Pharmaceutical compositions containing clostridium difficile toxoids a and b

DESCRIPTION

PHARMACEUTICAL COMPOSITIONS CONTAINING AEB TOXOIDS OF CLOSTRIDIUM DIFFICILE.

Field of the Invention

The present invention relates to compositions including Clostridium difficile toxoids and corresponding methods.

Background of the Invention

Clostridium difficile (C. difficile) toxins A and B are responsible for C. difficile-associated disease (CDAD), which manifests as nosocomial diarrhea and pseudomembranous colitis (Kuijper et al., Clinical Microbiology and Infection 12 (Suppl. 6 ): 2-18, 2006; Drudy et al.,

International Journal of Infectious Diseases 11 (1): 5-10, 2007; Warny et al., Lancet 366 (9491): 1079-1084, 2005; Dove et al. , Infection and Immunity 58 (2): 480-488, 1990;

Barroso et al., Nucleic Acids Research 18 (13): 4004, 1990).

Treatment of the toxins with formaldehyde results in the corresponding toxoids A and B, which are completely inactivated and retain at least partial immunogenicity (Torres et al., Infection and Immunity 63 (12): 4619-4627, 1995). Vaccination employing both toxoids has been shown to be effective in hamsters, healthy adults, and patients with recurrent CDAD (Torres et al., Infection and Immunity 63 (12): 4619-4627, 1995; Kotloff et al.,

Infection and Immunity 69 (2): 988-995, 2001; Sougioultzis et al. , Gastroenterology 128 (3): 764-770, 2005; Torres et al., Vaccine Research 5 (3): 149-162, 1996). Additionally, administration of both free and bound aluminum salt toxoids (adjuvant) leads to appropriate immune responses (Torres et al., Vaccine Research 5 (3): 149-162, 1996; Giannasca et al., Infection and Immunity 67). (2): 527538, 1999). Administration of both toxoids simultaneously is more effective than administration of individual proteins alone (Kim et al., Infection and Immunity 55 (12): 2984-2992, 1987). Both toxoids A and B are thus candidates for vaccine development. Improvement in their integrity and / or contumatic reduction in their tendency to aggregate is desirable to produce optimal storage stability.

US 6,214,341 describes passive immunization against Clostridium difficile disease.

Summary of the Invention

The invention provides compositions, such as pharmaceutical compositions (e.g. vaccine compositions), including a Clostridium difficile toxoid (e.g., a C. toxoid). difficile toxins A and / or B; with toxoids A and B present in a ratio of, for example, 5: 1 to 1: 5, for example 3: 2 (A: B)) and pharmaceutically acceptable excipients comprising (a) a buffer selected from a citrate buffer sodium or potassium salt and a sodium or potassium phosphate buffer; and sucrose, which reduce or retard toxoid aggregation, and / or increase the thermal stability of the toxoid with respect to a composition lacking pharmaceutically acceptable excipients. In one example, pharmaceutically acceptable excipients reduce or retard toxoid aggregation by 50% or more relative to a composition lacking pharmaceutically acceptable excipients. In another example, pharmaceutically acceptable excipients increase the thermal stability of the toxoid by 0.5 ° C or more over a composition that lacks pharmaceutically acceptable excipients. Optionally, the compositions of the invention may include an adjuvant (for example, an aluminum compound, such as an aluminum hydroxide compound, aluminum phosphate, or aluminum hydroxyphosphate). The compositions may be in liquid form, dry powder, freeze-dried, spray-dried, foam-dried.

Pharmaceutically acceptable excipients may, for example, be selected from the group consisting of buffers, tonicity agents, simple carbohydrates, sugars, carbohydrate polymers, amino acids, oligopeptides, polyamino acids, polyhydric alcohols and ethers thereof, detergents. , lipids, surfactants, antioxidants, salts, human serum albumin, gelatins, formaldehyde, or combinations thereof. Described as such, (i) the buffer is selected from the group consisting of citrate, phosphate, glycine, histidine, carbonate, and bicarbonate, and is in a concentration of 5-100 mM; (ii) the tonicity agent is mannitol at a concentration of 150 mM; (iii) sugar is selected from sorbitol, trehalose, and sucrose at a concentration of 1-30%; (iv) the amino acid, oligopeptide, or polyamino acid is present in a concentration of up to 100 mM; (v) polyhydric alcohol is selected from the group consisting of glycerol, polyethylene glycol, and ethers thereof of molecular weight 200-10,000, in a concentration of up to

20^: (vi) lipid detergents are selected from the group consisting of pluronic sodium deoxycholate, Tween 20, Tween 80, in concentrations of up to 0.5%; (vii) carbohydrate polymers are selected from dextran and cellulose; (viii) salts are selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, and magnesium acetate, up to 150 mM; and (ix) formaldehyde is present at 0.001-0.02%.

The excipients described include those listed in Table 1, Table 2, Table 8, or Table 9. In other examples, the compositions include sodium or potassium citrate, and / or sodium or potassium phosphate, optionally in combination with sucrose and / or formaldehyde. . Thus, in various examples, compositions include Clostridium difficile toxoids A and B, 5-100 mM (e.g., 10-30 mM, or 20 mM) sodium or potassium citrate (or phosphate), 220% (e.g. 2-10% or 5%) sucrose, and -0.020% (e.g. 0.016%) formaldehyde, pH 5.5-8.5 (e.g. 6.5-8.0, or 7, 5). A combination of sorbitol, dextrose, and / or Tween 80 is also described.

The invention also provides methods of preparing the compositions including a Clostridium difficile toxoid and pharmaceutically acceptable excipients comprising (a) a buffer selected from a sodium or potassium citrate buffer and a sodium or potassium phosphate buffer; and sucrose, which reduce or retard toxoid aggregation, and / or increase the thermal stability of the toxoid with respect to a composition lacking pharmaceutically acceptable excipients. Such methods include providing a Clostridium difficile toxoid and mixing a Clostridium difficile toxoid with pharmaceutically acceptable excipients, such as those described herein. The compositions may be stored in liquid or lyophilized form as described herein.

The invention also provides a composition of the invention for use in a method of preventing or treating C. difficile infection or disease in an individual.

The invention also provides for the use of a combination of

(a) a buffer selected from a sodium or potassium citrate buffer and a sodium or potassium phosphate buffer; and

b) sucrose in order to increase thermal stability and / or reduce or retard the aggregation of a Clostridium difficile toxoid in vitro.

Described herein are methods of inducing a C. difficile immune response in an individual, which involves administering to the individual a composition as described herein, in one example, the patient does not have, but is at risk of developing, C. difficile disease, and in another For example, the patient has C. difficile disease. The use of the compositions of the invention in inducing an immune response to C. difficile in an individual, or in preparing medicaments for use for this purpose is described.

The invention provides several advantages. For example, use of the excipients described herein may result in increased physical stability of C. difficile toxoids A and B, and / or decreased or delayed aggregation, which are important for the production of pharmaceuticals (eg vaccines) including toxoids. In addition, the use of ratios of the invention (eg 3: 2, A: B) and addition of adjuvant just prior to administration (except in the stored vaccine formulation) may lead to increased immunogenicity.

Other features and advantages of the invention will be apparent from the following detailed description, drawings, and claims.

Brief Description of the Drawings

Figure 1. Study of the effects of solute on structural stability of toxoid A (x) in the presence of 20% trehalose (□), 20% sucrose (), 10% sorbitol (o), 10% dextrose (·) , 20% glycerol (Δ), 0.05% tween 80 (Δ),

0.1% pluronic F68 (0): (a) CD signal at 208 nm; (b) intensity of ANS emission; (c) peak position of ANS emission; and (d) DSC thermogram. Thermal traces represent an average of 2 measurements, where each data point had a standard error of less than 0.5.

Figure 2. Study of the effects of solute on structural stability of toxoid B (x) in the presence of 20% trehalose (□), 20% sucrose (), 10% sorbitol (o), 10% dextrose (·) , 20% glycerol (Δ), 0.05% tween 80 (Δ),

0.1% pluronic F68 (0): (a) CD signal at 208 nm; (b) intensity of ANS emission; (c) peak position of ANS emission; and (d) DSC thermograms. Thermal traces represent an average of 2 measurements. Each data point had a standard error of less than 0.5.

Figure 3. Studies of the effect of solute combinations on the thermal stability of toxoid A (x) in the presence of 10% sorbitol and 0.05% tween 80 (η), 10% dextrose and

<td> 0,05%</td><td>in</td><td>tween</td><td> 80 ()</td><td> , 10%</td><td>in</td><td>sorbitol,</td><td>10% of</td><td>dextrose</td><td>and</td>

<td> 0,05%</td><td>in</td><td>tween</td><td>80 (o)</td><td> , 10%</td><td>in</td><td>dextrose</td><td>and 10%</td><td colspan="2">of sorbitol</td>

<td> (·) :</td><td>(The)</td><td colspan="2">monitored</td><td>fur</td><td colspan="2">CD signal on</td><td>2 0 8 nm</td><td>and (b) OD</td><td>The</td>

350 nm. Thermal traces represent an average of 2 measurements, in which each data point had a default error of less than 0.05.

Figure 4. Studies of solute combinations and their effects on the thermal stability of toxoid B (x) in the presence of 10% sorbitol and 0.05% tween 80 (□), 10% dextrose and 0.05% tween 80 (), 10% sorbitol, 10% dextrose, and 0.05% tween 80 (o), 10% dextrose and

10% sorbitol (·): (a) monitored by CD signal at 208 nm and (b) OD at 350 nm. Thermal traces represent an average of 2 measurements, where each data point had a standard error of less than 0.05.

Figure 5. Studies of tween 80 (□) properties in the presence of 10% dextrose (), 10% sorbitol (o), 10% dextrose and 10% sorbitol (·) as a function of temperature: 208 nm 0.05% tween 80 (a) and 0.1% tween 80 (b) CD signal; OD at 350 nm for 0.05% tween 80 (c) and 0.1% tween 80 (d); Diameter based on Number

MSD for hydrodynamic diameter (full square) and diameter based on Lognormal Number (full diamond) for 0.05% tween 80 (e) and 0.1% tween 80 (f). Sizes> 1 pm are not accurate given the nature of DLS measurements. Thermal traces represent an average of 2 measurements, where each data point had a standard error of less than 0.5.

Figure 6. Studies of the effect of solute concentration at the midpoint of the thermal transition (Tm) monitored with 208 nm CD signal for toxoid A (a) and toxoid B (b) in the presence of sorbitol and 0.05% tween 80 (♦), dextrose and 0.05% tween 80 (), sorbitol, dextrose, and 0.05% tween 80 (Δ), sorbitol, dextrose, and 0.1% tween 80 (χ), sorbitol and dextrose (0).

Figure 7. Hydrodynamic diameter as a temperature function for toxoid A (ac) and toxoid B (df) where filled squares represent diameter based on MSD number and filled diamonds represent diameter based on lognormal number. Sizes> 1 pm are not accurate given the nature of DLS measurements. (a, d) protein alone; (b, e) protein in the presence of 10% sorbitol and 10% dextrose; (c, f) protein in the presence of 10% sorbitol, 10% dextrose and 0.05% twe-en 80. Thermal traces represent an average of 2 measurements, where each data point had a standard error of less than 0.5.

Figure 8. Alhydrogel® (aluminum hydroxide adjuvant) binding studies: (a) absorption isotherm and (b) desorption isotherm in the presence of 2 M NaCl for toxoid A (0) and toxoid Β (A).

Figure 9. Study of secondary structure of toxoid A by circular dichroism (CD) spectroscopy in the pH range of

5.5 to 7.5.

Figure 10. Study of the secondary structure of toxoid B by circular dichroism (CD) spectroscopy in the pH range of

5.5 to 7.5.

Figure 11. Study of circular dichroism spectroscopy fusion toxoid A (CD) fusion in the pH range of

5.5 to 8.0.

Figure 12. Study of toxoid B melting temperature by circular dichroism (CD) spectroscopy in the pH range 5.0 to 7.5.

Figure 13. Study of aggregation at different pH values ​​over time at a fixed storage temperature.

Figure 14. Study of salt-dependent toxoid A aggregation at 37 ° C over time.

Figure 15. Study of salt-dependent toxoid B aggregation at 37 ° C over time.

Figure 16. Study of lyophilization parameters of vaccine formulations.

Detailed Description

The invention provides compositions including toxoids of

Acceptable Clostridium compositions below, pharmaceutically difficile and excipients which provide beneficial properties to For example, and as further described excipients included in the compositions of the invention may result in increased stability of one or more of the toxoid components of the compositions and / or decreased or delayed aggregation. of toxoids.

C. difficile toxoids which may be included in the compositions of the invention may be made using any of several methods known in the art. For example, methods involving formaldehyde inactivation may be used (see, for example, Kotloff et al., Infection and Immunity 69 (2): 988-995, 2001). Preferably, the compositions include both toxoid A and toxoid B, but compositions including only one of these toxoids are also included in the invention. A strain of C. An exemplary difficile that can be used as a source of toxins is ATCC 43255 (VPI 10463). Toxoids may be present in the compositions in varying ratios, for example, 5: 1 (A: B) to 1: 5 (A: B). In specific examples, the ratios may be 2: 1, 3: 1, or 3: 2 (A: B). The total amount of toxoid in the compositions of the invention may be, for example, 100 ng to 1 mg, 100 ng to 500 pg, 1 to 250 pg, 10 to 100 pg, 25 to 75 pg, or 50 pg. The compositions may optionally be stored in single unit dosage vials.

Described are compositions which include one or more compounds such as, for example, buffers (e.g., citrate, phosphate, glycine, histidine, carbonate, or bicarbonate; 5 to 100 mM; examples of citrate salts that may be used include sodium, potassium, magnesium and zinc); tonicity agents (e.g., mannitol; 1 to 50 mM); carbohydrates, such as sugars or sugar alcohols (for example, sorbitol, trehalose, or sucrose; 1 30%) or carbohydrate polymers (e.g., dextran and cellulose); amino acids, oligopeptides, or polyamino acids (up to 100 mM); polyhydric alcohols (e.g., glycerol, polyethylene glycols, or ethers thereof, of molecular weight 200 to 10,000, and concentrations of up to 20%); detergents, lipids, or surfactants (e.g., Tween 20, Tween 80, or pluronics, with concentrations up to 0.5%); antioxidants; salts (e.g. sodium chloride, potassium chloride, magnesium chloride, or magnesium acetate, up to 150 mM); albumin (e.g. human serum albumin); gelatines; formaldehyde (0.001 to 0.02%); or combinations thereof.

Described are excipients that include those listed in tables 1, 2, 8, and 9, below. In various examples, the excipients may be those which result in (i) increased thermal stability (e.g., at least 0.5 ° C, for example 0.5 to 5 ° C, 1 to 4 ° C, or 2 ° C). at 3 ° C) as measured by, for example, the assays described below (eg Differential Scanning Calorimetry (DSC)), and / or (ii) aggregation of toxoid A, toxoid B, or both decreased toxoid A and B or delayed by, for example, 50% or more (e.g. 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%), as measured, for example, by assays described below. Compositions including toxoid aggregates are also included in the invention.

Exemplary excipients and buffers thus include sodium citrate (e.g. 0.01 to 0.2 M, e.g. 0.02 to 01 M) and sucrose (e.g. 1 to 20% or 5 to 10 %). Described are excipients and buffers which include sorbitol (e.g. 4 to 20% or 5 to 10%), trehalose (e.g. 4 to 20% or 5 to 10%), tween 80 (e.g. 0.05 to 0 , 1%), diethanolamine (e.g. 0.3 M), histidine (e.g. 0.02 to 0.3 M), guanidine (e.g. 0.3 M), dextrose (e.g. 5-20) %), glycerol (e.g. 20%), albumin (e.g. 1 to 2.5%), lactose (e.g. 10 to 20%), mannitol (e.g. 10%), sucrose (e.g. 5 to 20%), F-68 pluronic acid (eg 0.1%), 2-OH propyl β-CD (eg 5 to 10%), dextran T40 (eg 0.03 to 0.08 mg / ml), Bri ] (e.g. 0.01 to 0.1%), lysine (e.g. 0.3 M), Tween 20 (e.g. 0.01 to 0.05%), and aspartic acid (e.g. 0 , 15 M) (see tables 1, 2, 8, and 9). These excipients may be used at the concentrations listed in the tables. Alternatively, the amounts may be varied and, for example, 0.1 to 10 times as is understood in the art. Other carbohydrates, sugar alcohols, surfactants, and amino acids that are known in the art may also be included in the compositions of the invention.

Excipients and buffers may be used individually or in combination. As an example of a combination, the compositions may include sodium citrate and sucrose, which have been shown to provide benefits with respect to toxoid stability. The amounts of such components may be, for example, 10 to 30 mM, 15 to 25 mM, or 20 mM sodium citrate; and sucrose ala 20% or 5 to 10%. In addition to such components, such compositions may include a low amount of formaldehyde, such as 0.001 to 0.020, 0.01 to 0.018, or 0.16% formaldehyde. The pH of such a composition may be, for example, 5.5 to 8.0 or 6.5 to 7.5, and the composition may be stored at, for example, 2 to 8 ° C, in liquid or lyophilized form. . In variations of this composition, sodium citrate may be substituted with sodium phosphate (10 to 30 mM, 15 to 25 mM, or 20 mM) and it is described that sucrose may be substituted with sorbitol (e.g. 4 to 20% or 5 to 10%), or trehalose (e.g., 4 to 20% or 5 to 10%). Other variations of the compositions are included in the invention, and involve the use of other components listed herein. Based on the above, an exemplary composition of the invention includes 20 mM sodium citrate, 5% sucrose, and 0.016% formaldehyde, pH 7.5.

Described are compositions which include sorbitol, dextrose and Tween 80, which is a combination that has been shown to provide benefits with respect to aggregation and stability (see below). The amounts of such components may be, for example, 5 to 15%, 8 to 12%, or 10% sorbitol; 5 to 15%, 8 to 12%, or 10% dextrose; and 0.01 to 1%, 0.025 to 0.5%, or 0.05 to 0.1% tween 80. Described is a composition in which these components are present in 10% (sorbitol and dextrose) and 0.05. 0.1% tween 80). Also described is that the excipients are dextrose (10%) and sorbitol (10%).

The compositions of the invention may be stored in liquid or dry form, with the latter including as examples freeze-dried powder form, freeze-dried form, spray-dried form, and foam-dried form. Thus, in addition to one or more excipients as described above, the compositions of the invention may include a liquid medium (e.g. saline or water) which may be buffered with, for example sodium phosphate (e.g. mM) containing NaCl (e.g. 150 mM). An exemplary pH range of the compositions of the invention is 5 to 10, for example 5 to 9, 5 to 8, 5.5 to 9, 6 to 7.5, or 6.5 to 7. In addition, the compositions may be include one or more stabilizing agents. In other examples, the compositions are in lyophilized form, and such compositions may be reconstituted by the use of a liquid medium (e.g., saline or water) prior to administration.

The compositions of the invention may optionally include one or more adjuvants in addition to the toxoid antigens and excipient (s) described above. Adjuvants which may be used in the invention include aluminum compounds such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxide phosphate. Antigen may be precipitated with or absorbed into the aluminum compound using standard methods. As a specific example, aluminum (e.g. Rehydragel LV®, Reheis, Inc., Berkeley Heights, New Jersey; up to, for example, 2 mg AlOH / dose, for example about 1.5 mg AlOH / dose Alhydrogel® (e.g. Alhydrogel® 2%; (aluminum hydroxide adjuvant)), Brenntag Biosectror, ​​Frederickssund, Denmark (AIOH3)) may be used. The amount of aluminum used may be, for example, 100-850 pg / dose, 200-600 pg / dose, or 300600 pg / dose.

One approach to formulation included in the invention involves formulating the toxoids and excipients together, and then adding an adjuvant, such as an aluminum adjuvant, just prior to administration. This approach has been found to increase immunogenicity, as also described below. In another approach, the adjuvant is included in the formulation before storage (in liquid or lyophilized form). Additional adjuvants that may be used in the compositions and methods of the invention include RIBI (ImmunoChem, Hamilton, MT), QS21 (Aquila), Bay (Bayer), and Polyphosphazene (Virus Research Institute, Cambridge, MA; WO 95/2415).

The invention also includes methods of preparing the compositions described herein, which involve the production of toxoids as described, for example, by Kotloff et al., Infection and Immunity 69 (2): 988-995, 2001, and combining the toxoids with one or more excipients as described herein using standard methods of pharmaceutical formulations. As described above, the compositions may be stored in liquid or lyophilized form. Lyophilization may be performed using standard methods (see, for example, examples below), and lyophilized material may be reconstituted in a sterile liquid (e.g., water, saline, or a solution including any desired excipient (s) ( s)), with or without an adjuvant, before administration.

Also, the invention includes the use of the compositions in the prevention and treatment of C. difficile infection or disease. Accordingly, the invention includes administering the compositions of the invention to prevent or treat C. difficile-associated disease (CDAD), such as recurrent CDAD, as well as features of CDAD including diarrhea (e.g., nosocomial diarrhea) and pseudomembranous colitis. As is known in the art, CDAD is often associated with treatment of individuals with antibiotics, such as individuals who are hospitalized. Accordingly, the compositions for use in the treatment methods of the invention may be used in the treatment of such patients. In addition, the treatment according to the invention may be combined with antibiotic treatment (e.g., vancomycin and / or metronidazole) and / or passive immunotherapy (see, for example, United States Patent No.

6,214,341). The administration methods of the invention may also be used in the generation of C. difficile immunoglobulin for use in passive immunization of patients (see, for example, United States Patent No.

6.214.341) .

Described are methods of identifying excipients that may be used to generate compositions including C. difficile toxins or toxoids having improved properties. Such methods involve screening assays, such as those also described below, which facilitate the identification of conditions resulting in decreased or delayed aggregation and / or increased stability of one or more of the toxin and / or toxoid components of the compositions. These methods include aggregation assays and stability assays as described also below. Also described is the use of other assays to identify desirable formulations, including solubility, immunogenicity, and viscosity assays.

The compositions of the invention may be administered by, for example, percutaneous (e.g., intramuscular, intravenous, or intraperitoneal) administration in such amounts and regimens as are appropriate to those skilled in the art. For example, 100 ng-1 mg, 100 ng-500 pg, 1250 pg, 10-100 pg, 25-75 pg, or 50 pg toxoid may be administered. For prophylaxis or therapy purposes, the vaccine may be administered, for example, 1, 2, 3, or 4 times. When multiple doses are administered, the doses may be separated from each other, for example for a week to a month. In another example, four doses of 50 pg each may be administered intramuscularly for any period of eight weeks.

Example I

To identify conditions that enhance the physical stability of Clostridium difficile toxoids A and B, the evaluation of compound stabilization was performed. Evaluation of 30 GRAS compounds (generally considered safe) at various concentrations and combinations was performed in two parts. First, a high throughput aggregation assay was used to evaluate for compounds that retard or prevent toxoid aggregation under stress conditions (toxoids at pH 5-5.5 were incubated at 55 ° C for 55 or 75). stabilized minutes). Both proteins have also been studied for their ability to slow down unfolding under conditions that lead to a presumably native folded state (pH 6.5). The thermal stability of toxoids on the surface of

Alhydrogel® (aluminum hydroxide adjuvant) was monitored with DSC and also showed significant improvement in the presence of certain excipients. Compounds that effectively inhibited the aggregation of both toxoids were also investigated for their ability to enhance protein structural stability. To identify stabilizing agents for adjuvant-bound toxoids, the selected excipients were also studied for their ability to enhance the thermal stability of adjuvant-bound toxoids. In conclusion, this study generated information regarding the behavior of free and adjuvant-bound toxoids over a range of conditions (temperatures and solutes) that can be used to designate pharmaceutical formulations of enhanced physical stability.

Experimental Materials and Methods

Materials

Toxoids A and B were produced in highly purified form using previously described methods (Kotloff et al., Infection and Immunity 69 (2): 988-995, 2001). Protein concentration was determined by UV absorption at 280 nm using absorption units of 1.173 for toxoid A and 0.967 for toxoid B at mg / mL concentrations, respectively. All reagents used were of analytical grade and were purchased from Sigma (St. Louis, MO). 0 Sodium phosphate buffer (5 mM, pH 5.0, 5.5, and 6.5) containing 150 mM NaCl was used for excipient evaluation studies. Sodium phosphate buffer (5 mM, pH 6.5) containing 150 mM NaCl was used for stirring and adjuvant studies. For buffer exchange, the protein was dialyzed at refrigerator temperature using Slide-A-Lyzer® Dialysis Cassettes, 10 kDa MWCO (Pierce, Rockford, IL).

Excipient Evaluation Studies

Aggregation test. Approximately 30 GRAS (generally considered safe) compounds in 58 concentration ranges and various combinations were evaluated for their ability to inhibit toxoid aggregation. Protein aggregation was monitored by optical density measurements at 350 nm (OD 350 nm) using a 96-well plate reader (Spectra Max M5, Molecular Devices, Sunnyvale, CA). The aggregation assay was performed at pH 5.5 for toxoid A (1.2 mg / ml) and at pH 5.0 for toxoid B (0.5 mg / ml) at 55 ° C. Under these conditions, the proteins are partially deployed and associate spontaneously. Thus, any excipient stabilizing influence that disturbs these two processes can potentially be detected. The protein was added to the wells of a 96-well plate containing excipients at the corresponding pH and the samples were incubated at 55 ° C for 75 minutes for toxoid A and 55 minutes for toxoid Β. The optical density of the solutions was monitored at 350 nm every 5 minutes. Controls of protein solutions without compounds and buffer added alone with the (blank) excipient (s) were examined simultaneously. Measurements were corrected for the behavior of excipient intrinsic buffer by subtracting the blanks prior to data analysis. Each sample was evaluated in triplicate. Percentage inhibition of aggregation was calculated using the following expression:

% inhibition of aggregation = 100 - [ΔθΡ / ϊ /. ©. _χ jqq 3 AOD₃₅o (C) 'where AOD350 (E) represents the change in OD at 350 nm of the protein in the presence of the excipient and AOD350 (C) the change in OD at 350 nm of the protein without excipient (Peek et al., Journal of Pharmaceutical Sciences 96 (1): 44-60, 2006).

Structural Stability Studies. Toxoid solutions were studied at a concentration of 0.2 mg / ml for CD measurements and 0.1 mg / ml for fluorescence and UV absorption analysis. No concentration dependencies were seen in this range. Each sample was evaluated in duplicate to ensure reproducibility of measurements.

Far-UV circular dichroism spectroscopy (CD). The CD spectra were acquired using a Jasco J-810 spectropolarimeter equipped with a Peltier 6 position temperature controller. The CD spectra were obtained from 260-190 nm with a scan speed of 20 nm / min, an accumulation of 2 and a response time of 2 seconds. The 208 nm CD signal was monitored every 0.5 ° C over a temperature range of 10 to 85 ° C employing a temperature ramp of 15 ° C / hour for study thermal transitions (fusion curves) of proteins ( in sealed cuvettes with 0.1 cm path). The CD signal was converted to molar ellipticity by Jasco Spectral Manager software. The center point temperature of the thermal transition was obtained from a sigmoidal fit of the melting curves using Origin software.

Fluorescence Spectroscopy ANS. The accessibility of supporting sites on the proteins was monitored by fluorescence emission of extrinsic probe 8-Anilino-1-naphthalene sulfonate (ANS). Each sample contained a 20-fold molar excess of SNA for protein. Emission spectra were collected from 400 to 600 nm with a step size of 2 nm and an integration time of 1 second after ANS excitation at 372 nm. Emission spectra were collected every 2.5 ° C at 5 minutes equilibrium over a temperature range of 10 to 85 ° C. The ANS buffer reference value at each corresponding pH was subtracted from the crude emission spectra. Peak positions of emission spectra were obtained from polynomial adjustments using Origin software.

High Resolution UV Absorption Spectroscopy. High resolution UV absorption spectra were acquired using an Agilent 8453 UV visible spectrophotometer. Protein aggregation was studied by monitoring the OD at 350 nm every 2.5 ° C in the temperature range 10 to 85 ° C. ° C with a 5 minute incubation (sufficient for equilibrium to be reached) at each temperature.

Light scattering from proteins to excipients) was light scattering

Dynamics. Average hydrodynamic diameter pH 6.5 (alone and in the presence of analyzed using a dynamics instrument (Brookhaven Instrument Corp.,

Holtzille, NY). The instrument was equipped with a diode pumped laser at 50 mW (λ = 532 nm) and scattered light was monitored at 90 ° for the incident beam. Autocorrelation functions were generated using a digital autocorrelator (BI-9000AT). Hydrodynamic diameter was calculated from the diffusion coefficient by the Stokes-Einstein equation using the cumulant method (based on lognormal number). Data were fitted to a non-negatively constrained least squares algorithm to produce multimodal distributions (MSD). The instrument was equipped with a temperature controlled circulating water bath RTE111 (Neslab, Newington, NH) and the hydrodynamic diameter was monitored over a temperature range of 10 to 85 ° C.

Differential Scanning Calorimetry (DSC). DSC was performed using a MicroCal VPDSC with automatic sample collector (MicroCal, LLC; Northampton, MA). Toxoid thermograms (0.5 mg / ml) alone and in the presence of excipient (s) were obtained from 10-90 ° C using a scan rate of 60 ° C / hour. Charged cells were equilibrated for 15 minutes at 10 ° C before starting each scan. Buffer thermograms alone were subtracted from each protein scan prior to analysis.

Agitation Studies. Toxoid solutions at a concentration of 0.4 mg / ml were studied in the presence and absence of excipients. Protein samples (0.4 ml) were placed in 1.5 ml centrifuge tubes and shaken on a rotator (Thermomixer R, Eppendorf AG, Hamburg, Germany) at 300 rpm for 72 hours at a constant temperature of 4 ° C. . Protein concentration and OD at 350 nm were measured before and after rotation to assess absorption and aggregation to the vessel walls. Samples were centrifuged for 10 minutes at a speed of 100,000g at 4 ° C and the concentration and OD at 350 nm of the supernatant were measured to detect insoluble aggregate formation. Protein structures were evaluated by CD. Each sample was measured in duplicate.

Adjuvant Studies

Absorption for Aluminum Hydroxide (Alhydrogel®) (aluminum hydroxide adjuvant). The ability of toxoids to adsorb to A-lhydrogel® (Brenntag

Biosectror, ​​Frederickssund, Denmark; aluminum hydroxide adjuvant) at various concentrations (0.025 1 mg / ml) was determined by constructing a binding isotherm. Protein solutions in the presence of 0.4 mg / ml Alhydrogel® (aluminum hydroxide adjuvant) were vortexed on an end-over-end tube rotator at refrigerator temperature for 20 minutes. The samples were centrifuged at 14,000xg for 30 seconds to pellet the adjuvant. The value of protein concentration remaining in the supernatants was used to construct binding curves. The ability of the protein to bind Alhydrogel® (aluminum hydroxide adjuvant) in the presence of excipients was determined by the same procedure. In this case Alhydrogel® (aluminum hydroxide adjuvant) was added to the protein-excipient solution.

Alhydrogel® toxoid desorption (aluminum hydroxide adjuvant). Desorption of Alhydrogel® (aluminum hydroxide adjuvant) proteins was assessed in the presence of 2 M NaCl. Alhydrogel® toxoid granules (aluminum hydroxide adjuvant) were prepared as described above. The pellets were washed with buffer (pH 6.5) to remove protein present in the supernatant prior to addition of NaCl solution. Alhydrogel® (aluminum hydroxide adjuvant) solutions were vortexed on a top-to-top tube rotator at refrigerator temperature for 20 minutes. Samples were centrifuged at 14,000xg for 30 seconds to granulate the adjuvant. Protein concentration in the supernatants was used to construct desorbing isotherms.

Stability of Alhydrogel®-bound Toxoids (aluminum hydroxide adjuvant). The thermal stability of Alhydrogel®-bound toxoids (aluminum hydroxide adjuvant) was monitored with DSC using a MicroCal VPAutoDSC (MicroCal, LLC, Northampton, MA). The 0.5 mg / ml toxoids were bound to 0.4 mg / ml Alhydrogel® (aluminum hydroxide adjuvant) by the procedure described above. Toxoid thermorgamas were obtained from 10 to 90 ° C with a scan rate of 60 ° C / hour. Samples were equilibrated for 15 minutes at 10 ° C before each scan. Alhydrogel® (aluminum hydroxide adjuvant) alone thermograms were subtracted from each protein / adjuvant scan prior to analysis.

Results and discussion

Excipient Evaluation Studies

To investigate the ability of GRAS compounds to prevent / retard aggregation, the toxoids were incubated alone and in the presence of excipients under stress conditions (55 ° C incubation). Toxoid aggregation was monitored in a high productivity manner by monitoring changes in OD at 350 nm during the incubation time. Turbidity changes were also used to calculate% inhibition of aggregation and are summarized in table 1 for toxoid A and table 2 for toxoid B.

High productivity toxoid A aggregation assays found that more than half of the excipients either retarded or prevented the increase in OD at 350 nm over time and led to inhibition of aggregation by 90% or more (Table 1). Among the excipients examined, 2.5% albumin, 2.5% α-cyclodextrin, 0.1% tween 80, 0.3 M histidine, and 0.3 M lysine led to OD values ​​at 350 ° C. instantaneously high, suggesting that toxoid A is insoluble under these conditions. Toxoid A aggregation was also significantly enhanced in the presence of 16 other excipients, among which the 25 and 50 mM arginine / glutamine mixture, 0.3 M arginine, and 0.3 M proline were especially potent.

<td colspan="2">The inhibition</td><td colspan="2">of aggregation</td><td>in</td><td>toxoid B</td><td>by 90% or more</td>

<td>it occurred</td><td>at</td><td>presence</td><td>in</td><td> 15</td><td>excipients</td><td>(table 2). THE</td>

<td>presence</td><td>in</td><td>histidine</td><td>at 0,</td><td>3 M</td><td>or citrate</td><td>0.2 M sodium</td>

<td>led to</td><td>OD</td><td>at 350 nm</td><td colspan="4">instantly high. Other 20</td>

compounds more gradually induced aggregation over the monitored time. Extremely high OD increases at 350 nm were observed in the presence of 0.015 M calcium chloride, 0.15 M ascorbic acid, and 0.3 M arginine.

In many cases, the aggregation of both toxoids was facilitated by the same excipients (Tables 1 and 2). In contrast, 5% 2-OH propyl γ-CD, 0.01% and 0.1% tween 20, 0.15 M aspartic acid, and 0.3 M guanidine facilitated toxoid B aggregation alone. In addition, aggregation in the presence of 0.015 M calcium chloride was much higher for toxoid B than toxoid A. This may be related to the C-terminal domain of known increased toxin A thermal stability in the presence of calcium chloride (Demarest et al., Journal of Molecular Biology 346 (5): 1197-1206, 2005). The dissimilarities between toxoids in their responses to solute-induced aggregation are presumably related to structural differences between corresponding toxins (Warny et al., Lancet 367 (9491): 1079-1084, 2005; Just et al. , Reviews of Physiology, Biochemistry and Pharmacology 152: 23-47, 2005). A lack of inositolphosphates among the compounds studied suggests that observed toxoid aggregation does not involve autocatalytic cleavage (Reineke et al., Nature (London, United Kingdom) 446 (7134): 415-419, 2007). Most carbohydrates, detergents, and cyclodextrins examined inhibited toxoid aggregation. The following excipients have been found to effectively inhibit aggregation of both toxoids: 20% trehalose, 20%

<td>sucrose, 10% of glycerol.</td><td>sorbitol,</td><td>10% of</td><td>dextrose,</td><td>and 20% of</td>

<td>The carbohydrates,</td><td>sorbitol,</td><td>glycerol,</td><td>it's two</td><td>surfactants</td>

<td>mentioned above</td><td> (0,05% /</td><td>0.1% of</td><td>Tween 80</td><td>and 0.1% of</td>

<td>pluronic F-68)</td><td colspan="4">have also been studied for their</td>

ability to stabilize the secondary and tertiary structure of proteins at pH 6.5 by monitoring ANS fluorescence, heating CD signal changes, and DSC (Figures 1 and 2). Toxoid A in the presence of 20% sucrose and 20% trehalose produced an early onset of secondary structure change, while the rest of the excipients delayed the thermal transition by ~ 2 ° C (Figure 1a). Surprisingly, toxoid B showed an early onset of secondary structure change only in the presence of 20% sucrose, while the rest of the excipients delayed the thermal transition by ~ 1 ° C (Figure 2a). The early onset of secondary structure alteration of toxoids in the presence of trehalose and / or sucrose can be explained by the stabilization of the partially unfolded state by the solutes. In addition, the possibility that toxoids are partially unfolded by binding to their C-terminal carbohydrate recognition sequence repeats by polysaccharides (Greco et al., Nature Structural &amp; Molecular Biology 3 (5): 460-461, 2006) cannot be deleted. In the case of structural destabilization by the second mechanism, the dissimilarity of behavior of the two toxoids in the presence of trehalose may be related to the structural differences between the toxoids (the C-terminal domain has 30 repeats in toxoid A and 19 repeats in toxoid B; Just et al., Reviews of Physiology, Biochemistry and Pharmacology 152: 23-47, 2005). It is interesting to note that the monosaccharide (dextrose) had a stabilizing effect on the secondary structure of both toxoids. Temperature-induced unfolding of both toxoids and associated ANS binding was not influenced by the presence of compounds (Figure 1b, 2b). The effect of detergents on toxoid temperature-induced unfolding was monitored with DSC and did not appear to be significant (Figure 1c, 2c, and Table 3). These observations suggest that excipients do not strongly stabilize the toxoid structure by the well-described preferential exclusion mechanism, but rather inhibit their aggregation by other mechanisms, such as direct blocking of protein / protein interactions that are responsible for protein association ( Timasheff, Proc Natl Acad Sci USA 99 (15): 9721-9726, 2002;

Timasheff, Advances in Protein Chemistry 51 (Linkage Thermodynamics of Macromolecular Interactions): 355-432, 1998).

To study the effect of combining most active agents on the secondary structure, the results of a mixture of sorbitol, dextrose, and Iween 80 were characterized by monitoring the thermal transitions of CD toxoids and OD aggregation at 350 nm (Figures 3 and 4). Heating of Iween 80 solutions (0.05% or 0.1%) alone or in the presence of sorbitol and / or dextrose led to changes in their micelle structures, which were manifested by a decrease in CD signal and dispersion. increased light when monitored by OD at 350 nm (Figure 5). Excipient concentration had an approximately linear effect on the temperature of thermal transitions (Figure 6). This supports the hypothesis that excipients prevent aggregation by directly inhibiting protein association. The effects of excipients on the thermal transition are summarized in Tables 4 and 5. The combination of 10% dextrose and 10% sorbitol in the presence or absence of 0.05% Tween 80 tends to retard the center point of the thermal transition of both. toxoids largely (~ 4 ° C for toxoid A and ~ 10 ° C for toxoid B) (Figure 3). This may be explained by a synergistic effect and / or a higher total concentration of the stabilizing compounds. In case of toxoid B, the initial transition temperature was not delayed in the presence of the combination of agents, but the central point of the thermal transition was significantly delayed. This may be related to a more gradual unfolding of toxoid B in the presence of two or more excipients. 0 toxoid A manifested a significant aggregation delay (monitored with OD at 350 nm) in the presence of stabilizing compounds (Figure 4a). The hydrodynamic diameter of toxoids in the presence and absence of excipients was also monitored by DLS (Figure 7). Toxoid A manifested a delayed onset of previously observed hydrodynamic diameter increase in the presence of excipients (Figure 7 ac), while a minor effect was seen with toxoid B (Figure 7 df). These observations suggest that the particular combination of potential vaccine excipients tested herein stabilizes protein structure both in a preferential hydration mechanism and in direct inhibition of protein association. The use of such stabilizing compounds can potentially increase the physical stability of toxoids during storage.

Agitation Studies

The effect of agitation on the physical stability of the toxoid was studied by monitoring protein absorption by storage vial walls, formation of insoluble aggregates, and changes in protein thermal stability. An insignificant change in protein concentration, OD at 350 nm, and in CD fusions in the presence and absence of excipients indicated that the toxoids do not undergo fundamental physical changes under the application of this stress based agitation.

Adjuvant Studies

Adjuvant binding isotherms revealed that Alhydrogel® at toxoid concentrations efficiently bound low (aluminum hydroxide adjuvant) saturated binding at the highest protein concentration (Figure 8a). The 0.5 mg / ml toxoids are 95% or more bound to Alhydrogel® (aluminum hydroxide adjuvant), which allows the use of DSC to directly monitor protein stability on the adjuvant surface. The absence of toxoid desorption in the 2 M NaCl addition indicates that the interaction of toxoids with Alhydrogel® (aluminum hydroxide adjuvant) is not only electrostatic since it is generally observed in protein / aluminum hydroxide interactions (Figure 8b Gupta et al., Pharmaceutical Biotechnology 6: 229-248, 1995; Seeber et al., Vaccine 9 (3): 201-203, 1991; White et al. , Developments in Biologicals (Basel, Switzerland) 103 (Physico-Chemical Procedures for the Characterization of Vaccines): 217-228, 2000).

On binding to Alhydrogel® (aluminum hydroxide adjuvant), toxoid A shows no detectable change in this thermal stability, while adjuvant-bound toxoid B shows a decrease in Tm at ~ 1.4 ° C. The Alhydrogel®-bound toxoid fraction (aluminum hydroxide adjuvant) is somewhat reduced in the presence of most excipients (Tables 6 and 7). This suggests that excipients partially interfere with toxoid binding to Alhydrogel® (aluminum hydroxide adjuvant) perhaps by direct interaction with the protein and / or adjuvant. The thermal stability of proteins bound to Alhydrogel® (aluminum hydroxide adjuvant) in the presence and absence of excipients is summarized in table 6 for toxoid A and table 7 for toxoid Β. The presence of excipients disrupted the thermal stability of adjuvant-linked toxoids by decreasing or increasing the transition temperature. A decrease in thermal stability was seen in both toxoids in the presence of 10% sorbitol, while the presence of 10% sorbitol and 10% dextrose decreases the thermal stability of toxoid B alone. Additionally, Tween 80 had a stabilizing effect only in the case of adjuvant-bound toxoid B. On the other hand, dextrose (10%) had a stabilizing effect on thermal stability of both toxoids. Interestingly, the combination of the three excipients (10% sorbitol, 10% dextrose, with 0.05% or 0.1% Tween 80) tends to increase the thermal transition of both adjuvant-linked toxoids to 3%. 4 ° C.

Conclusions

A systematic approach to stabilizer evaluation resulted in the identification of excipients that improved the thermal stability of both Clostridium difficile toxoids A and B. Studies of Alhydrogel®-bound toxoid (aluminum hydroxide adjuvant) in the presence of selected excipients identified conditions that produced improved physical stability of the adjuvant-bound proteins. This study also generated information regarding the physical behavior of toxoids over a range of conditions (temperature, solute) that can be used to designate enhanced storage stability formulations.

Example II

Additional changes to the C. difficile toxoid vaccine formulation were investigated in an effort to improve vaccine stability and immunogenicity profiles. Preclinical and clinical data generated with the vaccine to date have indicated that increased stability and immunogenicity profiles would be important to support future clinical studies.

pH

Determining a pH that yields maximum stability was part of the formulation improvement effort. Studies were performed on liquid samples maintained at -65 ° C, 5 ° C, 25 ° C, and 37 ° C for up to 28 days with pH ranging from 5.5-7.5. The following methods were employed to establish the stability profile:

1. circular dichroism (CD) spectroscopy (changes in secondary structure),

2. circular dichroism (CD) spectroscopy (changes in melting temperature, T_m) , and

3 spectrophotometry (OD350nm) and SEC-HPLC (aggregate formation).

There was no change in secondary structure observed in CD spectrum for toxoid A above pH 6.0 and for toxoid B across the total pH range tested (Figure 9 and Figure 10; Salnikova et al., J. Pharm. Sci. 97 (9): 3735-3752, 2008). Acquired melting point data from CD measurements revealed that the maximum stability of both toxoids is at a pH greater than 7.0 (Figure 11 and Figure 12).

Toxoid A and B aggregation states across a pH range of 6-7.5 varied little in the d-60 ° C range when analyzed by SEC-HPLC for aggregate formation (% monomer) and% recovery area. However, differences in aggregation states become more evident across the pH range when temperatures have been raised (particularly above lower pH by leaning towards aggregation levels. This is accompanied by an increase in optical density by 350 nm at lower pH values, as described in Salnikova et al. , J. Pharm. Know. 97 (9): 3735-3752, 2008.

5 ° C), with larger alteration values

When aggregation was evaluated at different pH values ​​over time at a fixed storage temperature (&lt;-60 ° C) (Figure 13), the results again indicated that aggregation states were very stable at ultra low temperatures with It is suggested that aggregation was promoted at low pH levels (&lt;pH 7.0). With these data in mind, the nominal pH for vaccine volume storage was adjusted to 7.5.

Ionic force

Determining the ionic strength that yields maximum stability was also part of the formulation improvement effort. Studies were performed on liquid samples maintained at -65 ° C, 5 ° C, 25 ° C, and 37 ° C for up to 28 days in 20 mM sodium citrate buffer, pH 7.25, with varying concentrations (0 -300 mM) sodium chloride. Also tested was NaCl replacement with 5% sucrose. The methods employed to establish the stability profile were SEC-HPLC, SDS-PAGE, and visual appearance.

No clear difference was discernible by SDS-PAGE or visible appearance. SEC-HPLC clearly showed toxoid B aggregation at higher salt concentrations. Aggregation in toxoid A appeared to be time and temperature dependent with the only noticeable effect to be seen in 50 mM NaCl. Data indicated that 050 mM sodium chloride or 5% sucrose should be added to achieve maximum toxoid stability (Figure 14 and Figure 15).

Buffer Change and Excipient Addition

A preliminary study was performed to evaluate the effect of buffer and excipient on toxoid stability. Data derived from H-PLC-SEC demonstrated that sorbitol sodium citrate buffer as an excipient provided the greatest stability as determined by the percent recovery of toxoids over time, as shown in table 8 and table 9.

Evaluation and Lyophilized Preparations

To select a lyophilized formulation that would be stable for Phase II clinical studies, we employed a hamster immunogenicity assay because it is the preclinical assay that demonstrates the greatest sensitivity for producing changes that relate to clinical immunogenicity. Data from preliminary studies led to a sodium citrate-based excipient evaluation study as a buffer and sorbitol as a stabilizing excipient. Sucrose was introduced as a stabilizing excipient as a replacement for sorbitol in lyophilized formulations because of the long lyophilization times and low Tg observed in sorbitol formulations. A second lyophilization / excipient evaluation study was also performed using potassium phosphate and trehalose buffer based on data from a parallel study. From these studies and data from previous experiments, three lead formulations, one liquid and two lyophilized, have emerged.

Lyophilized formulations were prepared and their stability evaluated under accelerated conditions and in real time. Toxoids A and B were stored separately to further study their individual stability profiles. Appearance data and hamster immunogenicity data of a formulation (lyophilized, 20 mM citrate, 5% sucrose, 0.016% formaldehyde, pH 7.5) after storage at -65, 5 or 42 ° C for three months are presented below. No to almost no difference in appearance is observed between formulations at baseline and after storage at -65, 5, or 42 ° C for 3 months (Table 10 and Table 11). In addition, the hamster's immune response is not significantly different between formulations stored at 5 ° and 42 ° C for 7 months, or between the same formulations with or without formaldehyde. Storage at 42 ° C is a highly stressed condition and, because no changes are observed during storage, the results indicate that the formulation would most likely be stable considerably longer at lower temperatures. However, a statistically detailed analysis of the data intended to estimate shelf life requires that any quantifiable change be seen so that a rate can be calculated. As no changes have been observed to date, no true rate can be calculated. In light of these data, it was planned to use a Lyophilized Drug Product Formulation consisting of C. difficile toxoids A and B in 20 mM citrate, 5% sucrose, 0.016% formaldehyde, pH 7.5, stored at 2 ° C. -8 ° C.

The formulations used in the stability studies detailed in this report have been prepared using the lyophilization cycle summarized in table 12. This cycle produced distinct white solid masses but was even close to completing primary dryness as determined by the Pirani vacuum reading. decreasing to the same as the gauge vacuum reading (Fiqura 16). The following critical changes have been made to the freeze drying cycle to address this issue and create a more scalable process:

1. The storage temperature was reduced to -35 ° C to ensure that the drug substance remained frozen during primary drying on a larger scale,

2. Primary drying has been extended to 4000 minutes to ensure completion of primary drying on a larger scale, and

3 The vacuum has been increased to 100 mT to expedite drying and allow a more scalable process.

The lyophilization cycle transferred to Althea Technologies, Inc. for clinical BPF batch processing is described in Table 13.

Summary of Physicochemical and Biological Properties

The main physicochemical and biological properties of the vaccine determined experimentally are summarized below.

Chemically, the vaccine is comprised of inactivated forms (toxoids) of C. difficile toxins A and B present in a 3: 2 ratio, respectively. C. difficile toxins A and B are large proteins, 308 kDa and 270 kDa, respectively which are similar but distinct in structure.

Physically, the vaccine is presented as a> 90% pure solution with no evidence of measurable aggregation.

Biochemically, immunologically specific for toxin A or B-reactive toxoids

A and B of the vaccine are their respective antibodies in Western blot analysis.

Biologically, the vaccine is immunogenic in hamsters, producing consistent and dose-dependent serum antibody responses. Vaccine toxoids A and B are devoid of cytotoxic activity. The toxoid A component of the vaccine maintains some receptor binding activity, such as that observed for native toxin A.

The vaccine is presented as a lyophilized form in a buffer composed of 20 mM sodium citrate, pH 7.5, 5% sucrose, 0.016% formaldehyde. The product is stored at 2-8 ° C.

Lyophilization Evaluation Studies

To evaluate the lyophilized formulations a hamster immunogenicity was employed for evaluation. Lyophilization occurred on a LyoStar II FTS. Freezing was completed by reducing the storage temperature low enough to force the product temperature to fall below Tg '. Primary drying began by pulling a vacuum and holding until free water was sublimated. The storage temperature was then increased to initiate secondary drying and also to keep the product dry by extracting the absorbed water. The formulations were stable under temperature conditions of 5, 25, and 42 ° C.

Table 1. Effect of GRAS excipients on toxoid aggregation A. Compounds that retard / prevent aggregation have% positive aggregation inhibition values; Compounding-inducing compounds have negative% aggregation inhibition values.

<td>Excipient Concentration</td><td>% inhibition of aggregation</td><td> 40</td><td>Excipient Concentration</td><td>% inhibition of aggregation</td>

<td>2.5% Albumin</td><td> 103*</td><td></td><td>Glycerol 10%</td><td> 88</td>

<td>α 2.5% cyclodextrin</td><td> 101*</td><td></td><td>2-OH propyl 10% γ-CD</td><td> 81</td>

<td>0.1% Tween 80</td><td> 100*</td><td></td><td>0.05% Tween 20</td><td> 73</td>

<td>0.3 M Diethanolamine</td><td> 100</td><td> 45</td><td>0.05% Tween 80</td><td> 67</td>

<td>0.1 M Sodium Citrate</td><td> 100</td><td></td><td>0.15 M Aspartic Acid</td><td> 65</td>

<td>10% sorbitol</td><td> 100</td><td></td><td>0.1% Tween 20</td><td> 64</td>

<td>0.3 M Histidine</td><td> 100*</td><td></td><td>Pluronic F - 68 to 0.05%</td><td> 50</td>

<td>10% sucrose</td><td> 100</td><td></td><td>0.01% Tween 20</td><td> 37</td>

<td>10% Trealose</td><td> 100</td><td> 50</td><td>0.04 mg / mL Dextran Sulfate</td><td> 30</td>

<td>0.3 M Guanidine</td><td> 99</td><td></td><td>0.05% Brij 35</td><td> 26</td>

<td>Sorbitol 20%</td><td> 99</td><td></td><td>Dextran Sulfate 0.004 mg / ml</td><td> 16</td>

<td>Dextrose 20%</td><td> 99</td><td></td><td>2-OH 5% y-CD propyl</td><td> 10</td>

<td>Dextrose 10%</td><td> 99</td><td></td><td>5% albumin</td><td> 9</td>

<td>Trealose 20%</td><td> 99</td><td> 55</td><td>Brij 35 at 0.01%</td><td> -2</td>

<td>0.2 M Sodium Citrate</td><td> 99</td><td></td><td>0.015M Calcium Chloride</td><td> -7</td>

<td>20% glycerol</td><td> 98</td><td></td><td>0.01% F-68 Pluronic</td><td> -14</td>

<td>0.01% Tween 80</td><td> 98</td><td></td><td>5% gelatin</td><td> -4 6</td>

<td>Albumin 1%</td><td> 98</td><td></td><td>0.15 M Malic Acid</td><td> -52</td>

<td>Lactose 20%</td><td> 98</td><td> 60</td><td>0.15 M lactic acid</td><td> -72</td>

<td>10% Mannitol</td><td> 97</td><td></td><td>2.5% Gelatin</td><td> -74</td>

<td>Sucrose 20%</td><td> 97</td><td></td><td>0.15 M Glutamic Acid</td><td> -77</td>

<td>0.1% F-68 Pluronic</td><td> 96</td><td></td><td>Dextran T40 at 0.003 mg / ml</td><td> -87</td>

<td>2-OH 10% β-CD propyl</td><td> 96</td><td></td><td>0.3 M Glycine</td><td> -88</td>

<td>2-OH 5% β-CD propyl</td><td> 96</td><td> 65</td><td>0.1 mg / ml Dextran Sulphate</td><td> -88</td>

<td>0.08 mg / mL dextran T40</td><td> 95</td><td></td><td>0.15 M Ascorbic Acid</td><td> -99</td>

<td>0.1% Brij 35</td><td> 95</td><td></td><td>0.3 M Proline</td><td> -112</td>

<td>Dextran T40 0.03 mg / ml</td><td> 93</td><td></td><td>0.3 M Arginine</td><td> -265</td>

<td>Lactose 10%</td><td> 92</td><td></td><td>Arg / Glu 50 mM each</td><td> -426</td>

<td>Excipient Concentration</td><td>% inhibition of aggregation</td><td> 40</td><td>Excipient Concentration</td><td>% inhibition of aggregation</td>

<td>0.3 M Lysine</td><td> 89*</td><td> 70</td><td>Arg / Glu at 25 mM each</td><td> -463</td>

Uncertainties are in the order of ± 1%. * 0D at 350 nm initial high.

Table 2. Effect of GRAS excipients on toxoid B aggregation. Compounds that retard / prevent aggregation have% positive aggregation inhibition values; Compounding-inducing compounds have negative% aggregation inhibition values.

<td>Excipient</td><td>% in</td><td></td><td>Excipient</td><td>% in</td>

<td>Concentration</td><td>Inhibition</td><td></td><td>Concentration</td><td>Inhibition</td>

<td></td><td>of aggregation</td><td></td><td></td><td>of aggregation</td>

<td></td><td>dog</td><td></td><td></td><td>dog</td>

<td></td><td></td><td> 40</td><td></td><td></td>

<td>α 2.5% cyclodextrin</td><td> 100</td><td></td><td>Dextran T40 0.03 mg / ml</td><td> 35</td>

<td>0.3 M Histidine</td><td> 100*</td><td></td><td>Dextran T40 0.08 mg / ml</td><td> 25</td>

<td>0.1% Tween 80</td><td> 100</td><td></td><td>2-OH propyl 10% γ-CD</td><td> 12</td>

<td>0.05% Tween 80</td><td> 100</td><td></td><td>0.05% Brij 35</td><td> 10</td>

<td>1% Albumin</td><td> 99</td><td> 45</td><td>0.05% F-68 Pluronic</td><td> 6</td>

<td>20% dextrose</td><td> 99</td><td></td><td>10% glycerol</td><td> 4</td>

<td>5% albumin</td><td> 98</td><td></td><td>0.04 mg / ml Dextran Sulfate</td><td> 3</td>

<td>0.2 M Sodium Citrate</td><td> 98*</td><td></td><td>Dextran Sulfate 0.004 mg / ml</td><td> 2</td>

<td>Trealose 20%</td><td> 98</td><td></td><td>0.05% Tween 20</td><td> 0</td>

<td>0.1 M Sodium Citrate</td><td> 97</td><td> 50</td><td>2-OH 5% propyl γ-CD</td><td> -5</td>

<td>20% sorbitol</td><td> 97</td><td></td><td>0.01% Tween 20</td><td> -5</td>

<td>20% sucrose</td><td> 96</td><td></td><td>0.01% F-68 Pluronic</td><td> -8</td>

<td>0.3 M Diethanolamine</td><td> 96</td><td></td><td>0.1% Tween 20</td><td> -13</td>

<td>10% dextrose</td><td> 95</td><td></td><td>Brij 35 at 0.01%</td><td> -25</td>

<td>10% sorbitol</td><td> 92</td><td> 55</td><td>0.3 M Glycine</td><td> -26</td>

<td>2.5% Albumin</td><td> 87</td><td></td><td>2.5% Gelatin</td><td> -36</td>

<td>2-OH 5% β-D propyl</td><td> 79</td><td></td><td>0.1 mg / ml Dextran Sulphate</td><td> -38</td>

<td>Excipient</td><td>% in</td><td></td><td>Excipient</td><td>% in</td>

<td>Concentration</td><td>Inhibition</td><td></td><td>Concentration</td><td>Inhibition</td>

<td></td><td>of aggregation</td><td></td><td></td><td>of aggregation</td>

<td></td><td>dog</td><td></td><td></td><td>dog</td>

<td>2-OH 10% β-D propyl</td><td> 78</td><td></td><td>Arg / Glu 50 mM each</td><td> -39</td>

<td>10% Mannitol</td><td> 76</td><td></td><td>0.15 M glutamic acid</td><td> -41</td>

<td>10% sucrose</td><td> 71</td><td> 60</td><td>Arg / Glu 25 mM each</td><td> -42</td>

<td>20% glycerol</td><td> 71</td><td></td><td>0.15 M Aspartic Acid</td><td> -50</td>

<td>10% Trealose</td><td> 69</td><td></td><td>5% gelatin</td><td> -56</td>

<td>0.1% F-68 Pluronic</td><td> 68</td><td></td><td>0.3 M Proline</td><td> -57</td>

<td>0.1% Brij 35</td><td> 63</td><td></td><td>Dextran T40 at 0.003 mg / ml</td><td> -59</td>

<td>Lactose 20%</td><td> 52</td><td> 65</td><td>0.15 M lactic acid</td><td> -80</td>

<td>0.15 M malic acid</td><td> 44</td><td></td><td>0.3 M Guanidine</td><td> -96</td>

<td>0.01% Tween 80</td><td> 40</td><td></td><td>0.015 M Calcium Chloride</td><td> -141</td>

<td>10% lactose</td><td> 39</td><td></td><td>0.15 M Ascorbic Acid</td><td> -223</td>

<td>0.3 M Lysine</td><td> 37</td><td></td><td>0.3 M Arginine</td><td> -280</td>

Uncertainties are in the order of ± 1%. * OD at 350 nm initial high.

Table 3. Effect of solutes toxoid A and thermal stability (Tm) were monitored by temperature corresponding to the thermal transition position.

(detergents) in a Β. The stability

DSC. Tm is the maximum peak of the

<td>Protein</td><td>Tm (° C)</td>

<td>Toxoid A</td><td> 59,8 ± 0,0</td>

<td>Toxoid A + 0.05% tween 80</td><td> 59,1 ± 0,4</td>

<td>Toxoid A + 0.1% pluronic F68</td><td> 59,1 ± 0,4</td>

<td>Toxoid B</td><td> 55,8 ± 0,0</td>

<td>Toxoid B + 0.05% tween 80</td><td> 56,1 ± 0,3</td>

<td>Toxoid B + 0.1% F68 pluronic</td><td> 58,0 ± 0,3</td>

Table 4. Effect of excipients at central point of toxoid A of thermal transition temperature (Tm). The thermal transition was monitored by the CD signal at 208 nm as a function of temperature. Each measurement was conducted in duplicate and has ~ 0.5 ° C uncertainty.

<td>Toxoid A in the presence of excipient (s)</td><td>Tm</td><td>Difference from Tm</td>

<td>Toxoid A</td><td> 59,8</td><td>O O</td>

<td>20% trehalose</td><td> 59, 4</td><td>1 O Cn</td>

<td>20% sucrose</td><td> 63,1</td><td> 3,3</td>

<td>20% glycerol</td><td> 62,4</td><td> 2,6</td>

<td>0.1% F68 pluronic</td><td> 61,1</td><td> 1,3</td>

<td>10% sorbitol</td><td> 62,4</td><td> 2,6</td>

<td>10% dextrose</td><td> 62,4</td><td> 2,6</td>

<td>0.05% Tween 80</td><td> 59, 9</td><td>O O</td>

<td>5% sorbitol + 0.05% Tween 80</td><td> 60,4</td><td> 0, 6</td>

<td>10% sorbitol + 0.05% Tween 80</td><td> 62,3</td><td> 2,5</td>

<td>15% sorbitol + 0.05% Tween 80</td><td> 62,4</td><td> 2,6</td>

<td>5% dextrose + 0.05% Tween 80</td><td> 60,2</td><td> 0,4</td>

<td>10% dextrose + 0.05% Tween 80</td><td> 62,3</td><td> 2,5</td>

<td>15% dextrose + 0.05% Tween 80</td><td> 62,5</td><td> 2,7</td>

<td>2.5% sorbitol + 2.5% dextrose + 0.05% from Tween 80</td><td> 60,2</td><td> 0,4</td>

<td>5% sorbitol + 5% dextrose + 0.05% Tween 80</td><td> 61,3</td><td> 1,5</td>

<td>10% sorbitol + 10% dextrose + 0.05% Tween 80</td><td> 63,5</td><td> 3,7</td>

<td>0.1% Tween 80</td><td> 59,0</td><td>1 O co</td>

<td>10% sorbitol + 0.1% Tween 80</td><td> 60, 6</td><td> 0,7</td>

<td>Toxoid A in the presence of excipient (s)</td><td>Tm</td><td>Difference from Tm</td>

<td>10% dextrose + 0.1% Tween 80</td><td> 61,5</td><td> 1,7</td>

<td>Sorbitol 2.5% + dextrose 2.5% + 0.1% dextrose Tween 80</td><td> 60,0</td><td> 0,2</td>

<td>5% sorbitol + 5% dextrose + 0.1% Tween 80</td><td> 60,5</td><td> 0,7</td>

<td>10% sorbitol + 10% dextrose + 0.1% Tween 80</td><td> 62,1</td><td> 2,3</td>

<td>2.5% sorbitol + 2.5% dextrose</td><td> 60,8</td><td> 0, 9</td>

<td>5% sorbitol + 5% dextrose</td><td> 61,7</td><td>I—¹co</td>

<td>10% sorbitol + 10% dextrose</td><td> 63, 9</td><td> 4,0</td>

<td>15% sorbitol + 15% dextrose</td><td> 64,6</td><td>4 ^ CO</td>

<td>20% sorbitol + 20% dextrose</td><td> 66, 6</td><td> 6,7</td>

<td>20% sorbitol + 10% dextrose</td><td> 64,9</td><td> 5,1</td>

<td>10% sorbitol + 20% dextrose</td><td> 50,1</td><td> -9,7</td>

Table 5. Effect of excipients at the central point of toxoid B of thermal transition temperature (Tm). The thermal transition was monitored by the CD signal at 208 nm as a function of temperature. Each measurement was conducted in duplicate and has ~ 0.5 ° C uncertainty.

<td>Toxoid B in the presence of excipient (s)</td><td>Tm</td><td>Difference from Tm</td>

<td>Toxoid B</td><td> 55,8</td><td>O O</td>

<td>20% trehalose</td><td> 60,3</td><td> 4,5</td>

<td>20% sucrose</td><td> -</td><td> -</td>

<td>20% glycerol</td><td> 58,6</td><td> 2,8</td>

<td>0.1% F68 pluronic</td><td> 56,2</td><td> 0,4</td>

<td>Toxoid B in the presence of excipient (s)</td><td>Tm</td><td>Difference from Tm</td>

<td>10% sorbitol</td><td> 56,6</td><td>O co</td>

<td>10% dextrose</td><td> 57,3</td><td> 1,6</td>

<td>0.05% Tween 80</td><td> 55,1</td><td> -0,7</td>

<td>5% sorbitol + 0.05% Tween 80</td><td> 56,7</td><td> 0, 9</td>

<td>10% sorbitol + 0.05% Tween 80</td><td> 63,2</td><td> 7,4</td>

<td>15% sorbitol + 0.05% Tween 80</td><td> 64,0</td><td> 8,2</td>

<td>5% dextrose + 0.05% Tween 80</td><td> 59,1</td><td> 3,3</td>

<td>10% dextrose + 0.05% Tween 80</td><td> 70,8</td><td> 15,0</td>

<td>2.5% sorbitol + 2.5% dextrose + 0.05% from Tween 80</td><td> 56,7</td><td> 0, 9</td>

<td>5% sorbitol + 5% dextrose + 0.05% Tween 80</td><td> 63,5</td><td> 7,7</td>

<td>10% sorbitol + 10% dextrose + 0.05% from Tween 80</td><td> 69,1</td><td> 13,3</td>

<td>0.1% Tween 80</td><td> 53,6</td><td> -2,2</td>

<td>10% sorbitol + 0.1% Tween80</td><td> 58,5</td><td> 2,7</td>

<td>10% dextrose + 0.1% Tween80</td><td> 62,6</td><td> 6, 9</td>

<td>2.5% sorbitol + 2.5% dextrose + 0.1% from Tween 80</td><td> 60,0</td><td> 4,2</td>

<td>5% sorbitol + 5% dextrose + 0.1% Tween 80</td><td> 56,5</td><td>O CO</td>

<td>10% sorbitol + 10% dextrose + 0.1% Tween 80</td><td> 60,8</td><td> 5,1</td>

<td>5% sorbitol + 5% dextrose</td><td> 56,5</td><td> 0,7</td>

<td>10% sorbitol + 10% dextrose</td><td> 65,3</td><td> 9, 6</td>

<td>15% sorbitol + 15% dextrose</td><td> 60,1</td><td> 4,4</td>

<td>20% sorbitol + 20% dextrose</td><td> 63,1</td><td> 7,3</td>

<td>20% sorbitol + 10% dextrose</td><td> 38,0</td><td> -17,8</td>

<td>Toxoid B in the presence of excipient (s)</td><td>Tm</td><td>Difference from Tm</td>

<td>10% sorbitol + 20% dextrose</td><td> 61,2</td><td> 5,5</td>

Table 6. Thermal stability of Alhydrogel®-bound toxoid A (aluminum hydroxide adjuvant) in the presence and absence of excipient (s) (unless otherwise specified). A thermal stability (Tm) was monitored by DSC at Tm indicating the temperature corresponding to the peak position of the thermal transition. The percentage of adjuvant-bound toxoid was measured under each condition with an uncertainty of 1%. Each condition was studied in duplicate.

<td>Alhydrogel®-bound toxoid A (hydroxide adjuvant of aluminum) in the presence of solutes</td><td>% in protein on</td><td>Tm</td><td>Difference from Tm</td>

<td>Toxoid A unbound</td><td> -</td><td> 58,8 ± 0,4</td><td> 0,1</td>

<td>Toxoid A</td><td> 96</td><td> 58,7 ± 0,3</td><td> -</td>

<td>10% sorbitol</td><td> 81</td><td> 52,4 ± 1,5</td><td> -6,2</td>

<td>10% dextrose</td><td> 86</td><td> 60,6 ± 0,5</td><td> 2,0</td>

<td>0.05% Tween 80</td><td> 93</td><td> 58,7 ± 0,1</td><td>O O</td>

<td>10% sorbitol + 10% dextrose</td><td> 77</td><td> 62,9 ± 0,0</td><td> 4,2</td>

<td>10% sorbitol + 10% dextrose + 0.05% Tween 80</td><td> 81</td><td> 63,1 ± 1,2</td><td> 4,2</td>

<td>10% sorbitol + 10% dextrose + 0.1% Tween 80</td><td> 74</td><td> 62,0 ± 1,3</td><td> 3,3</td>

<td>10% sorbitol + 0.05% Tween 80</td><td> 86</td><td> 59,3 ± 0,8</td><td> 0,7</td>

<td>10% dextrose + 0.05% Tween</td><td> 85</td><td> 59,5 ± 1,1</td><td>O co</td>

<td>Alhydrogel®-bound toxoid A</td><td>% in</td><td>Tm</td><td>Difference</td>

<td>(hydroxide adjuvant of</td><td>protein</td><td></td><td>from Tm</td>

<td>aluminum) in the presence of solutes</td><td>on</td><td></td><td></td>

<td> 80</td><td></td><td></td><td></td>

Table 7. The thermal stability of Alhydrogel®-bound toxoid B (aluminum hydroxide adjuvant) in the presence and absence of solute (s) (unless otherwise specified). The monitored by DSC.

thermal stability (Tm) was A Tm indicates the temperature corresponding to the peak position of the thermal transition. The percentage of adjuvant-bound protein was measured under each condition with an uncertainty of 1%. Each condition was studied in duplicate.

<td>Toxoid B linked to Alhydrogel®</td><td>% in</td><td>Tm</td><td>Difference</td>

<td>(hydroxide adjuvant of</td><td>protein</td><td></td><td>from Tm</td>

<td>aluminum) in the presence of solutes</td><td>on</td><td></td><td></td>

<td>Toxoid B not connected</td><td> -</td><td> 56,2 ± 0,4</td><td> 1,4</td>

<td>Toxoid B</td><td> 99</td><td> 54,8 ± 0,5</td><td> -</td>

<td>10% sorbitol</td><td> 92</td><td> 52,5 ± 1,4</td><td> -2,2</td>

<td>10% dextrose</td><td> 96</td><td> 57,9 ± 0,2</td><td> 3,1</td>

<td>0.05% Tween 80</td><td> 96</td><td> 58,2 ± 0,6</td><td> 3,4</td>

<td>10% sorbitol + 10% dextrose</td><td> 95</td><td> 54,2 ± 0,9</td><td>1 O Cn</td>

<td>10% sorbitol + 10% dextrose</td><td> 99</td><td> 58,0 ± 2,8</td><td> 3,3</td>

<td>+ 0.05% Tween 80</td><td></td><td></td><td></td>

<td>10% sorbitol + 10% dextrose</td><td> 77</td><td> 58,7 ± 1,0</td><td> 3,9</td>

<td>+ 0.1% Tween 80</td><td></td><td></td><td></td>

<td>10% sorbitol + 0.05% Tween</td><td> 96</td><td> 58,5 ± 1,0</td><td> 3, 8</td>

<td> 80</td><td></td><td></td><td></td>

<td>10% dextrose + 0.05% Tween</td><td> 92</td><td> 55,8 ± 2,5</td><td> 1,1</td>

<td>Toxoid B linked to Alhydrogel®</td><td>% in</td><td>Tm</td><td>Difference</td>

<td>(hydroxide adjuvant of</td><td>protein</td><td></td><td>from Tm</td>

<td>aluminum) in the presence of solutes</td><td>on</td><td></td><td></td>

<td> 80</td><td></td><td></td><td></td>

Table 8

Toxoid A

Months Stability% Toxoid Recovery

<img file="PT2198007T_D0001.tif" />

Table 9

Toxoid B

Months Stability% Toxoid Recovery

<img file="PT2198007T_D0002.tif" />

Table 10. Appearance of a Lyophilized Formulation after storage at different temperatures

<td rowspan="2">Molecule</td><td colspan="2"></td><td colspan="3">Appearance After 3 Months at:</td>

<td></td><td>Initiation</td><td></td><td>Ι !!!!! βϊ®ΙΙΪΙΪΙί</td><td></td>

<td rowspan="3">Toxoid A</td><td>Appearance of the mass</td><td>shrunk ment Minimum White</td><td>shrunk ment Minimum White</td><td>shrunk ment Minimum White</td><td>shrunk ment Minimum White</td>

<td>Time to Reconstitute tution</td><td>&lt;10 sec</td><td>&lt;1 min</td><td>&lt;1 min</td><td>&lt;1 min</td>

<td>Appearance Net</td><td>none particulate colorless, clear</td><td>none particularly side colorless, clear</td><td>none particulate colorless, clear</td><td>none particulate colorless, clear</td>

<td rowspan="3">Toxoid B</td><td>Appearance Net</td><td>shrunk ment Minimum White</td><td>Collapse Light, white on base</td><td>Collapse Light, white on base</td><td>shrunk ment Minimum White</td>

<td>Time to Reconstitute tution</td><td>&lt;10 sec</td><td>&lt;1 min</td><td>&lt;1 min</td><td>&lt;1 min</td>

<td>Appearance Net</td><td>none particulate colorless, clear</td><td>none particularly side colorless, clear</td><td>none particulate colorless, clear</td><td>none particulate colorless, clear</td>

Table 11. Immunogenicity of a Lyophilized Formulation after storage at different temperatures (serum IgG and anti-toxin A and B titrations in hamsters).

<td>Mo- lecu over there</td><td>Description of Formulation: Relationship A: B = 3: 2, Protein Total = 0.4 mg / mL</td><td colspan="3">20 mM citrate, 5% sucrose, pH 7.5 Freeze Dried</td><td colspan="3">20 mM citrate, 5% sucrose, 0.016% H₂CO, pH 7.5 Lyophilized</td>

<td></td><td>Temp. in Storage</td><td>5 ° C</td><td>25 ° C</td><td>42 ° C</td><td>5 ° C</td><td>25 ° C</td><td>42 ° C</td>

<td></td><td></td><td colspan="3">Average Logon</td><td colspan="3">Average Logon</td>

<td rowspan="5">All- xoi- from A</td><td>1 month</td><td> 5,34</td><td> -</td><td> 5,26</td><td> -</td><td> -</td><td> -</td>

<td>3 months</td><td> 5,57</td><td> -</td><td> 5,56</td><td> -</td><td> -</td><td> 5, 42</td>

<td>Five months</td><td> 5,33</td><td> 5,23</td><td> -</td><td> -</td><td> 5,30</td><td> -</td>

<td>7 months</td><td> -</td><td> 5,49</td><td> 5,41</td><td> 5,47</td><td> 5, 45</td><td> -</td>

<td>Log Change</td><td> -0,01</td><td> 0,26</td><td> 0,15</td><td> -</td><td> 0,15</td><td> -</td>

<td rowspan="5">All- xoi- from B</td><td>1 month</td><td> 5,49</td><td> -</td><td> 5, 62</td><td> -</td><td> -</td><td> -</td>

<td>3 months</td><td> 5,54</td><td> -</td><td> 5,42</td><td> -</td><td> -</td><td> 5,23</td>

<td>Five months</td><td> 5, 62</td><td> 5,34</td><td> -</td><td> -</td><td> 5, 49</td><td> -</td>

<td>7 months</td><td> -</td><td> 5,49</td><td> 5, 62</td><td> 5,70</td><td> 5, 78</td><td> -</td>

<td>Log Change</td><td> 0,13</td><td> 0,15</td><td> 0</td><td> -</td><td> 0,29</td><td> -</td>

H2CO = formaldehyde

Table 12

<td>|||||||||||||||||||||| B</td><td></td><td></td><td> 1</td><td>B</td><td> §</td><td>B</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

Drop in temperature from 1 ° C / min to 5 ° C. Kept for 30 minutes.

Drop in temperature from 1 ° C / min to -5 ° C. Kept for 30 minutes.

Temperature drop at 5 ° C / min to -50 ° C. Kept for 90 minutes.

<td>Connect the vacuum with a 60 mT attachment point.</td>

<td></td>

<td></td>

<td>Drop in temperature from 1 ° C / min to -40 ° C.</td>

<td>Kept for 300 minutes. Vacuum equal to 60 mT.</td>

<td>Temperature drop at 0.5 ° C / min to -34 ° C.</td>

<td>Kept for 1100 minutes.</td>

<td>60 mT vacuum</td>

<td> 11111111111111111111111¾</td>

<td>Temperature drop at 0.2 ° C / minute at 5 ° C. Kept for 480 minutes. Vacuum equal to 60 mT. *</td>

<td>Temperature drop at 0.1 ° C / minute at 25 ° C. Kept for 300 minutes. Vacuum equal to 60 mT.</td>

<td> 111111111!!!!!·</td>

<td>Temperature drop at 0.5 ° C / min at 0 ° C. Kept for 9999 minutes. Vacuum equal to 100 mT.</td>

<td>Illllllie</td>

<td>Reload at 600,000 mT with dry purified N2.</td>

<td></td>

Table 13

Drop in temperature from 1 ° C / min to 5 ° C. Kept for 30 minutes.

Temperature drop at 0.5 ° C / min to -45 ° C. Kept for 120 minutes.

Vacuum connected with 100 mT attachment point

Temperature drop at 0.2 ° C / min to -35 ° C. Kept for 4000 minutes. Vacuum attachment point at 100 mT

111111111111111111111111¾^

Temperature drop at 0.2 ° C / minute at 5 ° C. Kept for 480 minutes. Vacuum attachment point at 100 mT

Temperature drop at 0.2 ° C / min at 25 ° C. Kept for 300 minutes. Vacuum attachment point at 100 mT

11111111111111111111111¾^^^^

Temperature drop at 0.5 ° C / min at 0 ° C. Kept for 9999 minutes. Vacuum attachment point at 100 mT

SfiEf

Reload to 600,000 mT with dry purified N2

Use of singular forms here, such as a and / or, does not exclude the indication of the corresponding plural form unless the context indicates otherwise. Thus, for example, if a claim indicates the use of a toxin, toxoid, or excipient, it may also be interpreted as encompassing the use of more than one toxin, toxoid, or excipient, unless otherwise indicated. Other embodiments are in the following claims.