Appendix XV G. Composition of Polysaccharide Vaccines

Protein

Test solution Use a volumetric flask with a suitable volume for preparation of a solution containing about 5 mg per millilitre of dry polysaccharide. Transfer the contents of a container quantitatively to the flask and dilute to volume with water R. Place 1 mL of the solution in a glass tube and add 0.15 mL of a 400 g/L solution of trichloroacetic acid R. Shake, allow to stand for 15 min, centrifuge for 10 min at 5000 r/min and discard the supernatant. Add 0.4 mL of 0.1 M sodium hydroxide to the centrifugation residue.

Reference solutions Dissolve 0.100 g of bovine albumin R in 100 mL of 0.1 M sodium hydroxide (stock solution containing 1 g of protein per litre). Dilute 1 mL of the stock solution to 20 mL with 0.1 M sodium hydroxide (working dilution 1: 50 mg of protein per litre). Dilute 1 mL of the stock solution to 4 mL with 0.1 M sodium hydroxide (working dilution 2: 250 mg of protein per litre). Place in 6 glass tubes 0.10 mL, 0.20 mL and 0.40 mL of working dilution 1 and 0.15 mL, 0.20 mL and 0.25 mL of working dilution 2. Make up the volume in each tube to 0.40 mL using 0.1 M sodium hydroxide.

Prepare a blank using 0.40 mL of 0.1 M sodium hydroxide.

Add 2 mL of cupri-tartaric solution R3 to each tube, shake and allow to stand for 10 min. Add to each tube 0.2 mL of a mixture of equal volumes of phosphomolybdotungstic reagent R and water R, prepared immediately before use. Stopper the tubes, mix by inverting and allow to stand in the dark for 30 min. The blue colour is stable for 60 min. If necessary, centrifuge to obtain clear solutions.

Measure the absorbance (2.2.25) of each solution at 760 nm using the blank as the compensation liquid. Draw a calibration curve from the absorbances of the 6 reference solutions and the corresponding protein contents and read from the curve the content of protein in the test solution.

Nucleic Acids

Test solution Use a volumetric flask with a suitable volume for preparation of a solution containing about 5 mg per millilitre of dry polysaccharide. Transfer the contents of a container quantitatively to the flask and dilute to volume with water R.

Dilute the test solution if necessary to obtain an absorbance value suitable for the instrument used. Measure the absorbance (2.2.25) at 260 nm using water R as the compensation liquid.

The absorbance of a 1 g/L solution of nucleic acid at 260 nm is 20.

Phosphorus

Test solution Use a volumetric flask with a suitable volume for preparation of a solution containing about 5 mg per millilitre of dry polysaccharide. Transfer the contents of a container quantitatively to the flask and dilute to volume with water R. Dilute the solution so that the volume used in the test (1 mL) contains about 6 µg of phosphorus. Transfer 1.0 mL of the solution to a 10 mL ignition tube.

Reference solutions Dissolve 0.2194 g of potassium dihydrogen phosphate R in 500 mL of water R to give a solution containing the equivalent of 0.1 mg of phosphorus per millilitre. Dilute 5.0 mL of the solution to 100.0 mL with water R. Introduce 0.5 mL, 1.0 mL and 2.0 mL of the dilute solution into 3 ignition tubes.

Prepare a blank solution using 2.0 mL of water R in an ignition tube.

To all the tubes add 0.2 mL of sulfuric acid R and heat in an oil bath at 120 °C for 1 h and then at 160 °C until white fumes appear (about 1 h). Add 0.1 mL of perchloric acid R and heat at 160 °C until the solution is decolorised (about 90 min). Cool and add to each tube 4 mL of water R and 4 mL of ammonium molybdate reagent R. Heat in a water-bath at 37 °C for 90 min and cool. Adjust the volume to 10.0 mL with water R. The blue colour is stable for several hours.

Measure the absorbance (2.2.25) of each solution at 820 nm using the blank solution as the compensation liquid. Draw a calibration curve with the absorbances of the 3 reference solutions as a function of the quantity of phosphorus in the solutions and read from the curve the quantity of phosphorus in the test solution.

O-Acetyl Groups

Test solution Use a volumetric flask with a suitable volume for preparation of a solution containing about 5 mg per millilitre of dry polysaccharide. Transfer the contents of a container quantitatively to the flask and dilute to volume with water R. Dilute the solution so that the volumes used in the test contain 30 µg to 600 µg of acetylcholine chloride (O-acetyl). Introduce 0.3 mL, 0.5 mL and 1.0 mL in duplicate into 6 tubes (3 reaction solutions and 3 correction solutions).

Reference solutions Dissolve 0.150 g of acetylcholine chloride R in 10 mL of water R (stock solution containing 15 g of acetylcholine chloride per litre). Immediately before use, dilute 1 mL of the stock solution to 50 mL with water R (working dilution 1: 300 µg of acetylcholine chloride per millilitre). Immediately before use, dilute 1 mL of the stock solution to 25 mL with water R (working dilution 2: 600 µg of acetylcholine chloride per millilitre). Introduce 0.1 mL and 0.4 mL of working dilution 1 in duplicate (reaction and correction solutions) in 4 tubes and 0.6 mL and 1.0 mL of working dilution 2 in duplicate (reaction and correction solutions) in another 4 tubes.

Prepare a blank using 1 mL of water R.

Make up the volume in each tube to 1 mL with water R. Add 1.0 mL of 4 M hydrochloric acid to each of the correction tubes and to the blank. Add 2.0 mL of alkaline hydroxylamine solution R to each tube. Allow the reaction to proceed for exactly 2 min and add 1.0 mL of 4 M hydrochloric acid to each of the reaction tubes. Add 1.0 mL of a 100 g/L solution of ferric chloride R in 0.1 M hydrochloric acid to each tube, stopper the tubes and shake vigorously to remove bubbles.

Measure the absorbance (2.2.25) of each solution at 540 nm using the blank as the compensation liquid. For each reaction solution, subtract the absorbance of the corresponding correction solution. Draw a calibration curve from the corrected absorbances for the 4 reference solutions and the corresponding content of acetylcholine chloride and read from the curve the content of acetylcholine chloride in the test solution for each volume tested. Calculate the mean of the 3 values.

1 mole of acetylcholine chloride (181.7 g) is equivalent to 1 mole of O-acetyl (43.05 g).

Hexosamines

Test solution Use a volumetric flask with a suitable volume for preparation of a solution containing about 5 mg per millilitre of dry polysaccharide. Transfer the contents of a container quantitatively to the flask and dilute to volume with water R. Dilute the solution so that the volumes used in the test contain 125 µg to 500 µg of glucosamine (hexosamine). Introduce 1.0 mL of the diluted solution into a graduated tube.

Reference solutions Dissolve 60 mg of glucosamine hydrochloride R in 100 mL of water R (stock solution containing 0.500 g of glucosamine per litre). Introduce 0.25 mL, 0.50 mL, 0.75 mL, and 1.0 mL of the working dilution into 4 graduated tubes.

Prepare a blank using 1 mL of water R.

Make up the volume in each tube to 1 mL with water R. Add 1 mL of a solution of hydrochloric acid R (292 g/L) to each tube. Stopper the tubes and place in a water-bath for 1 h. Cool to room temperature. Add to each tube 0.05 mL of a 5 g/L solution of thymolphthalein R in alcohol R; add a solution of sodium hydroxide R (200 g/L) until a blue colour is obtained and then 1 M hydrochloric acid until the solution is colourless. Dilute the volume in each tube to 10 mL with water R (neutralised hydrolysates).

In a second series of 10 mL graduated tubes, place 1 mL of each neutralised hydrolysate. Add 1 mL of acetylacetone reagent (a mixture, prepared immediately before use, of 1 volume of acetylacetone R and 50 volumes of a 53 g/L solution of anhydrous sodium carbonate R) to each tube. Stopper the tubes and place in a water-bath at 90 °C for 45 min. Cool to room temperature. Add to each tube 2.5 mL of alcohol R and 1.0 mL of dimethylaminobenzaldehyde solution (immediately before use dissolve 0.8 g of dimethylaminobenzaldehyde R in 15 mL of alcohol R and add 15 mL of hydrochloric acid R) and dilute the volume in each tube to 10 mL with alcohol R. Stopper the tubes, mix by inverting and allow to stand in the dark for 90 min. Measure the absorbance (2.2.25) of each solution at 530 nm using the blank as the compensation liquid.

Draw a calibration curve from the absorbances for the 4 reference solutions and the corresponding content of hexosamine and read from the curve the quantity of hexosamine in the test solution.

Methylpentoses

Test solution Use a volumetric flask with a suitable volume for preparation of a solution containing about 5 mg per millilitre of dry polysaccharide. Transfer the contents of a container quantitatively to the flask and dilute to volume with water R. Dilute the solution so that the volumes used in the test contain 2 µg to 20 µg of rhamnose (methylpentoses). Introduce 0.25 mL, 0.50 mL and 1.0 mL of the diluted solution into 3 tubes.

Reference solutions Dissolve 0.100 g of rhamnose R in 100 mL of water R (stock solution containing 1 g of methylpentose per litre). Immediately before use, dilute 1 mL of the stock solution to 50 mL with water R (working dilution: 20 mg of methylpentose per litre). Introduce 0.10 mL, 0.25 mL, 0.50 mL, 0.75 mL and 1.0 mL of the working dilution into 5 tubes.

Prepare a blank using 1 mL of water R.

Make up the volume in each tube to 1 mL with water R. Place the tubes in iced water and add dropwise and with continuous stirring to each tube 4.5 mL of a cooled mixture of 1 volume of water R and 6 volumes of sulfuric acid R. Warm the tubes to room temperature and place in a water-bath for a few minutes. Cool to room temperature. Add to each tube 0.10 mL of a 30 g/L solution of cysteine hydrochloride R, prepared immediately before use. Shake and allow to stand for 2 h.

Measure the absorbance (2.2.25) of each solution at 396 nm and at 430 nm using the blank as compensation liquid. For each solution, calculate the difference between the absorbance measured at 396 nm and that measured at 430 nm. Draw a calibration curve from the absorbance differences for the 5 reference solutions and the corresponding content of methylpentose and read from the curve the quantity of methylpentose in the test solution for each volume tested. Calculate the mean of the 3 values.

Uronic Acids

Test solution Use a volumetric flask with a suitable volume for preparation of a solution containing about 5 mg per millilitre of dry polysaccharide. Transfer the contents of a container quantitatively to the flask and dilute to volume with water R. Dilute the solution so that the volumes used in the test contain 4 µg to 40 µg of glucuronic acid (uronic acids). Introduce 0.25 mL, 0.50 mL and 1.0 mL of the diluted solution into 3 tubes.

Reference solutions Dissolve 50 mg of sodium glucuronate R in 100 mL of water R (stock solution containing 0.4 g of glucuronic acid per litre). Immediately before use, dilute 5 mL of the stock solution to 50 mL with water R (working dilution: 40 mg of glucuronic acid per litre). Introduce 0.10 mL, 0.25 mL, 0.50 mL, 0.75 mL, and 1.0 mL of the working dilution into 5 tubes.

Prepare a blank using 1 mL of water R.

Make up the volume in each tube to 1 mL with water R. Place the tubes in iced water and add dropwise and with continuous stirring to each tube 5.0 mL of borate solution R. Stopper the tubes and place in a water-bath for 15 min. Cool to room temperature. Add 0.20 mL of a 1.25 g/L solution of carbazole R in ethanol R to each tube. Stopper the tubes and place in a water-bath for 15 min. Cool to room temperature. Measure the absorbance (2.2.25) of each solution at 530 nm using the blank as the compensation liquid.

Draw a calibration curve from the absorbances for the 5 reference solutions and the corresponding content of glucuronic acid and read from the curve the quantity of glucuronic acid in the test solution for each volume tested. Calculate the mean of the 3 values.

Sialic Acid

Test solution Transfer quantitatively the contents of one or several containers to a volumetric flask of a suitable volume that will give a solution with a known concentration of about 250 µg per millilitre of polysaccharide and dilute to volume with water R. Using a syringe, transfer 4.0 mL of this solution to a 10 mL ultrafiltration cell suitable for the passage of molecules of relative molecular mass less than 50 000. Rinse the syringe twice with water R and transfer the rinsings to the ultrafiltration cell. Carry out the ultrafiltration, with constant stirring, under nitrogen R at a pressure of about 150 kPa. Refill the cell with water R each time the volume of liquid in it has decreased to 1 mL and continue until 200 mL has been filtered and the remaining volume in the cell is about 2 mL. Using a syringe, transfer this residual liquid to a 10 mL volumetric flask. Wash the cell with 3 quantities, each of 2 mL, of water R, transfer the washings to the flask and dilute to 10.0 mL with water R (test solution). In each of 2 test-tubes place 2.0 mL of the test solution.

Reference solutions Use the reference solutions prescribed in the monograph.

Prepare 2 series of 3 test-tubes, place in the tubes of each series 0.5 mL, 1.0 mL and 1.5 mL respectively, of the reference solution corresponding to the type of vaccine to be examined and adjust the volume in each tube to 2.0 mL with water R.

Prepare blank solutions using 2.0 mL of water R in each of 2 test-tubes.

To all the tubes add 5.0 mL of resorcinol reagent R. Heat at 105 °C for 15 min, cool in cold water and transfer the tubes to a bath of iced water. To each tube add 5 mL of isoamyl alcohol R and mix thoroughly. Place in the bath of iced water for 15 min. Centrifuge the tubes and keep them in the bath of iced water until the examination by absorption spectrophotometry. Measure the absorbance (2.2.25) of each supernatant solution at 580 nm and 450 nm using isoamyl alcohol R as the compensation liquid. For each wavelength, calculate the absorbance as the mean of the values obtained with 2 identical solutions. Subtract the mean value for the blank solution from the mean values obtained for the other solutions.

Draw a graph showing the difference between the absorbances at 580 nm and 450 nm of the reference solutions as a function of the content of N-acetylneuraminic acid and read from the graph the quantity of N-acetylneuraminic acid (sialic acid) in the test solution.

Ribose

Test solution Use a volumetric flask with a suitable volume for preparation of a solution containing about 5 mg per millilitre of dry polysaccharide. Transfer the contents of a container quantitatively to the flask and dilute to volume with water R. Dilute the solution so that the volumes used in the test contain 2.5 µg to 25 µg of ribose. Introduce 0.20 mL and 0.40 mL of the diluted solution into tubes in triplicate.

Reference solutions Dissolve 25 mg of ribose R in water R and dilute to 100.0 mL with the same solvent (stock solution containing 0.25 g/L of ribose). Immediately before use, dilute 1 mL of the stock solution to 10.0 mL with water R (working dilution: 25 mg/L of ribose). Introduce 0.10 mL, 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL and 1.0 mL of the working dilution into 6 tubes.

Prepare a blank using 2 mL of water R.

Make up the volume in each tube to 2 mL with water R. Shake. Add 2 mL of a 0.5 g/L solution of ferric chloride R in hydrochloric acid R to each tube. Shake. Add 0.2 mL of a 100 g/L solution of orcinol R in ethanol R. Place the tubes in a water-bath for 20 min. Cool in iced water. Measure the absorbance (2.2.25) of each solution at 670 nm using the blank as the compensation liquid. Draw a calibration curve from the absorbance readings for the 6 reference solutions and the corresponding content of ribose and read from the curve the quantity of ribose in the test solution for each volume tested. Calculate the mean of the 3 values.