentration of TcdA and/or TcdB is from 0.1 to 10 mg/ml, preferably 0.2 to 2 mg/ml, more preferably 0.3 to 1 mg/ml, most preferably 0.3 mg/ml. Stabilizing agents

Stabilizing agents may also be added before, during or after the inactivation reaction. Suitable stabilizing agents include NaCI, KCI, sorbitol, mannitol, arginine, glycine, glycerol, mannitol, gelatine and others well known in the art.

In one or more exemplary embodiments, NaCI at a concentration of 0.1 to 2 M is added to the reaction solution comprising, protein, destabilizing agent, oxidizing agent and transition metal ion.

In one or more exemplary embodiments, NaCI at a concentration of 0.1 to 2 M is added to the reaction solution comprising, protein, destabilizing agent, oxidizing agent, transition metal ion and antioxidant.

In one or more exemplary embodiments, NaCI at a concentration of 0.1 M, or 0.2 M, or 0.3 M, or 0.4 M is added to the reaction solution comprising, protein, destabilizing agent, oxidizing agent and transition metal ion.

In one or more exemplary embodiments, NaCI at a concentration of 0.1 M, or 0.2 M, or 0.3 M, or 0.4 M is added to the reaction solution comprising, protein, destabilizing agent, oxidizing agent and transition metal ion and antioxidant.

In one or more exemplary embodiments, NaCI at a concentration of 0.5 M, or 0.6 M, or 0.7 M, or 0.8 M is added to the reaction solution comprising, protein, destabilizing agent, oxidizing agent and transition metal ion.

In one or more exemplary embodiments, NaCI at a concentration of 0.5 M, or 0.6 M, or 0.7 M, or 0.8 M is added to the reaction solution comprising, protein, destabilizing agent, oxidizing agent and transition metal ion and antioxidant.

In one or more exemplary embodiments, NaCI at a concentration of 0.9 M, or 1 M, or 1.5 M, or 2M is added to the reaction solution comprising, protein, destabilizing agent, oxidizing agent and transition metal ion.

In one or more exemplary embodiments, NaCI at a concentration of 0.9 M, or 1 M, or 1.5 M, or 2 M is added to the reaction solution comprising, protein, destabilizing agent, oxidizing agent and transition metal ion and antioxidant.

In one or more exemplary embodiments, glycerol at a concentration of 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15% or 20% is added to the reaction solution comprising, protein, destabilizing agent, oxidizing agent and transition metal ion.

In one or more exemplary embodiments, glycerol at a concentration of 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15% or 20% is added to the reaction solution comprising, protein, destabilizing agent, oxidizing agent and transition metal ion and antioxidant. Lyophilizing

In one or more exemplary embodiments, the method further comprises lyophilizing the composition, thereby producing an immunogenic composition comprising an inactivated protein.

In one or more exemplary embodiments, the method further comprises lyophilizing the composition, thereby producing an immunogenic composition comprising inactivated TcdA and/or TcdB.

Immunogenic composition

In one or more exemplary embodiments of the present invention provides an immunogenic composition produced according to the method of the present invention.

In one or more exemplary embodiments of the present invention provides an immunogenic composition comprising inactivated TcdA and/or TcdB produced according to the method of the present invention.

Vaccine composition

In one or more exemplary embodiments of the present invention provides a vaccine composition comprising an immunogenic composition of the present invention.

In one or more exemplary embodiments, the vaccine composition further comprises an adjuvant and/or a pharmaceutically acceptable diluent or carrier.

Method of eliciting an immune response

In one or more exemplary embodiments of the present invention provides a method of eliciting an immune response against a protein, the method comprising preparing an immunogenic composition using a method according to the present disclosure; and administering the immunogenic composition to a human and/or animal subject, thereby eliciting an immune response against the protein in the subject.

In one or more exemplary embodiments, the present invention provides a method of eliciting an immune response against TcdA and/or TcdB, the method comprising preparing an immunogenic composition using a method according to the present disclosure; and administering the immunogenic composition to a human and/or animal subject, thereby eliciting an immune response against TcdA and/or TcdB in the subject.

Purification of native C. difficile TcdA and TcdB

TcdA and TcdB from Clostridioides difficile Ribotype 027 (NCTC 13366) was produced using the dialysis bag method. Briefly, 50 ml of overnight C. difficile culture in 24g/L tryptone, 12g/L yeast extract, 10g/L mannitol, 1 g/L glycerol medium was inoculated into 1 liter of sterile 0.9% saline in a dialysis bag suspended in 4 liters of 30g/L tryptone, 20g/L yeast extract, 1 g/L sodium thioglycolate medium. Media were prereduced with nitrogen and autoclaved before inoculation. Cultures were grown for 72 hours at 37 °C, centrifuged and dialyzed using a Quattro 1000

Ultrafiltration/Diafiltration with 50 kDa cutoff filters in 50 mM TRIS-HCI (pH 7.5) buffer. Separation of the exotoxins (TcdA and TcdB) from the dialyzed supernatants was achieved using a HiTrap Q anion-exchange column, integrated on a fast protein liquid chromatograph (FPLC). Both toxins were eluted with a linear 0 - 1 M NaCI gradient, with TcdA eluting around 150 - 200 mM NaCI, and TcdB around 400 - 450 mM NaCI. Fractions with protein sizes corresponding to either TcdA or TcdB on SDS-PAGE were pooled and further purified using a HiPrep 16/60 Sephacryl S-300 size- exclusion column. For final polishing, a high resolution anion-exchange MonoQ 10/100 GL column was used resulting in more than 90 % pure TcdA and TcdB, as seen in Fig. 1. Purified toxins were dialyzed thoroughly and transferred into a suitable buffer e.g. TRIS, PBS, HEPES or others well known in the art, and aliquoted. They were able to be stored in -80 °C for several months.

Chemical inactivation of C. difficile TcdA and TcdB

In one or more exemplary embodiments, after purification of TcdA and TcdB as described supra, the toxins were chemically inactivated for a period of time sufficient to reduce the cytotoxicity more than 2 log-io relative to native toxin. Protein concentrations of around 0.1 to 10 mg/ml, preferably 0.2 to 2 mg/ml, more preferably 0.3 to 1 mg/ml, most preferably 0.5 mg/ml were used in a suitable buffer such as TRIS, MOPS, PBS, HEPES, sodium acetate, citrate or others well known in the art. The reaction pH could be varied from about pH 6 to 8.5, preferably pH 6.5 to 8, more preferably pH 7 to 8, and most preferably pH 7.5 at a temperature between 0 to 55°C, preferably 20 to 40°C, more preferably 37°C for 1 minute to 24 hours, preferably 30 to 360 minutes, more preferably 60 to 180 minutes, most preferably 120 minutes. Chemical inactivation of the toxins was achieved by using above mentioned parameters including at least one destablizing agent in combination with at least one oxidizing agent and at least one transition metal ion.

In one or more exemplary embodiments, after purification of TcdA and TcdB as described supra, the toxins were chemically inactivated for a period of time sufficient to reduce the cytotoxicity more than 3 log-io relative to native toxin. Protein concentrations of around 0.1 to 10 mg/ml, preferably 0.2 to 2 mg/ml, more preferably 0.3 to 1 mg/ml, most preferably 0.3 mg/ml were used in a suitable buffer such as TRIS, PBS, HEPES or others well known in the art. The reaction pH could be varied from about pH 6 to 8.5, preferably pH 6.5 to 8, more preferably pH 7 to 8, and most preferably pH 7.5 at a temperature between 0 to 50°C, preferably 4 to 37°C, more preferably 4 to 22°C, most preferably 4°C for 1 minute to 7 days, preferably 30 minutes to 48 hours, more preferably 60 minutes to 24 hours, most preferably 24 hours. Chemical inactivation of the toxins was achieved by using above mentioned parameters including at least one destablizing agent in combination with at least one oxidizing agent, at least one transition metal ion and at least one antioxidant.

Removing destabilizing agents and other reaction components

When the inactivation of protein has reached the desired level, the reaction has to be quenched and excess destabilizing agents and other reaction components such as oxidizing agents, transition metal ions, antioxidants or other reaction additives are removed. Quenching agents

In one or more exemplary embodiments, the reaction rate can be slowed down or quenched by the addition of chelating agents such as ethylenediaminetetraacetate (EDTA), DTPA or other chemical quenching agents known in the art, or the reaction can be quenched by the addition of catalase, sodium thiosulfate, sodium sulfite, methanol, ethanol or other hydroxyl scavengers known in the art.

In one or more exemplary embodiments, EDTA was used as the reaction quenching agent at a concentration of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 4 mM or 5 mM, preferably 2 mM.

Non-ionic detergents

In one or more exemplary embodiments, non-ionic detergents such as polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), n-dodecyl b-D-maltoside (DDM), Brij 56, n-octyl-p-D-glucoside,

Nonidet P40, Triton X-100 or the like well known in the art were used to remove ionic destabilizing agents from the protein. Addition of non-ionic detergents mentioned supra and the like well known in the art can bind the destabilizing ionic detergents in the reaction mixture such as SDS or the like, and remove them from the protein solution with a subsequent dialysis or buffer exchange.

In one or more exemplary embodiments, polysorbate 20 (Tween 20) at a concentration of 0.1 to 20 mM, preferably 0.5 to 10 mM, more preferably 1 to 5 mM, most preferably 3 mM was added after the inactivation reaction was completed to the reaction solution comprising, protein, ionic detergent, oxidizing agent and transition metal ion in order to remove the ionic detergent.

In one or more exemplary embodiments, polysorbate 20 (Tween 20) at a concentration of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, or 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM was added after the inactivation reaction was completed to the reaction solution comprising, protein, ionic detergent, oxidizing agent and transition metal ion in order to remove the ionic detergent.

Polymers

In one or more exemplary embodiments, polymers such as a-cyclodextrin, b-cyclodextrin, g- cyclodextrin or the like well known in the art were used to remove destabilizing agents from the protein. Addition of polymers mentioned supra and the like well known in the art can bind the destabilizing ionic detergents in the reaction mixture such as SDS or the like, and remove them from the protein solution with a subsequent dialysis or buffer exchange.

In one or more exemplary embodiments, b-cyclodextrin at a concentration of 0.1 to 10 mM, preferably 0.3 to 6 mM, more preferably 0.5 to 4 mM was added after the inactivation reaction was completed to the reaction solution comprising, protein, ionic detergent, oxidizing agent and transition metal ion in order to remove the ionic detergent. In one or more exemplary embodiments, b-cyclodextrin at a concentration of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM,

3.5 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM was added after the inactivation reaction was completed to the reaction solution comprising, protein, ionic detergent, oxidizing agent and transition metal ion in order to remove the ionic detergent.

Removing agents

In one or more exemplary embodiments, the method further comprises removing the destabilizing agent and/or oxidizing agent and/or transition metal ion and/or antioxidant from the composition.

In one or more exemplary embodiments, the inactivation reaction may be quenched by removing the reaction components by gel filtration chromatography on a FPLC/HPLC system or on a medium such as Sephadex G-25, e.g. PD spintrap G-25, PD minitrap G-25 or the like.

In one or more exemplary embodiments, the inactivation reaction may be quenched and reaction components e.g. SDS and/or urea and/or oxidizing agents removed by dialysis or by centrifugal filter devices.

Lyophilization

In one or more exemplary embodiments, the inactivation reaction may be quenched by freeze drying or lyophilization.

In one or more exemplary embodiments, potassium chloride, potassium phosphate or other potassium salts known in the art are used to remove destabilizing agents such as SDS or the like from the reaction mixture by means of precipitation.

In one or more exemplary embodiments, trichloroacetic acid precipitation, cold acetone precipitation, chloroform-methanol-water (C/M/W) precipitation, ammonium sulfate precipitation or other precipitation techniques well known in the art are used to remove destabilizing agents, such as SDS or the like, from the reaction mixture by means of precipitation.

Aliphatic alcohols

In one or more exemplary embodiments, amphipathic cosolvents such as 2-methyl-2,4-pentanediol (MPD), 1 -propanol, 1 -butanol, 2-butanol, 2-methyl-1 -propanol , 2-methyl-2-butanol, 1 ,2-butanediol, 1 ,2-pentanediol, 1 ,6-hexanediol, 2,5-hexanediol, 2,4-dimethyl-2,4-pentanediol or other aliphatic alcohols are used to remove destabilizing agents, such as SDS or the like, from the protein and recover a native-like refolded structure.

Detergent removal kits

In one or more exemplary embodiments, detergent removing kits such as SDS-Out Precipitation Kit (ThermoFischer), Detergent removal spin column (Pierce), ProteoSpin detergent clean-up kit (Norgen biotek) and others well known in the art are used to remove destabilizing detergents, such as SDS or the like, from the protein solution.

In vitro cytotoxicity testing of inactivated toxins

Cytotoxicity testing of native and inactivated toxins was carried out using Vera kidney cell culture (5x10⁴ cells/mL) from Cercopithecus aethiops. As an alternative IMR90 cells, a human diploid lung fibroblast cell line or other cell lines well known in the art could be used. Briefly, 150 pL cell culture in DMEM, or other cell culture medium well known in the art, was added to each well in a 96-well microtiter plate and incubated in a HeraCell 150i CO2 incubator at 36.5 °C, 5% CO2 for 24 h prior to cytotoxicity testing. 10 pL of T cd A or TcdB toxin or toxoid (0.3 to 0.5 mg/ml) was added to the first well in each row, followed by serial dilution. Plates were incubated for 48 h, emptied for media and washed twice with 200 pL/well PBS. After washing, 200 pL/well 4 % formaldehyde was added followed by incubation at room temperature for 10 min, followed by another washing step. Finally, plates were stained using 0.1 % crystal violet (200 pL/well), incubated at room temperature for 10 min and washed gently with water.

In vitro epitope recognition by monoclonal antibodies

Chemical inactivation treatments may adversely affect key antigenic epitopes of the inactivated toxins. To evaluate the structural integrity of antigenic epitopes, neutralizing monoclonal antibody recognition of the inactivated toxins was tested by indirect ELISA. Maxisorp plates were coated with 1 pg/ml inactivated TcdA or TcdB in 100 pL (0.05 M Na2CC>3, 0.05 M NaHCC>3, pH 9.6) coating buffer and incubated at 5°C overnight. After incubation, wells were blocked with 1 % BSA in PBS- Tween20 (0.05% v/v) for 1 h at 37 °C. Equal volumes (100 pL) of each mouse monoclonal antibody in 0.5% BSA-PBS-Tween20 (0.05% v/v) were added in duplicates with a concentration of 0.25 pg/ml, followed by incubation for 1 h at 37 °C. HRP-conjugated rabbit anti-mouse IgG (1 :5000) in 100 pL 0.5% BSA-PBS-Tween20 (0.05% v/v) was added to each well, followed by incubation for 1 h at 37 °C. The antibody binding was visualized by the addition of 100 pL TMB PLUS2 substrate for up to 10 minutes, and the reaction was stopped by adding 100 pL of 0.2 M H2SO4. Absorbance was measured at 450 nm using a plate reader. Plates were washed 5 times with 250 pL washing buffer (PBS, pH 7.4, containing 0.05% (v/v) Tween 20) between each step. SDS-PAGE Analysis

Ted A and TcdB samples were visualized by reducing SDS-PAGE using 4-20% TGX Stain -free™ mini-protein gels (BIO-RAD, USA). 8 pL of protein sample (0.5-1 p M/well) was mixed with 8 pL of 2 x Laemmli Sample Buffer (BIO-RAD, USA) in the presence of 0.35 M b-mercaptoethanol and incubated for 30 min at room temperature. Electrophoresis was carried out using TGS SDS-Buffer (BIO-RAD, USA) for 30 min. at 200 V, 500 mA. 5 pL/well of BIORAD Precision Plus Protein Standard was used as a molecular weight marker. Preparation of adsorbed inactivated TcdA and/or TcdB

For use as antigens in a vaccine, inactivated proteins are generally adsorbed to a suitable adjuvant such as an aluminium adjuvant or other adjuvants known in the art. For the present invention, Alhydrogel was used, but other adsorbants such as aluminium hydroxide, aluminium phosphate, calcium phosphate hydroxide, or salts produced de novo or other classes of adjuvants could also be equally well employed. The choice of adjuvant is dependent on each specific protein and the type of the desired immune response, and can therefore be different for each protein. Described and used for the present invention is the method of adsorption of inactivated TcdA and/or TcdB to Alhydrogel (aluminium hydroxide gel), but the present invention is not dependent on this specific adjuvant nor on any other adjuvant. Proteins that are adsorbed to adjuvants have been shown to induce a stronger immune response in animals and humans, however this is not a necessity, and an inactivated protein by itself without adjuvant can also be immunogenic and may induce a strong and sufficient immune response. Between 10 and 200 pg of protein is adsorbed per milligram of aluminium. Generally, adsorption is performed in suitable buffers such as TRIS, PBS, HEPES or other buffers well known in the art, at a suitable pH between 6 to 8.5, preferably about pH 7.5, at temperatures between 0 to 25 °C preferably about 5 °C, with agitation. Adsorption time may be between 5 min to 48 hours preferably about 24 hours. Of course, other adsorbants, adjuvants, buffers, pH ranges, temperatures and adsorption times can be employed, as the adsorption of inactivated TcdA and/or TcdB to adjuvant in itself is not a critical factor for the present invention. ELISA quantification of serum antitoxin titer

To quantify the level of antitoxin in the serum of immunised animals, indirect ELISA was conducted on serum titrations. Maxisorp plates were coated with 1 pg/ml inactivated TcdA or TcdB in 100 pL (0.05 M Na2CC>3, 0.05 M NaHCC>3, pH 9.6) coating buffer and incubated at 5 °C overnight. After incubation, wells were blocked with 1% BSA in PBS for 1 h at 37 °C. Equal volumes (100 pL) of each serum in 0.5% BSA-PBS-Tween20 (0.05% v/v) were added in triplicates in a 3-fold serial dilution and incubated for 1 h at 37 °C. HRP-conjugated rabbit anti-mouse IgG (1 :5000) in 100 pL 0.5% BSA-PBS-Tween20 (0.05% v/v) was added to each well, followed by incubation for 1 h at 37 °C. The antibody binding was visualized by the addition of 100 pL TMB PLUS2 substrate for up to 15 min, and the reaction was stopped by adding 100 pL of 0.2 M H₂SO₄. Absorbance was measured at 450 nm using a plate reader. Plates were washed 5 times with 250 pL washing buffer (PBS, pH 7.4, containing 0.05% (v/v) Tween 20) between each step.

RESULTS

Example 1 : Inactivation of TcdA and TcdB with SDS (0.17 mM), H202 and FeS04

After purification, approximately 0.5 mg/ml native TcdA or TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters as shown in Table 1. The parameters for inactivation were an inactivation time of 2 hours at 37 °C using 0.17 mM SDS (0.005 w/v), 10 mM H2O2, 0.05 mM FeS0₄. The reaction was quenched by the addition of EDTA, Tween 20 and b-cyclodextrin to a final concentration of 2 imM, 3 imM and 1 imM respectively, and incubated at 20 °C for 30 min, and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using Amicon centrifugal filter device with 30 or 100k MW cut-off or a Sephadex G-25 column. After the inactivation reaction, the cytotoxic activity of Ted A or TcdB was reduced more than 6 log-io and 5.2 log-io respectively relative to corresponding native toxin, as shown in Table 2.

Table 1

Table 2

Example 1A: Inactivation of TcdA with SDS (0.35 mM), H2O2, FeS04 and ascorbate

Approximately 0.3 mg/ml native TcdA in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 1A. The parameters for inactivation were an inactivation time of 1 hour at 37 °C using 0.35 mM SDS (0.01 % w/v), 1 mM H2O2, 0.01 mM FeSC and 1 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using a dialysis membrane or Amicon centrifugal filter device with 30k MW cut-off. After the inactivation reaction, the cytotoxic activity of TcdA was reduced more than 5 log-io relative to native TcdA, as shown in Table 2A.

Table 1A

Table 2A

Example 2: Inactivation of TcdA and TcdB with SDS (0.17 mM), H2O2 and Fe2(S04)3 After purification, approximately 0.5 mg/ml native TcdA or TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters as shown in Table 3. The parameters for inactivation were an inactivation time of 2 hours at 37 °C using 0.17 mM SDS (0.005 w/v), 10 mM H2O2, 0.05 mM Fe₂(SC>₄)3. The reaction was quenched by the addition of EDTA, Tween 20 and b-cyclodextrin to a final concentration of 2 mM, 3 mM and 1 mM respectively, and incubated at 20 °C for 30 min, and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using Amicon centrifugal filter device with 30 or 100k MW cut-off or a Sephadex G-25 column. After the inactivation reaction, the cytotoxic activity of TcdA or TcdB was reduced more than 5 log-io and 5 log-io respectively relative to corresponding native toxin, as shown in Table 4. Table 3

Table 4

Example 2A: Inactivation of TcdA with SDS (0.5 mM), H2O2, FeS04 and ascorbate

Approximately 0.3 mg/ml native TcdA in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 3A. The parameters for inactivation were an inactivation time of 1 hour at 37 °C using 0.5 mM SDS (0.014% w/v), 3 mM H2O2, 0.01 mM FeSC and 1 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using a dialysis membrane or Amicon centrifugal filter device with 30k MW cut-off. After the inactivation reaction, the cytotoxic activity of TcdA was reduced more than 6 log-io relative to native TcdA, as shown in Table 4A. Table 3A

Table 4A

Example 3: Inactivation of TcdA and TcdB with SDS (0.17 mM), H2<02 and CuS04

After purification, approximately 0.5 mg/ml native TcdA or TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters as shown in Table 5. The parameters for inactivation were an inactivation time of 2 hours at 37 °C using 0.17 mM SDS (0.005 w/v), 10 mM H2O2, 0.05 mM CuSC The reaction was quenched by the addition of EDTA, Tween 20 and b-cyclodextrin to a final concentration of 2 mM, 3 mM and 1 mM respectively, and incubated at 20 °C for 30 min, and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using Amicon centrifugal filter device with 30 or 100k MW cut-off or a Sephadex G-25 column. After the inactivation reaction, the cytotoxic activity of TcdA or TcdB was reduced more than 6 log-io and 5.2 log-io respectively relative to corresponding native toxin, as shown in Table 6. Table 5

Table 6

Example 3A: Inactivation of TcdA with SDS (0.5 mM), H2O2, FeS04 and ascorbate at 22 °C

Approximately 0.3 mg/ml native TcdA in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 5A. The parameters for inactivation were an inactivation time of 1 hour at 22 °C using 0.5 mM SDS (0.014% w/v), 3 mM H2O2, 0.01 mM FeSC and 1 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using a dialysis membrane or Amicon centrifugal filter device with 30k MW cut-off. After the inactivation reaction, the cytotoxic activity of TcdA was reduced more than 6 log-io relative to native TcdA, as shown in Table 6A. Table 5A

Table 6A

Example 4: Inactivation of TcdA or TcdB with SDS (3.5 mM), H2O2 and FeS04

After purification, approximately 1 mg/ml native TcdA and TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters as shown in Table 7. The parameters used for inactivation were an inactivation time of 20 min and 30 min for TcdA and TcdB respectively at 37 °C using 3.5 mM SDS (0.1% w/v), 10 mM H2O2, 0.1 mM FeSC . The reaction was quenched by the addition of EDTA, Tween 20 and b-cyclodextrin to a final concentration of 2 mM, 6 mM and 10 mM respectively, and incubated at 20 °C for 30 min, and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using Amicon centrifugal filter device with 30 or 100k MW cut-off or a Sephadex G-25 column. After the inactivation reaction, the cytotoxic activity of TcdA or TcdB was reduced more than 6 logic and 7.2 logic, respectively relative to corresponding native toxin, see Table 8. Table 7

Table 8

Example 4A: Inactivation of TcdA with SDS (0.5 mM), H2O2, FeS04 and ascorbate at 4 °C

Approximately 0.5 mg/ml native TcdA in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 7A. The parameters for inactivation were an inactivation time of 24 hours at 4 °C using 0.5 mM SDS (0.014% w/v), 3 mM H2O2, 0.01 mM FeSC and 1 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using a dialysis membrane or Amicon centrifugal filter device with 30k MW cut-off. After the inactivation reaction, the cytotoxic activity of TcdA was reduced more than 5 log-io relative to native TcdA, as shown in Table 8A. Table 7 A

Table 8A

Example 5: Inactivation of TcdA or TcdB with SDS (0.35 mM), H2O2 and FeS04

After purification, approximately 0.3 mg/ml native TcdA or TcdB in 50 mM sodium acetate pH 4.5 was inactivated according to the parameters as shown in Table 9. The parameters used for inactivation were an inactivation time of 2 hours at 37 °C using 0.35 mM SDS (0.01% w/v), 5 mM H2O2, 0.05 mM FeSC . The reaction was quenched by the addition of EDTA, Tween 20 and b- cyclodextrin to a final concentration of 2 mM, 3 mM and 1 mM respectively, and incubated at 20 °C for 30 min, and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using Amicon centrifugal filter device with 30 or 100k MW cut-off or a Sephadex G-25 column. After the inactivation reaction, the cytotoxic activity of TcdA or TcdB was reduced more than 6 log 10 and 6 log-io respectively relative to corresponding native toxin, as shown in Table 10. Table 9

Table 10

Example 5A: Inactivation of TcdA with Urea (2 M), H2O2, FeS04 and ascorbate at 4 °C

Approximately 0.3 mg/ml native TcdA in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 9A. The parameters for inactivation were an inactivation time of 24 hours at 4 °C using 2 M Urea, 3 mM H2O2, 0.01 mM FeSC and 1 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using a dialysis membrane or Amicon centrifugal filter device with 30k MW cut-off. After the inactivation reaction, the cytotoxic activity of TcdA was reduced more than 5 log 10 relative to native TcdA, as shown in Table 10A. Table 9A

Table 10 A

Example 6: Inactivation of TcdA or TcdB with urea (2 M), H2O2 and FeS04

After purification, approximately 0.5 mg/ml native TcdA or TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 11. The parameters for inactivation were an inactivation time of 1 hour at 37 °C using 2 M urea, 10 mM H2O2, 0.04 mM FeSC and 1 M NaCI. The reaction was quenched by the addition of EDTA, and thereafter dialyzed at 5 °C into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using Amicon centrifugal filter device with 30 or 100k MW cut-off or a Sephadex G-25 column. After the inactivation reaction, the cytotoxic activity of TcdA or TcdB was reduced more than 6 log-io and 6 log 10 respectively relative to corresponding native toxin, as shown in Table 12. Table 11

Table 12

Example 6A: Inactivation of TcdB with Urea (2 M), H2O2, FeS04 and ascorbate at 22 °C

Approximately 0.3 mg/ml native TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 1 1 A. The parameters for inactivation were an inactivation time of 2 hours at 22 °C using 2 M Urea, 3 mM H2O2, 0.1 mM FeSC and 1 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using a dialysis membrane or Amicon centrifugal filter device with 30k MW cut-off. After the inactivation reaction, the cytotoxic activity of Ted A was reduced more than 6 log-io relative to native TcdB, as shown in Table 12A. Table 11 A

Table 12A

Example 7: Inactivation of Diphtheria toxin with SDS (3.5 mM), H202 and FeS04

Purified native Diphtheria toxin, approximately 0.3 mg/ml in 50 mM TRIS-HCI pH 7.5, was inactivated according to the parameters shown in Table 13. The parameters for inactivation were an inactivation time of 2 hours at 37 °C using 3.5 mM SDS (0.1 % w/v), 50 mM H2O2, 0.5 mM FeSC . The reaction was quenched by the addition of EDTA, Tween 20 and b-cyclodextrin to a final concentration of 2 mM, 6 mM and 10 mM respectively, and incubated at 20 °C for 30 min, and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using Amicon centrifugal filter device or a Sephadex G-25 column. After the inactivation reaction, the cytotoxic activity of Diphtheria toxin is reduced more than 6 log-io relative to native toxin, see Table 14.

Table 13

Table 14

Example 7 A Inactivation of TcdB with Urea (2 M), H2O2, FeS04 and ascorbate at 37 °C

Approximately 0.3 mg/ml native TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 13A. The parameters for inactivation were an inactivation time of 1 hour at 37 °C using 2 M Urea, 3 mM H2O2, 0.1 mM FeSC and 1 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using a dialysis membrane or Amicon centrifugal filter device with 30k MW cut-off. After the inactivation reaction, the cytotoxic activity of Ted A was reduced more than 7 log 10 relative to native TcdB, as shown in Table 14A. Table 13A

Table 14A

Example 8: Removing SDS from inactivation reaction mixture

After the inactivation reaction of proteins, the destabilizing agents such as SDS or urea, and/or oxidizing agents such as H2O2 should be removed from the protein solution. Urea can be removed by dialysis, ultrafiltration or buffer exchange, but ionic detergents like SDS are more difficult to remove this way. Ionic detergents such as SDS can be removed from the protein by using a nonionic detergent such as polysorbate 20 (Tween 20) and/or a polymer such as b-cyclodextrin. The removal of the destabilizing agent, in this example SDS, was tested at different parameters, see Table 15. Approximately 0.7 mg/ml inactivated Ted A in 50 mM TRIS-HCI pH 7.5 containing the reaction components; 3.5 mM SDS, 1 mM H2O2, 0.12 mM FeSC and 2 mM EDTA was added 6 mM Tween 20 and/or 10 mM b-cyclodextrin and incubated for 1 hour at 20 °C. After incubation, the reaction mixture was dialyzed thoroughly, for example using an Amicon centrifugal filter device with 30 or 100k MW cut-off or Sephadex G-25 column to remove the reaction components herein including SDS. Incubation of the inactivation reaction mixture with Tween 20 prior to dialysis also significantly reduced protein loss during dialysis. Dialyzing the SDS-containing inactivation mixture of T cd A without the presence of Tween 20, showed a significant loss of protein during the dialysisprocess. In the presence of Tween 20 however, recovery of Ted A protein after dialysis is > 90%. Protein loss is also reduced in the presence of n-octyl- -D-glucoside, also a non-ionic detergent, however not as significant as with Tween 20 in this example. The same effect was also seen for TcdB. Table 15

15

Example 9: Inactivated TcdA has preserved antigenic epitopes

Inactivated TcdA was tested for antibody recognition by a neutralizing antibody directed specifically against native TcdA, thereby demonstrating preserved antigenic epitopes after chemical inactivation treatment, see Table 16.

Table 16

Example 9A: Inactivated TcdB has preserved antigenic epitopes

Inactivated TcdB was tested for antibody recognition by a neutralizing antibody directed specifically against native TcdB, thereby demonstrating preserved antigenic epitopes after chemical inactivation treatment, as shown in Table 16A.

Table 16A

Example 10: Inactivation of TcdB with Urea (2 M), H2O2, FeS04 and ascorbate at 4 °C

Approximately 0.5 mg/ml native TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 17. The parameters for inactivation were an inactivation time of 24 hours at 4 °C using 2 M Urea, 3 mM H2O2, 0.1 mM FeSC and 1 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using a dialysis membrane or Amicon centrifugal filter device with 30k MW cut-off. After the inactivation reaction, the cytotoxic activity of Ted A was reduced more than 6 log-io relative to native TcdB, as shown in Table 17A.

Table 17

Table 17 A

Example 10A: Inactivation of TcdB with SDS (0.5 mM), H2O2, FeS04 and ascorbate

Approximately 0.3 mg/ml native TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 18. The parameters for inactivation were an inactivation time of 1 hour at 37 °C using 0.5 mM SDS (0.014% w/v), 3 mM H2O2, 0.1 mM FeSC and 1 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM and thereafter dialyzed into a 50 mM TRIS-HCI pH 7.5 buffer to remove the reaction components. For example, using a dialysis membrane or Amicon centrifugal filter device with 30k MW cut-off. After the inactivation reaction, the cytotoxic activity of TcdB was reduced more than 5.9 log-io relative to native TcdB, as shown in Table 18A.

Table 18

Table 18 A

Example 11 : Immunisation of mice with TcdA and TcdB toxoids

Female C57BL/6J mice (8-10 weeks old) were immunised intramuscularly (IM) with an immunogen containing 5 pg TcdA toxoid and 5 pg TcdB toxoid, to assess the immunogenicity of toxoids produced by the present invention. The immunogen was formulated in neutral TRIS-HCL pH 7.5 buffer and mixed extensively with aluminium hydroxide (AI(OH)3). Each mouse was injected with 50 mI immunogen comprising 0.1 mg/ml T cdA toxoid and 0.1 mg/ml TcdB toxoid and 2 mg/ml aluminium hydroxide. The chemically inactivated toxoids that were assessed in this mice study, herein TcdA and TcdB were inactivated according to the method described in Example 2. Groups of 8 mice were immunised on day 0 and 21 with an immunogen according to Table 19, where after they were orally given a lethal dose of Clostridioides difficile Ribotype 027 (NCTC 13366) on day 56.

Table 19

¹ Chemical inactivation: 3.46 mM SDS (0.1 % w/v), 10 mM H2O2, 0.16 mM FeSC for 20/30 minutes at 37°C.

² Challenge dose: Each mouse was given 250 mI of 0.3 x 10⁷ CFU/ml C. difficile on day 56.

Result: There were no adverse reactions in the mice following each administration of the vaccine. As illustrated in Fig. 6, after two doses of a vaccine containing chemically inactivated TcdA and TcdB toxoids, all mice in Group 2 survived a lethal dose of C. difficile infection. In Group 1 , that only received the adjuvant, 3/8 mice died within day 3, showing that only the vaccinated mice were protected against the disease symptoms caused by C. difficile. All the mice in Group 1 also showed significant weight loss at day 3 (Fig. 7), compared to the vaccinated mice in Group 2, which were not affected at all. Furthermore, serum antitoxin IgG responses were measured for all mice at day 49 to assess the immunogenicity of the chemically inactivated TcdA and TcdB toxoids. The vaccinated mice in Group 2 show significantly increased levels of serum antitoxin against native TcdA (Fig. 8) and native TcdB (Fig. 9), compared to the unvaccinated Group 2 which shows no antitoxin response neither against TcdA nor TcdB.

Example 11 A: Inactivation of TcdA with SDS (0.35 mM), H2O2, C11SO4 and ascorbate

Approximately 0.3 mg/ml native TcdA in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 19A. The parameters for inactivation were an inactivation time of 0.5 hour at 37 °C using 0.35 mM SDS (0.01 % w/v), 0.70 mM H2O2, 0.01 mM CuS04 and 0.70 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of T cd A was reduced more than 5.2 log-io relative to native Ted A, as shown in Table 20.

Table 19 A

Table 20

Example 12: Inactivation of TcdA with Urea (2 M), H2O2, CuS04 and ascorbate

Approximately 0.3 mg/ml native TcdA in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 21 . The parameters for inactivation were an inactivation time of 0.5 hour at 37 °C using 2M urea, 0.70 mM H2O2, 0.05 mM CuSC and 0.70 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of TcdA was reduced more than 5.2 log 10 relative to native TcdA, as shown in Table 22. Table 21

Table 22

Example 13: Inactivation of TcdB with SDS (0.35 mM), H2O2, CuS04 and ascorbate

Approximately 0.3 mg/ml native TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 23. The parameters for inactivation were an inactivation time of 2 hours at 37 °C using 0.35 mM SDS (0.01% w/v), 0.5 mM H2O2, 0.01 mM CuSC and 0.5 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of TcdB was reduced more than 6.7 logic relative to native TcdB, as shown in Table 24.

Table 23

Table 24

Example 14: Inactivation of TcdB with urea (2 M), H2O2, CuS04 and ascorbate

Approximately 0.3 mg/ml native TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 25. The parameters for inactivation were an inactivation time of 2 hours at 37 °C using 2 M urea, 0.5 mM H202, 0.01 mM CuS04 and 0.5 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of TcdB was reduced more than 8 logio relative to native TcdB, as shown in Table 26.

Table 25

Table 26

Example 15: Effect of varying ascorbate concentration on inactivation of TcdB with SDS, H2O2, FeS0₄

Approximately 0.3 mg/ml native TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters as shown in Table 27. The parameters used for inactivation were an inactivation time of 1 hour at 37 °C using 0.35 mM SDS (0.01% w/v), 3 mM H2O2, 0.1 mM FeSC and either 0 mM, 0.5 mM, 1 mM or 3 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of TcdB is shown in Table 28.

Table 27

Table 28

Example 16: Effect of varying ascorbate concentration on inactivation of TcdB with Urea, H₂0_2> FeS04

Approximately 0.3 mg/ml native TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters as shown in Table 29. The parameters used for inactivation were an inactivation time of 1 hour at 37 °C using 2 M urea, 3 mM H2O2, 0.1 mM FeSC and either 0 mM, 0.5 mM, 1 mM or 3 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final

concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of TcdB is shown in Table 30. Table 29

Table 30

Example 17: Effect of varying ascorbate concentration on inactivation of TcdA with SDS, H2O2, FeS0₄

Approximately 0.3 mg/ml native TcdA in 50 mM Tris-HCI pH 7.5 was inactivated according to the parameters as shown in Table 31. The parameters used for inactivation were an inactivation time of 30 min at 37 °C using 0.35 mM SDS (0.01% w/v), 1 mM H2O2, 0.01 mM FeSC and either 0 mM, 0.5 mM or 1 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of T cd A is shown in Table 32. Table 31

Table 32

Example 18: Effect of Urea on inactivation of TcdB with H2O2, FeS04 and ascorbate Approximately 0.3 mg/ml native TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters as shown in Table 33. The parameters used for inactivation were an inactivation time of 1 hour at 37 °C using 3 mM H2O2, 0.1 mM FeSC , 1 mM sodium ascorbate and either 0 M, 1.5 M or 2 M urea. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of TcdB is shown in Table 34. Table 33

Table 34

Example 19: Effect of Urea on inactivation of TcdA with H2O2, FeS04

Approximately 0.3 mg/ml native TcdA in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters as shown in Table 35. The parameters used for inactivation were an inactivation time of 2 hours at 37 °C using 10 mM H2O2, 0.01 mM FeSC and either 1 .25 M, 1.5 M, 1 .75 M or 2 M urea. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of TcdA is shown in Table 36. Table 35

Example 20: Effect of pH on inactivation of TcdA with SDS, H2O2, FeS04 and ascorbate

Approximately 0.3 mg/ml native TcdA in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters as shown in Table 37. The parameters used for inactivation were an inactivation time of 0.5 hour at 37 °C using 0.35 mM SDS (0.01 % w/v), 2 mM H2O2, 0.01 mM FeSC , 1 mM sodium ascorbate at either pH 7.5, 8, 8.5 or 9. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of T cd A is shown in Table 38. Table 37

Table 38

Example 21 : Effect of pH on inactivation of TcdB with urea, H2O2, FeS04 and ascorbate Approximately 0.3 mg/ml native TcdB in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters as shown in Table 39. The parameters used for inactivation were an inactivation time of 1 hour at 37 °C using 2 M urea, 3 mM H2O2, 0.1 mM FeSC , 1 mM sodium ascorbate at either pH 7.5, 8, 8.5 or 9. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of TcdB is shown in Table 40. Table 39

Table 40

Example 22: Inactivated TcdA has preserved antigenic epitopes

Inactivated TcdA (according to example 4A) was tested for antibody recognition in an ELISA by TcdA-specific monoclonal antibodies and compared to formaldehyde-inactivated TcdA¹ , thereby demonstrating preserved antigenic epitopes after chemical inactivation treatment (Table 41 ).

Table 41

¹ Formaldehyde inactivation of TcdA was performed with 4.25 mg/ml formaldehyde, 4.25 mg/ml lysine in 100 mM phosphate buffer pH 7.4 and incubated for 18 days in 4°C. After 18 days, the inactivated TcdB was dialyzed into 50 mM phosphate buffer pH 7.4, 100 mM NaCI, 0.16 mg/ml formaldehyde.

Example 23: Inactivated TcdB has preserved antigenic epitopes

Inactivated TcdB (according to example 10) was tested for antibody recognition in an ELISA by TcdB-specific monoclonal antibodies and compared to formaldehyde-inactivated TcdB¹ , thereby demonstrating preserved antigenic epitopes after chemical inactivation treatment (Table 42). Table 42

¹ Formaldehyde inactivation of TcdB was performed with 4.25 mg/ml formaldehyde, 4.25 mg/ml lysine in 100 mM phosphate buffer pH 7.4 and incubated for 18 days in 4°C. After 18 days, the inactivated TcdB was dialyzed into 50 mM phosphate buffer pH 7.4, 100 mM NaCI, 0.16 mg/ml formaldehyde.

Example 24: Inactivation of TcsL (C. sordellii) with urea, H2O2, FeS04 and ascorbate

Approximately 0.3 mg/ml recombinant TcsL in 50 mM TRIS-HCI pH 7.5 was inactivated according to the parameters shown in Table 43. The parameters for inactivation were an inactivation time of 1 hour at 37 °C using 2 M urea, 3 mM H2O2, 0.01 mM FeSC and 1 mM sodium ascorbate. The reaction was quenched by the addition of EDTA to a final concentration of 2 mM. After the inactivation reaction, the cytotoxic activity of TcsL was reduced more than 8 log 10 relative to untreated recombinant TcsL, as shown in Table 44. Table 43

Table 44

Example 25: Secondary structural features of inactivated TcdA monitored by circular dichroism

FarllV (200-260 nm) circular dichroism (CD) were monitored to depict the secondary structural features of native and inactivated TcdA (fig. 2). For farUV CD measurements a protein concentration between 0.2 - 0.3 mg/ml in a volume of 200 pL was used.

Example 26: Tertiary structural features of inactivated TcdA monitored by circular dichroism

NearllV (250-320 nm) CD were monitored to depict the tertiary structural features of native and inactivated TcdA (fig. 3). For nearllV CD measurements a protein concentration between 0.3 - 0.5 mg/ml in a volume of 100 pL was used.

Example 27: Secondary structural features of inactivated TcdB monitored by circular dichroism

FarllV (200-260 nm) CD were monitored to depict the secondary structural features of native and inactivated TcdB (fig. 4). For farUV CD measurements a protein concentration between 0.2 - 0.3 mg/ml in a volume of 200 pL was used. Example 28: Tertiary structural features of inactivated TcdB monitored by circular dichroism

NearllV (250-320 nm) CD were monitored to depict the tertiary structural features of native and inactivated TcdB (fig. 5). For nearllV CD measurements a protein concentration between 0.3 - 0.5 mg/ml in a volume of 100 pL was used.

CHEMICALS USED

Sodium dodecyl sulfate (SDS), (Sigma-Aldrich, USA) (Lot. no. 68H0123)

Hydrogen peroxide 30%, stabilized (J.T. Baker, USA) (Batch no. 0000181466)

lron(ll)sulfate heptahydrate (Sigma-Aldrich, USA) (Batch no. 057K0735)

lron(lll)sulfate heptahydrate (Sigma-Aldrich, USA) (Lot. no. STBH5302)

Copper(ll)sulfate (Sigma-Aldrich, USA) (Lot. no. 54H3717)

Urea (Merck, Germany) (Lot. no. K45969887 531 )

Dithiothreitol (DTT), (Sigma, USA) (Lot. no. 92H0145)

Ethylenediaminetetraacetic acid disodium salt (2Na-EDTA), (Sigma-Aldrich, USA) (Lot. no.

1 12K0765)

Polysorbate 20 (Tween 20), (Merck, Germany) (Lot. No. S7450184 740)

b-cyclodextrin (Sigma-Aldrich, USA) (Lot. no. 016K07 221 )

Mouse monoclonal anti-TcdA antibody (Abeam, England)

Mouse monoclonal anti-TcdB antibody (Abeam, England)

Polyclonal Rabbit anti-Mouse IgG (H+L), HRP (SouthernBiotech, USA)

TMB PLUS2 substrate (Kem-En-Tec Nordic, Denmark) (Lot. no. 180104)

Alhydrogel (Brenntag Biosector, Denmark)

Tryptone (Formedium, England) (Batch no. 19/MFM/2287)

Yeast Extract (Formedium, England) (Batch no. 19/MFM/2267)

Trizma base (Sigma-Aldrich, USA) (Lot. no. SLBQ2142V)

Sodium thioglycolate (Sigma-Aldrich, USA) (Lot. no. STBH8882)

D(-)-Mannitol (Merck, Germany) (Lot. No. M148882 124)

Glycerol 85% (Merck, Germany) (Lot. No. Z0346094 521 )

Spectra/Por 1 50mm dialysis tube, MWCO 6-8 kDa (Repligen, USA) (Lot. no. 9200875) Crystal violet solution (Sigma-Aldrich, USA) (Lot. no. SLBJ3080V)

ITEMS OF THE INVENTION

1. A method for producing an immunogenic composition comprising an inactivated protein, the method comprising

contacting the protein with a solution comprising at least one destabilizing agent and at least one oxidizing agent.

2. The method of item 1 , further comprising contacting the protein with a solution comprising at least one transition metal ion.

3. The method of item 1 or item 2, wherein the destabilizing agent is a detergent.

4. The method of item 1 or item 2, wherein the destabilizing agent is a chaotropic agent.

5. The method of item 3, wherein the detergent is an ionic detergent.

6. The method of item 5, wherein the ionic detergent is selected from the group consisting of Sodium Dodecyl Sulfate (SDS), sodium deoxycholate, sodium cholate, sodium lauroyl sarcosinate, dioctyl sulfosuccinate and cetyltrimethylammonium bromide.

7. The method of item 5, wherein the ionic detergent is Sodium Dodecyl Sulphate (SDS).

8. The method of item 4, wherein the chaotropic agent is selected from the group consisting of urea, thiourea, guanidinium chloride, ethanol, n-butanol, lithium perchlorate, lithium acetate, phenol and 2-propanol.

9. The method of item 4, wherein the chaotropic agent is urea.

10. The method of any one of items 1-9, wherein the oxidizing agent is selected from the group consisting of hydrogen peroxide (H202), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4- methylbenzenesuifonamide sodium salt and dioxaneperoxide.

11. The method of item 10, wherein the oxidizing agent is hydrogen peroxide (H202).

12. The method of any one of items 2-11 , wherein the transition metal ion is from ferrous sulfate (FeS04), ferric sulfate (Fe2(S04)3), ferrous chloride, ferric chloride, copper dichloride, copper chloride, copper sulfate, silver nitrate, cobalt chloride, and chromium chloride.

13. The method of item 12, wherein the transition metal ion is from ferrous sulfate (FeS04).

14. The method of any one of the previous items, wherein the protein is a toxic protein.

15. The method of any one of the previous items, wherein the protein is a recombinant protein.

16. The method of any one of the previous items, wherein the protein is a bacterial toxin. 17. The method of any one of items 1-15, wherein the protein is from a fungus, a virus, a venom or a plant.

18. An immunogenic composition produced according to the method of any one of items 1-17.

Claims

1. A method for producing an immunogenic composition comprising inactivated TcdA and/or TcdB, the method comprising contacting TcdA and/or TcdB with a solution comprising at least one destabilizing agent and at least one oxidizing agent.

2. The method of claim 1 , further comprising contacting TcdA and/or TcdB with a solution comprising at least one transition metal ion.

3. The method of claim 1 or claim 2, further comprising contacting TcdA and/or TcdB with a solution comprising at least one antioxidant.

4. The method of claim 1 or claim 2, wherein the destabilizing agent is a detergent.

5. The method of claim 1 or claim 2, wherein the destabilizing agent is a chaotropic agent.

6. The method of claim 4, wherein the detergent is an ionic detergent.

7. The method of claim 6, wherein the ionic detergent is selected from the group consisting of Sodium Dodecyl Sulfate (SDS), sodium deoxycholate, sodium cholate, sodium lauroyl sarcosinate, dioctyl sulfosuccinate and cetyltrimethylammonium bromide.

8. The method of claim 6, wherein the ionic detergent is Sodium Dodecyl Sulphate (SDS).

9. The method of claim 5, wherein the chaotropic agent is selected from the group consisting of urea, thiourea, guanidinium chloride, ethanol, n-butanol, lithium perchlorate, lithium acetate, phenol and 2-propanol.

10. The method of claim 5, wherein the chaotropic agent is urea.

1 1. The method of any one of claims 1-10, wherein the oxidizing agent is selected from the group consisting of hydrogen peroxide (H2O2), sodium peroxide, performic acid, periodic acid, sodium permanganate, potassium permanganate, sodium hypochlorite, oxygen, ozone, N-chloro-4- methylbenzenesulfonamide sodium salt and dioxaneperoxide.

12. The method of claim 1 1 , wherein the oxidizing agent is hydrogen peroxide (H2O2).

13. The method of any one of claims 2-12, wherein the transition metal ion is from ferrous sulfate (FeSC ), ferric sulfate (Fe₂(SC>₄)3), ferrous chloride, ferric chloride, copper dichloride, copper chloride, copper sulfate, silver nitrate, cobalt chloride, and chromium chloride.

14. The method of claim 13, wherein the transition metal ion is from ferrous sulfate (FeSC ).

15. The method of claim 13, wherein the transition metal ion is from copper sulfate (CuSC ).

16. The method of claim 3, wherein the antioxidant is ascorbic acid or mineral salts thereof.

17. An immunogenic composition produced according to the method of any one of claims 1 -16.