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 Iglass containers,and store at a temperature between 2
and 8.Do not freeze.
and 8.Do not freeze.
Expiration date—
The expiration date is 18months 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 Specificationsin the section Reagents,Indicators,and Solutions)in water containing 100g trichloroacetic acid per 100mLof the solution.
Sample buffer—
Prepare a solution containing 141mMtris(hydroxymethyl)aminomethane,106mMtris(hydroxymethyl)aminomethane hydrochloride,0.51mMedetate disodium,2%(w/v)dodecyl lithium sulfate,10%(v/v)glycerol,0.22mM Coomassie blue G-250,and 0.175mMphenolsulfonphthalein.If necessary,adjust with hydrochloric acid or sodium hydroxide to a pHof 8.5.
Running buffer—
Prepare a solution containing 25mMtris(hydroxymethyl)aminomethane,192mMglycine,and 0.1%(w/v)dodecyl sodium sulfate (see Reagent Specificationsin the section Reagents,Indicators,and Solutions)in water.If necessary,adjust with hydrochloric acid or sodium hydroxide to a pHof 8.5.
Transblotting buffer—
Prepare a solution containing 12.5mMtris(hydroxymethyl)aminomethane,96mMglycine,and 10%(v/v)methanol.If necessary,adjust with hydrochloric acid or sodium hydroxide to a pHof 8.0.
Blocking buffer—
Prepare a solution containing 10mMmonobasic sodium phosphate,150mMsodium chloride,5%(w/v)nonfat dry milk,and 0.05%(w/v)Polysorbate 20.Adjust with sodium hydroxide to a pHof 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 anthracisin 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 IgGsolution 1:1000with Blocking buffer.
Chromogenic visualization solution—
Prepare a 150mg per mLsolution of 4-chloro-1-naphthol in water.
Test solution—
Use Anthrax Vaccine Filtrate as is.
Procedure—
In a suitable centrifuge tube transfer 30/cmLof the Test solution,where cis the total protein concentration,in µg per mL,of the solution as determined in the test for Total protein.Add 16.5/cmLof Trichloroacetic acid solution,and incubate for at least 10minutes.Centrifuge at 9000gfor about 10minutes,decant off the supernatant,and hold the tube inverted to drain on a filter paper.Dissolve the pellet in about 60µLof 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 100for 5minutes.Allow the solution to cool to room temperature,and centrifuge at 10,000gfor 15seconds to collect the liquids.In a suitable device for polyacrylamide-gel electrophoresis (see Electrophoresis á726ñand the section Polyacrylamide Gel Electrophoresisunder Biotechnology-Derived Articles—Tests á1047ñ)add appropriate volumes of the Running bufferin 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 bufferin the upper buffer chamber.Apply about 20-µLaliquots 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 40mA.When the dye-front is about 1cm from the bottom of the gel (about 40minutes),stop the current,and remove the gel from the gel assembly.[Note—Do not touch the gel with bare hand.Use gloves.]
for 5minutes.Allow the solution to cool to room temperature,and centrifuge at 10,000gfor 15seconds to collect the liquids.In a suitable device for polyacrylamide-gel electrophoresis (see Electrophoresis á726ñand the section Polyacrylamide Gel Electrophoresisunder Biotechnology-Derived Articles—Tests á1047ñ)add appropriate volumes of the Running bufferin 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 bufferin the upper buffer chamber.Apply about 20-µLaliquots 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 40mA.When the dye-front is about 1cm from the bottom of the gel (about 40minutes),stop the current,and remove the gel from the gel assembly.[Note—Do not touch the gel with bare hand.Use gloves.]
Place 3–4filter 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 1–2mm on each side,and “wet”the membrane by immersing it into the Transblotting bufferfor about 15seconds,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 bufferon 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 2–3additional 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 250mA,and continue transfer for 90minutes.
Remove the membrane,and wash it quickly by immersing into water for 15seconds.[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 EFat the top.Place each strip in a heat-sealable bag,add 5mLof Blocking buffer,and seal the bag.Incubate for 30minutes with constant agitation.Open each bag,and pour out the Blocking buffer.Add 9mLof the diluted Primary antibody solution against PAto the bag containing the strip marked PA.Similarly,add 9mLof the diluted Primary antibody solution against LFand EFto the bags containing strips labeled LFand EF,respectively.Seal the bags,and incubate under agitation for 2hours at room temperature or overnight at 2to 8.Remove the strips from the plastic bags,and place in separate plastic boxes.Add sufficient Blocking bufferso that each strip is completely immersed.Agitate for at least 30minutes at room temperature with two changes of Blocking buffer.Remove the strips,and place each strip in a new heat-sealable plastic bag.Add 9mLof the Secondary antibody solutionto each plastic bag.Seal the bags,and incubate for 1hour at room temperature under agitation.Remove the strips from the plastic bags,and place in separate plastic boxes.Add sufficient Blocking bufferso that each strip is completely immersed.Agitate for at least 30minutes at room temperature with two changes of the Blocking buffer.Transfer each strip into a new heat-sealable plastic bag,add 9mLChromogenic visualization solution,10µLof 30%(v/v)hydrogen peroxide,and seal the bags.Incubate for about 30minutes under agitation.Transfer the strips into separate plastic boxes,and remove the excess 4-chloro-1-naphthol by incubating with water under agitation for 10minutes.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).
to 8.Remove the strips from the plastic bags,and place in separate plastic boxes.Add sufficient Blocking bufferso that each strip is completely immersed.Agitate for at least 30minutes at room temperature with two changes of Blocking buffer.Remove the strips,and place each strip in a new heat-sealable plastic bag.Add 9mLof the Secondary antibody solutionto each plastic bag.Seal the bags,and incubate for 1hour at room temperature under agitation.Remove the strips from the plastic bags,and place in separate plastic boxes.Add sufficient Blocking bufferso that each strip is completely immersed.Agitate for at least 30minutes at room temperature with two changes of the Blocking buffer.Transfer each strip into a new heat-sealable plastic bag,add 9mLChromogenic visualization solution,10µLof 30%(v/v)hydrogen peroxide,and seal the bags.Incubate for about 30minutes under agitation.Transfer the strips into separate plastic boxes,and remove the excess 4-chloro-1-naphthol by incubating with water under agitation for 10minutes.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).
83kDAprotein—
Trichloroacetic acid solution,Sample buffer,Running buffer,and Test solution—
Prepare as directed under Identification.
Staining solution—
Prepare a solution of Coomassie blue G-250having a concentration of 1.25g per Lin 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 14to 200kDa,according to manufacturer's instruction.Dilute the solution with Sample buffersuch that the concentration of each protein in the solution is about 0.5µg per µL.
Procedure—
In a suitable centrifuge tube transfer 10/cmLof the Test solution,where cis the total protein concentration,in µg per mL,of the solution as determined by the test for Total protein(see below).Add 5.5/cmLof Trichloroacetic acid solution,and incubate for at least 10minutes.Centrifuge at 9000gfor about 10minutes,decant off the supernatant,and hold the tube inverted to drain on a filter paper.Dissolve the pellet in 20µLof Sample buffer,and transfer the solution to a polypropylene microfuge tube with a lid.Transfer 20µLof Protein molecular weight standard solutionto another polypropylene microfuge tube with a lid.Close the lids tightly,secure with lid-locks,and heat both solutions at 100for 5minutes.Allow the solutions to cool to room temperature,and centrifuge at 10,000gfor 15seconds 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 á726ñand the section Polyacrylamide Gel Electrophoresisunder Biotechnology-Derived Articles—Tests á1047ñ).When the dye-front is about 1cm from the bottom of the gel (about 40minutes),stop the current,and remove the gel from the gel assembly.Soak the gel in a suitable volume of the Staining solutionfor at least 1hour,such that the gel is completely immersed in the Staining solutionduring 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 (MWabout 83kDa)in the Test solutionlane.[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 83kDa band is not less than 35%of the total peak area.
for 5minutes.Allow the solutions to cool to room temperature,and centrifuge at 10,000gfor 15seconds 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 á726ñand the section Polyacrylamide Gel Electrophoresisunder Biotechnology-Derived Articles—Tests á1047ñ).When the dye-front is about 1cm from the bottom of the gel (about 40minutes),stop the current,and remove the gel from the gel assembly.Soak the gel in a suitable volume of the Staining solutionfor at least 1hour,such that the gel is completely immersed in the Staining solutionduring 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 (MWabout 83kDa)in the Test solutionlane.[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 83kDa 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 Specificationsin the section Reagents,Indicators,and Solutions)in water to obtain a known concentration of about 2.0mg per mL.
Standard solutions B,C,D,andE—
Dilute Standard solution Awith water to obtain solutions having protein concentrations of 4,8,16,and 24µg per mL,respectively.
Test solution—
Use Anthrax Vaccine Filtrate as is.
Procedure (See Biotechnology-Derived Articles—Tests á1047ñ,Total Protein Assay,Method 3)—
To a series of test tubes transfer 800µLeach of Standard solutions B,C,D,and Eand the Test solution.Also transfer 800µLof water to be used as the blank.Add 200µLof Coomassie blue G-250dye solution (see Reagent Specificationsin the section Reagents,Indicators,and Solutions)to each tube,and mix without foaming.Determine absorbances of the solutions at 595nm using a suitable spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ),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 Eand by drawing a best-fit straight line using the linear regression method.From the standard curve,determine the total protein concentration of the Test solutionusing the absorbance value.The protein concentration is between 5and 20µg per mL.
FINAL PRODUCT—
Aluminum—
Standard solutions—
Prepare as directed for Standard Preparations under Aluminum á206ñ,except to prepare solutions containing 10,20,30,40,and 50µg per mLof aluminum.
Test solution—
Mix Anthrax Vaccine Adsorbed,Final Product well,and transfer 0.2mLto a 10-mLvolumetric flask.Add 0.5mLof concentrated sulfuric acid and 0.5mLof concentrated nitric acid,and mix gently.Incubate at room temperature for 30minutes or until the solution becomes essentially clear.Dilute with water to volume.
Procedure—
Proceed as directed for Procedure under Aluminum á206ñ.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 0.8and 1.5mg per mL.
Safety—
It meets the requirements when tested as directed in the section Safety Tests—Biologicalsunder Biological Reactivity Tests,In Vivo á88ñ.
Sterility á71ñ—
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.
pHá791ñ:
between 7.5and 8.5.
Sodium chloride—
Standard solutions Aand B—
Prepare two solutions of sodium chloride in water having concentrations of 0.2mMand 2.0mM,respectively.
Test solution—
Transfer 0.5mLof Anthrax Vaccine Adsorbed,Final Product to a 50-mLvolumetric flask.Dilute with water to volume.
Procedure—
Determine the voltage readings of Standard solutions Aand Band the Test solutionusing 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 Aand B,and draw a straight line joining the points.Calculate the concentration of chloride ion in the Test solutionfrom 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 2.5g of potassium ferricyanide in about 100mLof water,and mix.
Phenylhydrazine hydrochloride solution—
Dissolve 4g of phenylhydrazine hydrochloride in 100mLof absolute alcohol,add 2mLof water,and mix.
Standard stock solution—
To prepare a stock solution,proceed as directed in the Assayunder Formaldehyde Solutionto determine the concentration of formaldehyde in percent (w/v).
Standard solutions—
Dilute the Standard stock solutionin 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 1.0mLeach of water,the Standard solutions,and the Test solution.To each tube add 1.0mLof Potassium ferricyanide solution,4.0mLof 18%(w/v)hydrochloric acid and 2.0mLof Phenylhydrazine hydrochloride solution.Mix after each addition.Incubate for 50to 60minutes at room temperature.Centrifuge the solutions at 10,000gfor at least 10minutes,and measure absorbances of the supernatants at 540nm using a suitable spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ).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 25g of citric acid monohydrate in about 60mLof water,and adjust with a solution of sodium hydroxide to a pHof 4.5.Transfer the solution to a 100-mLvolumetric flask.Dilute with water to volume,and mix.
Dye solution—
Dissolve 50mg of 2¢,4¢,5¢,7¢-tetrabromofluorescein in about 100mLwater,and mix.Dilute 1mLof this solution to 100mLwith water.
Docusate sodium solution—
Dissolve 50mg of docusate sodium in 1Lof water.
Standard solution A—
Transfer about 0.5g,accurately weighed,of benzethonium chloride to a 100-mLvolumetric flask,dissolve in about 60mLwater,dilute with water to volume,and mix.
Standard solutions B,C,D,and E—
DiluteStandard solution Awith 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 4.0mLeach of Standard solutions B,C,D,and Eand the Test solutionto suitable glass centrifuge tubes.Add 1.0mLCitrate bufferand 0.4mLof the Dye solutionto each tube,and mix.Add 4.0mLof 1,1,2,2-tetrachloroethane to each tube,and vigorously mix on a vortex mixer for 1minute.Centrifuge at about 1000gfor at least 15minutes to separate the organic layer from the aqueous layer.Transfer 2.0mLof the organic layer from the tubes to another set of glass tubes.Add 4.0mLof water and 0.5mLof Citrate buffer to each tube,and mix on a vortex mixer for approximately 1minute.Titrate the benzethonium chloride-dye complex in each tube with the Docusate sodium solution(see Titrimetry á541ñ)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 solutionrequired 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 solutionfrom the volume of Docusate sodium solutionrequired 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 1:25aseptically with a sterile 0.9%sodium chloride solution.
Test solutions—
Dilute Anthrax Vaccine Adsorbed,Final Product 1:1.6,1:4,1:10,and 1:25aseptically with a sterile 0.9%sodium chloride solution.
Procedure—
Assign each dilution to a set of 12randomly selected guinea pigs,strain Mdh:S(RA),6males and 6females,each weighing 315to 385g on the day of vaccination.Inject the animals subcutaneously in the ventral abdomen with 0.5mLof the assigned dilutions.On the 14th day post-vaccination,challenge the animals with approximately 1000spores of Bacillus anthracisVollum 1B,and record the deaths daily for a 10-day observation period.Record the numbers of surviving animals for each of the Standard solutionsand the Test solutionsat the end of the test.Perform calculations by estimating best-fit lines for the Standard solutionsand the Test solutionsusing 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 solutionsand the Test solutionsfor 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 0.53and 1.79,both values inclusive.
Auxiliary Information—
Staff Liaison:Tina S.Morris,Ph.D.,Senior Scientist
Expert Committee:(VVI)Vaccines,Virology,and Immunology
USP28–NF23Page 162
Pharmacopeial Forum:Volume No.29(4)Page 1002
Phone Number:1-301-816-8397