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Anthrax Vaccine Adsorbed
» Anthrax Vaccine Adsorbed is a sterile, milky-white suspension made from cell-free filtrates of microaerophilic cultures of an avirulent, nonencapsulated strain of Bacillus anthracis. The final product contains no dead or live bacteria. The production cultures are grown in a chemically defined protein-free medium containing amino acids, vitamins, inorganic salts, and sugars. The sterile filtrate is adsorbed on sterile aluminum hydroxide, concentrated 10-fold, and resuspended in sterile physiological saline containing formaldehyde with benzethonium chloride as a preservative. Sublots may be combined to produce final lots. The product meets potency requirements when tested against the U.S. Reference Standard Anthrax Vaccine, in accordance with approved procedures (guinea pig intracutaneous challenge models).
Packaging and storage— Preserve in multiple-dose tight Type I glass containers, and store at a temperature between 2 and 8. Do not freeze.
Expiration date— The expiration date is 18 months from the date of manufacture.
Labeling— Label it to state that it is to be well shaken before use and that it is not to be frozen.
FILTRATE—
Identification—
Trichloroacetic acid solution— Prepare a solution of trichloroacetic acid (see Reagent Specifications in the section Reagents, Indicators, and Solutions) in water containing 100 g trichloroacetic acid per 100 mL of the solution.
Sample buffer— Prepare a solution containing 141 mM tris(hydroxymethyl)aminomethane, 106 mM tris(hydroxymethyl)aminomethane hydrochloride, 0.51 mM edetate disodium, 2% (w/v) dodecyl lithium sulfate, 10% (v/v) glycerol, 0.22 mM Coomassie blue G-250, and 0.175 mM phenolsulfonphthalein. If necessary, adjust with hydrochloric acid or sodium hydroxide to a pH of 8.5.
Running buffer— Prepare a solution containing 25 mM tris(hydroxymethyl)aminomethane, 192 mM glycine, and 0.1% (w/v) dodecyl sodium sulfate (see Reagent Specifications in the section Reagents, Indicators, and Solutions) in water. If necessary, adjust with hydrochloric acid or sodium hydroxide to a pH of 8.5.
Transblotting buffer— Prepare a solution containing 12.5 mM tris(hydroxymethyl)aminomethane, 96 mM glycine, and 10% (v/v) methanol. If necessary, adjust with hydrochloric acid or sodium hydroxide to a pH of 8.0.
Blocking buffer— Prepare a solution containing 10 mM monobasic sodium phosphate, 150 mM sodium chloride, 5% (w/v) nonfat dry milk, and 0.05% (w/v) Polysorbate 20. Adjust with sodium hydroxide to a pH of 7.4.
Primary antibody solutions— Prepare suitable monoclonal antibodies raised against the Protective Antigen (PA), the Lethal Factor (LF), and the Edema Factor (EF), respectively, of Bacillus anthracis in murine ascites cells, harvested, and used without further purification. Immediately before use, dilute each of the murine ascites fluids containing the monoclonal antibodies 1:1000 (v/v) with the Blocking buffer.
Secondary antibody solution— Immediately before use, dissolve according to the manufacturer's instructions, if necessary, and dilute the stock horseradish peroxidase conjugated to goat anti-mouse IgG solution 1:1000 with Blocking buffer.
Chromogenic visualization solution— Prepare a 150 mg per mL solution of 4-chloro-1-naphthol in water.
Test solution— Use Anthrax Vaccine Filtrate as is.
Procedure— In a suitable centrifuge tube transfer 30/c mL of the Test solution, where c is the total protein concentration, in µg per mL, of the solution as determined in the test for Total protein. Add 16.5/c mL of Trichloroacetic acid solution, and incubate for at least 10 minutes. Centrifuge at 9000g for about 10 minutes, decant off the supernatant, and hold the tube inverted to drain on a filter paper. Dissolve the pellet in about 60 µL of Sample buffer, and transfer the solution to a polypropylene microfuge tube that has a lid. Close the lid tightly, secure with a lid-lock, and heat at 100 for 5 minutes. Allow the solution to cool to room temperature, and centrifuge at 10,000g for 15 seconds to collect the liquids. In a suitable device for polyacrylamide-gel electrophoresis (see Electrophoresis 726 and the section Polyacrylamide Gel Electrophoresis under Biotechnology-Derived Articles—Tests 1047) add appropriate volumes of the Running buffer in the upper and the lower buffer chambers. Attach a 4%–20% gradient tris-glycine polyacrylamide slab gel sandwiched between two glass plates, such that the wells for sample application are exposed to the Running buffer in the upper buffer chamber. Apply about 20-µL aliquots of the treated Test solution in three alternate lanes. [Note—Do not apply any solution in the outside lanes.] Connect the lower buffer chamber electrode to the positive terminal and the upper buffer chamber electrode to the negative terminal of a suitable power supply unit, and carry out the electrophoresis at a constant current of about 40 mA. When the dye-front is about 1 cm from the bottom of the gel (about 40 minutes), stop the current, and remove the gel from the gel assembly. [Note—Do not touch the gel with bare hand. Use gloves.]
Place 3–4 filter papers, cut to the size of the gel and soaked in the Transblotting buffer, on the anode plate of a suitable semidry electroblotter. Cut a nitrocellulose membrane to the same size as the gel plus 1–2 mm on each side, and “wet” the membrane by immersing it into the Transblotting buffer for about 15 seconds, such that there is no air-bubble between the buffer and the membrane. Place the “wet” membrane immediately on the stack of filter papers, and remove all air bubbles between the membrane and filter paper by rolling a pipet, or equivalent, gently over the surface of the membrane. Place a few drops of the Transblotting buffer on the membrane, and then carefully place the gel on it. Gently roll a pipet, or equivalent, over the surface of the gel to ensure intimate contact between the gel and the membrane, making sure that there are no air bubbles in between. Place a filter paper cut to the size of the gel and soaked in the Transblotting buffer, such that there is no air-bubble between the filter paper and the gel. Place 2–3 additional filter papers, prepared in a similar manner, on the top, and complete the transfer stack by placing the cathode plate on the top. Apply a current of about 250 mA, and continue transfer for 90 minutes.
Remove the membrane, and wash it quickly by immersing into water for 15 seconds. [Note—Do not touch the membrane with bare hand. Use gloves.] Cut the membrane into three strips such that each strip contains a lane containing the Test solution, and mark the strips as PA, LF, and EF at the top. Place each strip in a heat-sealable bag, add 5 mL of Blocking buffer, and seal the bag. Incubate for 30 minutes with constant agitation. Open each bag, and pour out the Blocking buffer. Add 9 mL of the diluted Primary antibody solution against PA to the bag containing the strip marked PA. Similarly, add 9 mL of the diluted Primary antibody solution against LF and EF to the bags containing strips labeled LF and EF, respectively. Seal the bags, and incubate under agitation for 2 hours at room temperature or overnight at 2 to 8. Remove the strips from the plastic bags, and place in separate plastic boxes. Add sufficient Blocking buffer so that each strip is completely immersed. Agitate for at least 30 minutes at room temperature with two changes of Blocking buffer. Remove the strips, and place each strip in a new heat-sealable plastic bag. Add 9 mL of the Secondary antibody solution to each plastic bag. Seal the bags, and incubate for 1 hour at room temperature under agitation. Remove the strips from the plastic bags, and place in separate plastic boxes. Add sufficient Blocking buffer so that each strip is completely immersed. Agitate for at least 30 minutes at room temperature with two changes of the Blocking buffer. Transfer each strip into a new heat-sealable plastic bag, add 9 mL Chromogenic visualization solution, 10 µL of 30% (v/v) hydrogen peroxide, and seal the bags. Incubate for about 30 minutes under agitation. Transfer the strips into separate plastic boxes, and remove the excess 4-chloro-1-naphthol by incubating with water under agitation for 10 minutes. Visual observation indicates a strong positive band on the strip labeled PA (Protective Antigen), a faintly detectable band on the strip labeled LF (Lethal Factor), and no detectable band on the strip labeled EF (Edema Factor).
83 kDA protein—
Trichloroacetic acid solution, Sample buffer, Running buffer, and Test solution— Prepare as directed under Identification.
Staining solution— Prepare a solution of Coomassie blue G-250 having a concentration of 1.25 g per L in a mixture of water, methanol, and acetic acid (5:4:1, v/v).
Protein molecular weight standard solution— Reconstitute a vial of protein molecular weight standard mixture containing proteins of molecular weights at least in the range of 14 to 200 kDa, according to manufacturer's instruction. Dilute the solution with Sample buffer such that the concentration of each protein in the solution is about 0.5 µg per µL.
Procedure— In a suitable centrifuge tube transfer 10/c mL of the Test solution, where c is the total protein concentration, in µg per mL, of the solution as determined by the test for Total protein (see below). Add 5.5/c mL of Trichloroacetic acid solution, and incubate for at least 10 minutes. Centrifuge at 9000g for about 10 minutes, decant off the supernatant, and hold the tube inverted to drain on a filter paper. Dissolve the pellet in 20 µL of Sample buffer, and transfer the solution to a polypropylene microfuge tube with a lid. Transfer 20 µL of Protein molecular weight standard solution to another polypropylene microfuge tube with a lid. Close the lids tightly, secure with lid-locks, and heat both solutions at 100 for 5 minutes. Allow the solutions to cool to room temperature, and centrifuge at 10,000g for 15 seconds to collect the liquids. Apply the solutions to two consecutive lanes of a 4%–20% gradient tris-glycine polyacrylamide slab gel [NOTE—Do not apply any solution in the outside lanes.], and electrophorese as directed under Identification (see Electrophoresis 726 and the section Polyacrylamide Gel Electrophoresis under Biotechnology-Derived Articles—Tests 1047). When the dye-front is about 1 cm from the bottom of the gel (about 40 minutes), stop the current, and remove the gel from the gel assembly. Soak the gel in a suitable volume of the Staining solution for at least 1 hour, such that the gel is completely immersed in the Staining solution during staining. [NOTE—Do not touch the gel with bare hand. Use disposable gloves.] Destain the gel with a large volume of water under constant agitation with repeated changes of water until the background of the gel is completely color free. Using the molecular weights of the proteins in Protein molecular weight standard solution, identify the band corresponding to the Protective Antigen (MW about 83 kDa) in the Test solution lane. [Note—This band is also the single most predominant band in the lane of the Test solution.] Scan the gel, and determine the relative amount (by peak area) of the 83-kDa band by densitometry in the lane of the Test solution. The content of 83 kDa band is not less than 35% of the total peak area.
Total protein—
Standard solution A— Prepare a solution of albumin bovine serum (see Reagent Specifications in the section Reagents, Indicators, and Solutions) in water to obtain a known concentration of about 2.0 mg per mL.
Standard solutions B, C, D, and E— Dilute Standard solution A with water to obtain solutions having protein concentrations of 4, 8, 16, and 24 µg per mL, respectively.
Test solution— Use Anthrax Vaccine Filtrate as is.
Procedure (See Biotechnology-Derived Articles—Tests 1047, Total Protein Assay, Method 3)— To a series of test tubes transfer 800 µL each of Standard solutions B, C, D, and E and the Test solution. Also transfer 800 µL of water to be used as the blank. Add 200 µL of Coomassie blue G-250 dye solution (see Reagent Specifications in the section Reagents, Indicators, and Solutions) to each tube, and mix without foaming. Determine absorbances of the solutions at 595 nm using a suitable spectrophotometer (see Spectrophotometry and Light-Scattering 851 ), using the blank to set the instrument to zero. [NOTE—Do not use quartz (silica) spectrophotometer cells; the dye binds to silica.] Construct a standard curve by plotting the absorbances versus protein concentrations, in µg per mL, of Standard solutions B, C, D, and E and by drawing a best-fit straight line using the linear regression method. From the standard curve, determine the total protein concentration of the Test solution using the absorbance value. The protein concentration is between 5 and 20 µg per mL.
FINAL PRODUCT—
Aluminum—
Standard solutions— Prepare as directed for Standard Preparations under Aluminum 206, except to prepare solutions containing 10, 20, 30, 40, and 50 µg per mL of aluminum.
Test solution— Mix Anthrax Vaccine Adsorbed, Final Product well, and transfer 0.2 mL to a 10-mL volumetric flask. Add 0.5 mL of concentrated sulfuric acid and 0.5 mL of concentrated nitric acid, and mix gently. Incubate at room temperature for 30 minutes or until the solution becomes essentially clear. Dilute with water to volume.
Procedure— Proceed as directed for Procedure under Aluminum 206. Plot the absorbances versus the content of aluminum, in µg per mL, for the Standard solutions, and draw a best-fit straight line through the points using a linear regression model. Calculate the amount of aluminum in Anthrax Vaccine Adsorbed, in mg per mL. The aluminum concentration is between 0.8 and 1.5 mg per mL.
Safety— It meets the requirements when tested as directed in the section Safety Tests—Biologicals under Biological Reactivity Tests, In Vivo 88.
Sterility 71— It meets the requirements when tested as directed for Direct Inoculation of the Culture Medium method under Test for Sterility of the Product to be Examined.
pH 791: between 7.5 and 8.5.
Sodium chloride—
Standard solutions A and B— Prepare two solutions of sodium chloride in water having concentrations of 0.2 mM and 2.0 mM, respectively.
Test solution— Transfer 0.5 mL of Anthrax Vaccine Adsorbed, Final Product to a 50-mL volumetric flask. Dilute with water to volume.
Procedure— Determine the voltage readings of Standard solutions A and B and the Test solution using an ion-specific electrode specific for the chloride ion electrically coupled with a standard silver–silver chloride reference electrode. Plot the voltage readings versus concentration of chloride, in mg per mL, for Standard solutions A and B, and draw a straight line joining the points. Calculate the concentration of chloride ion in the Test solution from the voltage reading. Assuming that the chloride ion comes entirely from sodium chloride, calculate the concentrations of sodium chloride in the Test solution. The concentration of sodium chloride in Anthrax Vaccine Adsorbed is between 0.75% and 0.95% (w/v).
Formaldehyde—
Potassium ferricyanide solution— Dissolve 2.5 g of potassium ferricyanide in about 100 mL of water, and mix.
Phenylhydrazine hydrochloride solution— Dissolve 4 g of phenylhydrazine hydrochloride in 100 mL of absolute alcohol, add 2 mL of water, and mix.
Standard stock solution— To prepare a stock solution, proceed as directed in the Assay under Formaldehyde Solution to determine the concentration of formaldehyde in percent (w/v).
Standard solutions— Dilute the Standard stock solution in water to obtain solutions having concentrations of 0.005%, 0.01%, and 0.02% (w/v).
Test solution— Use Anthrax Vaccine Adsorbed, Final Product as is.
Procedure— To suitable glass centrifuge tubes transfer 1.0 mL each of water, the Standard solutions, and the Test solution. To each tube add 1.0 mL of Potassium ferricyanide solution, 4.0 mL of 18% (w/v) hydrochloric acid and 2.0 mL of Phenylhydrazine hydrochloride solution. Mix after each addition. Incubate for 50 to 60 minutes at room temperature. Centrifuge the solutions at 10,000g for at least 10 minutes, and measure absorbances of the supernatants at 540 nm using a suitable spectrophotometer (see Spectrophotometry and Light-Scattering 851). Plot the absorbances versus concentrations of formaldehyde, in mg per mL, in the Standard solutions, and draw the best-fit straight line through the points. Calculate the amount of formaldehyde in the sample in percent (w/v). The concentration of formaldehyde in Anthrax Vaccine Adsorbed is less than 0.02% (w/v).
Benzethonium chloride—
Citrate buffer— Dissolve 25 g of citric acid monohydrate in about 60 mL of water, and adjust with a solution of sodium hydroxide to a pH of 4.5. Transfer the solution to a 100-mL volumetric flask. Dilute with water to volume, and mix.
Dye solution— Dissolve 50 mg of 2¢,4¢,5¢,7¢-tetrabromofluorescein in about 100 mL water, and mix. Dilute 1 mL of this solution to 100 mL with water.
Docusate sodium solution— Dissolve 50 mg of docusate sodium in 1 L of water.
Standard solution A— Transfer about 0.5 g, accurately weighed, of benzethonium chloride to a 100-mL volumetric flask, dissolve in about 60 mL water, dilute with water to volume, and mix.
Standard solutions B, C, D, and E— Dilute Standard solution A with water to obtain solutions having concentrations of 0.001%, 0.002%, 0.003%, and 0.004% (w/v), respectively.
Test solution— Use Anthrax Vaccine Adsorbed, Final Product as is.
Procedure— Transfer 4.0 mL each of Standard solutions B, C, D, and E and the Test solution to suitable glass centrifuge tubes. Add 1.0 mL Citrate buffer and 0.4 mL of the Dye solution to each tube, and mix. Add 4.0 mL of 1,1,2,2-tetrachloroethane to each tube, and vigorously mix on a vortex mixer for 1 minute. Centrifuge at about 1000g for at least 15 minutes to separate the organic layer from the aqueous layer. Transfer 2.0 mL of the organic layer from the tubes to another set of glass tubes. Add 4.0 mL of water and 0.5 mL of Citrate buffer to each tube, and mix on a vortex mixer for approximately 1 minute. Titrate the benzethonium chloride-dye complex in each tube with the Docusate sodium solution (see Titrimetry 541) to the colorimetric endpoint indicated by the disappearance of the pink color of the organic layer. [Note—Vigorously mix the solution on a vortex mixer after each addition of the Docusate sodium solution.] Plot the volumes of Docusate sodium solution required versus the concentrations of benzethonium chloride in Standard solutions B, C, D, and E, and draw a best-fit straight line through the points. Determine the concentration of benzethonium chloride in the Test solution from the volume of Docusate sodium solution required to titrate the Test solution. The concentration of benzethonium chloride in Anthrax Vaccine Adsorbed is between 0.0015% and 0.0030% (w/v).
Relative potency—
Standard solutions— Dilute approved U.S. Reference Standard Anthrax Vaccine 1:1.6, 1:4, 1:10, and 1:25 aseptically with a sterile 0.9% sodium chloride solution.
Test solutions— Dilute Anthrax Vaccine Adsorbed, Final Product 1:1.6, 1:4, 1:10, and 1:25 aseptically with a sterile 0.9% sodium chloride solution.
Procedure— Assign each dilution to a set of 12 randomly selected guinea pigs, strain Mdh:S(RA), 6 males and 6 females, each weighing 315 to 385 g on the day of vaccination. Inject the animals subcutaneously in the ventral abdomen with 0.5 mL of the assigned dilutions. On the 14th day post-vaccination, challenge the animals with approximately 1000 spores of Bacillus anthracis Vollum 1B, and record the deaths daily for a 10-day observation period. Record the numbers of surviving animals for each of the Standard solutions and the Test solutions at the end of the test. Perform calculations by estimating best-fit lines for the Standard solutions and the Test solutions using a logistic regression model that utilizes the number of animals that survived at the end of the test and the time to death for the animals that died. Evaluate statistically the lines corresponding to the Standard solutions and the Test solutions for parallelism. Determine the common slope, and draw the parallel lines using the common slope. The relative potency of Anthrax Vaccine Adsorbed with respect to the corresponding Approved U.S. Reference Standard Anthrax Vaccine is the antilog of the horizontal distance between the two parallel lines. The relative potency of Anthrax Vaccine Adsorbed is acceptable if it is between 0.53 and 1.79, both values inclusive.
Auxiliary Information— Staff Liaison : Tina S. Morris, Ph.D., Senior Scientist
Expert Committee : (BBVV05) Biologics and Biotechnology - Vaccines and Virology
USP29–NF24 Page 177
Pharmacopeial Forum : Volume No. 29(4) Page 1002
Phone Number : 1-301-816-8397