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GENERAL TESTING METHODS FOR BIOLOGICAL PRODUCTS

Determination of Aluminium

      For adsorbed vaccines containing aluminium, not more than 1.25 mg per dose, unless otherwise stated in the monograph, determined by the following method.

      Homogenize the preparation being examined and transfer a quantity expected to contain between 5 and 6 mg of aluminium to a 50-ml Kjeldahl flask. Add 1 ml of sulfuric acid, 0.3 ml of nitric acid and some glass beads. Heat the solution until dense, white fumes are evolved. If charring occurs, add a few more drops of nitric acid and continue boiling until the colour disappears. Allow to cool for a few minutes, carefully add 10 ml of water and boil until a clear solution is obtained. Allow to cool, add 0.1 ml of methyl orange TS and neutralize with 10 M sodium hydroxide (about 6.5 to 7.0 ml). If a precipitate forms, dissolve it by adding, dropwise, sufficient 1 M sulfuric acid. Transfer the solution to a flask, rinsing the Kjeldahl flask with 25 ml of water. Add 25.0 ml of 0.02 M disodium edetate VS, 10 ml of acetate buffer pH 4.4 and a few glass beads and boil gently for 3 minutes. Add 0.25 ml of pyridylazonaphthol TS and titrate the excess of disodium edetate in the hot solution with 0.02 M copper(II) sulfate VS until the colour changes to purplish brown. Carry out a blank titration omitting the vaccine (Appendix 6.17). The difference between the titrations represents the volume of 0.02 M disodium edetate VS equivalent to the aluminium present. Each ml of 0.02 M disodium edetate is equivalent to 0.5396 mg of Al.

Determination of Calcium

      For adsorbed vaccines containing calcium, not more than 1.3 mg per dose, unless otherwise stated in the monograph, determined by the following method.

      Homogenize the preparation being examined. Add 0.2 ml of hydrochloric acid to 1.0 ml and dilute to 3.0 ml with water. Determine the content of calcium by the “Atomic Spectrometry: Emission and Absorption” (Appendix 2.3), measuring at 620 nm and using calcium solution ASp, diluted if necessary with water, for the preparation of the standard solutions. 

Determination of Formaldehyde

      For vaccines containing formaldehyde, not more than 0.02 per cent w/v of free formaldehyde (CH2O).Use test A unless otherwise prescribed.

      Test B is suitable for vaccines where sodium metabisulfite has been used to neutralize excess formaldehyde.

Test A

      To 1 ml of a tenfold dilution of the vaccine in water add 4 ml of water and 5 ml of acetylacetone reagent. Warm in a water-bath at 40º and allow to stand for 40 minutes. The solution is not more intensely coloured than a reference solution prepared at the same time and in the same manner using 1 ml of a solution containing 0.002 per cent w/v of formaldehyde, CH2O, in place of the dilution of the vaccine. The comparison should be made by examining the tubes down their vertical axes.

Test B

      Test solution​

      Prepare a 1 in 200 dilution of the vaccine being examined with water. If the vaccine is an emulsion, prepare an equivalent dilution using the aqueous phase separated by a suitable procedure. If one of the methods described below is used for separation of the aqueous phase, a 1 in 20 dilution of the latter is used.

      Reference solutions

      Prepare solutions containing 0.25 g/l, 0.50 g/l, 1.00 g/l and 2.00 g/l of CH2O by dilution of formaldehyde solution with water.

      Procedure

      To 0.5 ml of the test solution and of each of the reference solutions in test-tubes, add 5.0 ml of a freshly prepared 0.05 per cent w/v solution of 3-methyl-2- benzothiazolinone hydrazone hydrochloride hydrate. Close the tubes, shake and allow to stand for 60 minutes. Add 1 ml of iron(III) chloride-sulfamic acid TS and allow to stand for 15 minutes. Measure the absorbance (Appendix 2.2) of the solutions at 628 nm. Calculate the content of formaldehyde in the vaccine being examined from the calibration curve established using the reference solutions. The test is invalid if the correlation coefficient (r) of the calibration curve is less than 0.97.

      Emulsions

       If the vaccine being examined is an emulsion, the aqueous phase is separated using a suitable procedure and used for preparation of the test solution. The following procedures have been found suitable.

      (a) Add 1.0 ml of the vaccine being examined to 1.0 ml of isopropyl myristate and mix. Add 1.3 ml of 1 M hydrochloric acid, 2.0 ml of chloroform and 2.7 ml of saline TS. Mix thoroughly. Centrifuge at 15,000 × g for 60 minutes. Transfer the aqueous phase to a 10-ml volumetric flask and dilute to volume with water. If this procedure fails to separate the aqueous phase, repeat the procedure but use a 10 per cent w/v solution of polysorbate 20 in saline TS instead of saline TS and centrifuge at 22,500 × g. 

      (b) Add 1.0 ml of the vaccine being examined to 1.0 ml of a 10 per cent w/v solution of sodium chloride and mix. Centrifuge at 1000 × g for 15 minutes. Transfer the aqueous phase to a 10-ml volumetric flask and dilute to volume with water.

      (c) Add 1.0 ml of the vaccine being examined to 2.0 ml of a 10 per cent w/v solution of sodium chloride and 3.0 ml of chloroform and mix. Centrifuge at 1000 × g for 15 minutes. Transfer the aqueous phase to a 10-ml volumetric flask and dilute to volume with water.

Determination of Phenol

      For biological products containing phenol as preservative, not more than 0.25 per cent w/v, unless otherwise stated in the monograph, determined by the following method.

      Homogenize the preparation being examined. Dilute an appropriate volume with water to give a solution expected to contain 15 μg of phenol per ml. To 5.0 ml of the resulting solution add 5 ml each of borate buffer pH 9.0, aminophenazone TS and a 5 per cent w/v solution of potassium hexacyanoferrate(III). Allow to stand for 10 minutes and measure the absorbance at 546 nm (Appendix 2.2). Calculate the phenol content from the absorbance obtained, using a calibration curve prepared by repeating the operation using 5.0 ml of each of a series of reference solutions containing 5, 10, 15, 20, and 30 μg of phenol per ml, respectively.

Determination of Thiomersal1

      For biological products containing thiomersal as preservative, not less than 0.005 per cent w/v and not more than 0.02 per cent w/v of thiomersal, determined by the following method.

      Wash separators with nitric acid and rinse with tap water and water. Transfer two portions of the preparation being examined each of 1.0 ml to individual separators. Add a 1 per cent w/v solution of ammonium acetate to make 10 ml. To each separator, add 10.0 ml of dithizone standard solution. Shake vigorously for 45 seconds. Carefully separate the resulting chloroform layer, and measure the absorbance at 520 nm (Appendix 2.2), using dithizone standard solution as a blank. Calculate the thiomersal content from the absorbance obtained using a calibration curve prepared from Thiomersal RS by repeating the operation using 1.0 ml of each of a series of reference solutions containing 50, 75 and 100 μg of thiomersal per ml, respectively.


1Biological products available in some countries may contain thiomersal. Risk-benefit should be considered when medical problem exists.

MONOGRAPHS • GENERAL TESTING METHODS FOR BIOLOGICAL PRODUCTS
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หมายเหตุ / Note : TP II 2011 PAGE 178-179