Haemophilus influenzae type b Tetanus Toxoid Conjugate Summary Validation Report

February 18, 2012

________________
1. Executive Summary a. Goals: The reference procedure described herein was developed for the MC monographfor Haemophilus influenzae type b Tetanus Toxoid Conjugate b. Back ground i. Haemophilus Influenzae Type B Tetanus Toxoid Conjugate is a sterile and colorless liquid of a purified capsular polysaccharide manufactured from a suitable strain of haemophilus influenzae type b. The capsular polysaccharide, polyribosylribitol phosphate, referred to as PRP, is a linear copolymer composed of repeated units of 3--d-ribofuranosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size.. ii. The drug is available as final bulk (Subject for future MC monograph). 2. Reference Procedures Identification: A. Immunochemical method TM Proceed as directed in the Wellcogen Haemophilus influenzae type b kit procedure. (Make: Remel Europe TM Ltd., Wellcogen Haemophilus influenzae type b) i. Solution A: Control latex ii. Solution B: Polyvalent positive control iii. Solution C: Negative control iv. Solution D: Test latex v. Sample solution: Haemophilus influenzae type b Tetanus Toxoid Conjugate solution vi. Procedure: Control sample: Transfer one drop of Solution A and Solution B in one circle on a reaction card Positive control: Transfer one drop of Solution D and Solution B in one circle on a reaction card. Negative control: Transfer one drop of Solution D and Solution C in one circle on a reaction card. Sample solution 1: Transfer one drop of Solution D and one drop of Sample solution in one circle on a reaction card. [Note: Ensure that to dispense an accurate drop.] Sample solution 2: Transfer one drop of Solution A and one drop of Sample solution in one circle on a reaction card. [Note: Ensure that to dispense an accurate drop.] Mix the card slowly and observe for agglutination for 3 min. vii. Results: Control sample: Agglutination should not observe Positive control: Agglutination should observe Negative control: Agglutination should not observe Sample solution 1: Agglutination should observe Sample solution 2: Agglutination should not observe B. Identification of tetanus toxoid by ouchterlony double immunodiffusion i. Solution A: 0.9% Sodium chloride in water ii. Solution B: 0.4% Agarose in Solution A iii. Solution C: Methanol iv. Solution D: Glacial acetic acid v. Solution E: Solution C, Solution D and water (4:1:5) vi. Solution F (Blank solution): Formulation buffer

vii. Solution G (Negative control): 0.3 mg/mL of USP Haemophilus influenzae type b PRP RS solution in water viii. Solution H (Positive control): 0.3 mg/mL of USP Tetanus Toxoid RS solution in water ix. Solution I: 0.3 mg/mL of Haemophilus influenzae type b Tetanus Toxoid Conjugate solution in water [Note: Based on protein concentration] x. Solution J: 100 Lf/mL of Tetanus antiserum xi. Solution K: Coomassie brilliant blue G-250 dye solution (Make: Bio Rad) xii. Agarose gel slide: Heat Solution B until clear liquid appears; pour the solution on a microscopic slide. Allow the slide at room temperature for a period of 10 min to solidify. [Note: Ensure that the solution shall spread equally over the slide]. After solidification, punch four wells on the microscopic slide and one well in between the four wells. xiii. Procedure: Load 20 L of Solution F, Solution G, Solution H and Solution I to designated wells on the microscopic slide and Solution J in the center well. Allow the slide at room temperature for a period of 2 hr. After 2 hr keep the slide in a airtight container and containing pre-wetted filter paper sheet upside down and incubate the slide at room temperature for a period of 48 hr. After 48 hr, place the slide in the Solution E for a period of 30 min. Remove the Solution E and place the slide for staining in the Solution K for 1 hr. [Note: Ensure that the gel slide have immersed properly in the Solution K]. After staining remove the Solution K and place the slide in water. xiv. Analysis Samples: Sample solution Observe the precipitin band formation (blue color between the wells) under a bright surface in Sample solution ASSAY Procedure1: Polysaccharide composition Ribose content by orcinol assay i. Solution A: 10% Orcinol in ethanol ii. Solution B: 10% Ferric chloride in water iii. Solution C: Concentrated hydrochloric acid iv. Solution D: Solution B and Solution C (1:199) v. Solution E: Solution A and Solution D (1:9) vi. Standard solution: 0.1 mg/mL of D-Ribose-5-phosphate in water vii. Sample solution: Haemophilus influenzae type b Tetanus Toxoid Conjugate solution in water viii. Procedure: Transfer 10 L, 20 L, 40 L, 60 L, 80 L and 100 L of Standard solution and transfer 200 L of Sample solution to designated test tubes. Make up to 0.2 mL in all the test tubes with water. Transfer 0.2 mL of water to the blank test tubes. Add 0.2 mL of Solution E to all test tubes and mix well. Incubate the test tubes at 90 for 40 min. After incubation cool the test tubes for 10 min at room temperature and transfer 0.2 mL of solution in a 96 well microtitre plate. Determine the ribose content of the test measuring the absorbance at 670 nm. [Note: If the absorbance of sample solution falls outside the values of the standard curve, the assay should be repeated using a more appropriate pre-dilution for the sample solution.] ix. System suitability Sample: Use Standard solution calibration curve Suitability requirements Regression of calibration curve: NLT 0.98 %RSD between the replicates: NMT 10.0% x. Analysis Samples: Sample solution Calculate the content (mg/mL) of PRP in the Sample solution Result = CR (Mr1/Mr2) CR = Concentration of Ribose, mg/mL Mr1 = Molecular weight of PRP, 368 Mr2 = Molecular weight of D-Ribose-5-phosphate, 132
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CR = (AS I) / S DF AS = Absorbance of the Sample solution I = Intercept S = Slope DF = Dilution factor Procedure 2: High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) i. Solution A: Water ii. Solution B: 0.5 M Sodium acetate trihydrate in water iii. Solution C: 0.2 M Sodium hydroxide in water iv. Solution D: 2 M Sodium hydroxide in water v. Solution E: Dissolve 11.7 g of sodium chloride, 0.214 g of sodium phosphate dibasic and 1.17 g of sodium phosphate monobasic monohydrate in 800 mL of water. Adjust the pH to 6.5 with 1N HCl and make up the final volume to 1 L with water. vi. Solution F: 0.04 mg/mL of USP Haemophilus influenzae type b PRP RS in Solution E vii. Standard solution 1: 1 g/mL of USP Haemophilus influenzae type b PRP RS from Solution F, in Solution E viii. Standard solution 2: 2 g/mL of USP Haemophilus influenzae type b PRP RS from Solution F, in Solution E ix. Standard solution 3: 4 g/mL of USP Haemophilus influenzae type b PRP RS from Solution F, in Solution E x. Standard solution 4: 8 g/mL of USP Haemophilus influenzae type b PRP RS from Solution F, in Solution E xi. Standard solution 5: 10 g/mL of USP Haemophilus influenzae type b PRP RS from Solution F, in Solution E xii. Standard solution 6: 15 g/mL of USP Haemophilus influenzae type b PRP RS from Solution F, in Solution E xiii. Hydrolyzed standard solution 1-6: Transfer 1 mL of the Standard solution 1-6 to a 1.5 mL micro centrifuge tube, add 100 L of Solution D to the micro centrifuge tubes and heat for 3 hr at 55 in dry bath oven. Cool to room temperature and analyse the solution. xiv. Sample solution: Haemophilus influenzae type b Tetanus Toxoid Conjugate in Solution E. [Note: Dilute the Sample solution that falls into the Standard curve range.] xv. Hydrolyzed sample solution: Hydrolyze the Sample solution similar to that of the Hydrolyzed standard solution 1-6. [Note: Prepare the Hydrolyzed standard solution and Hydrolyzed sample solution at the same time, using the same stock of reagents and concentrations.] xvi. System suitability solution: Six replicates from the Standard solution 4 xvii. Mobile phase: Solution A, Solution B and Solution C (2:1:1) xviii. Chromatographic system (See Chromatography<621>, System Suitability.) 1. Mode: HPIC 2. Detector: Pulsed amperometric detector (electrochemical), set to the following wave form, See Table 1 Table 1 Step 1 2 3 Time (s) 0.00 0.20 0.40 Potential (V) 0.1 0.1 0.1 Integration off Begins off

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4 5 6 7 8

0.41 0.42 0.43 0.44 0.50

-2.0 -2.0 0.6 -0.1 -0.1

off off off off off

xix.

xx.

3. Electrode: Carbohydrate disposable working electrode 4. Reference electrode: AgCl electrode 5. Column: 4.0 mm 25-cm; packing L53, (similar to Dionex, Carbopac PA10 in series with a 4.0 mm 5-cm; guard column; packing L53, similar to Dionex, Carbopac PA10) 6. Flow rate: 1 mL/min 7. Temperatures Column oven: 30 Auto sampler: 15 8. Injection volume: 100 L 9. Run time: 20 min System suitability: Samples: System suitability solution Suitability requirements RSD: NMT 2.0% RSD Analysis: Samples: Hydrolyzed standard solution 1-6 and Hydrolyzed sample solution Calculate the total PRP content (g/mL) in the Sample solution. Result = (RS - I) / S DF RS = Response from the Sample solution I = Intercept S = Slope DF = Dilution factor

Procedure 3: Total protein i. Solution A: 0.1 M Sodium hydroxide in water ii. Solution B: 2% w/v Sodium carbonate monohydrate in Solution A iii. Solution C: 2% w/v Disodium tartrate dehydrate in water iv. Solution D: 1% w/v Copper sulphate pentahydrate in water v. Solution E: Solution C, Solution D and Solution B (1:1:98). vi. Solution F: Folin-ciocalteu's phenol solution and water (1:1) vii. Standard stock solution: 0.3 mg/mL of bovine serum albumin (BSA) standard solution in water viii. Standard solution 1: 0.05 mg/mL of BSA in water, from the Standard stock solution ix. Standard solution 2: 0.01 mg/mL of BSA in water, from the Standard stock solution x. Standard solution 3: 0.15 mg/mL of BSA in water, from the Standard stock solution xi. Standard solution 4: 0.2 mg/mL of BSA in water, from the Standard stock solution xii. Standard solution 5: 0.25 mg/mL of BSA in water, from the Standard stock solution xiii. Standard solution 6: 0.3 mg/mL of BSA in water, from the Standard stock solution xiv. Sample solution: Haemophilus influenzae type b Tetanus Toxoid Conjugate solution in water xv. System suitability solution: 0.175 mg/mL of BSA in water from the Standard stock solution. xvi. Procedure: Transfer 0.2 mL of Standard solutions 1-6, System suitability solution and Sample solution to designated micro tubes. Transfer 0.2 mL of water to the blank test tubes. Add 1 mL of Solution E to all
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xvii.

xviii.

tubes and mix well. Allow to stand at room temperature for 10 2 min. Add 0.1 mL of Solution F to all tubes, mix each tube immediately, and allow to stand at room temperature for 30 2 min. Transfer 0.2 mL of the solutions from each micro tube in to 96 well microtitre plate. Determine the absorbance at 750 nm with a suitable spectrophotometer. System suitability Sample: System suitability solution and Standard solutions 1-6 Suitability requirements 2 R of calibration curve: NLT 0.99, Standard solutions 1-6 Sample recovery: 90%110%, System suitability solution Analysis Samples: Sample solution Calculate the protein concentration (mg/mL) in the Sample solution: Result = (CS / 1000) DF CS = Concentration of the Sample solution, g/mL DF = Dilution factor CS = (AS I) / S DF AS = Absorbance of the Sample solution I = Intercept S = Slope DF = Dilution factor

IMPURITIES Free PRP A. Orcinol Assay i. Solution A: 10% Orcinol in ethanol ii. Solution B: 10% Ferric chloride in water iii. Solution C: Concentrated hydrochloric acid iv. Solution D: 1.0 M Hydrochloric acid in water v. Solution E: 1% Deoxycholic acid in water at pH 6.8 vi. Solution F: Solution B and Solution C (1:199) vii. Solution G: Solution A and Solution F (1:9) viii. Solution H: 0.1 mg/mL of Haemophilus influenzae type b Tetanus Toxoid Conjugate solution ix. Standard solution: 0.02 mg/mL of D-Ribose-5-phosphate in water x. Sample solution: Mix 0.1 mg of Solution H, 80 L of Solution E and incubate at 2 to 8 for 30 min. Add 50 L of Solution D and centrifuge at 2 to 8 with 7500 rpm for 15 min. Collect the supernatant for analysis. xi. Precipitation blank solution: Mix 870 L of water, 80 L of Solution E and incubate at 2 to 8 for 30 min. Add 50 L of Solution D and centrifuge at 2 to 8 with 7500 rpm for 15 min. Collect the supernatant for analysis. xii. Procedure: Transfer 5 L, 10 L, 25 L, 50 L, 100 L, and 200 L of Standard solution and transfer 200 L each of Sample solution and precipitation blank solution to designated test tubes. Make up to 0.2 mL in all the test tubes with water. Transfer 0.2 mL of water to the blank test tubes. Add 0.2 mL of Solution G to all test tubes and mix well. Incubate the test tubes at 90 for a period of 40 min. After incubation cool the test tubes for 10 min at room temperature. Transfer 0.2 mL of solutions from each micro tube in to 96 well microtitre plate for absorbance. Determine the ribose content of the test measuring the absorbance at 670 nm. [Note: Determine total PRP content for each sample solution prior to deoxycholic acid precipitation.] xiii. System suitability Sample: Use Standard solution calibration curve Suitability requirements Regression of calibration curve: NLT 0.98 %RSD between the replicates: NMT 10.0% xiv. Analysis Samples: Sample solution Calculate the percentage of free PRP in the Sample solution Result = (MF / MP) 100
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MF = Measured free PRP content, mg/mL MP = Measured PRP content, mg/mL [Note: proceed as directed in the Assay, Procedure 1 ] MF = (CR - CP) (Mr1/Mr2) CR = Concentration of ribose in Sample solution, mg/mL CP = Concentration of ribose in Precipitation blank solution, mg/mL Mr1 = Molecular weight of PRP, 368 Mr2 = Molecular weight of D-Ribose-5-phosphate, 132 CR = (AS I) / S DF AS = Absorbance of the Sample solution I = Intercept S = Slope DF = Dilution factor CP = (AS I) / S DF AS = Absorbance of the Precipitation blank solution I = Intercept S = Slope DF = Dilution factor Procedure 2: High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) i. Solution A: Water ii. Solution B: 0.5 M Sodium acetate trihydrate in water iii. Solution C: 0.2 M Sodium hydroxide in water iv. Solution D: 2 M Sodium hydroxide in water v. Solution E: 1 M Hydrochloric acid in water vi. Solution F: 1% Deoxycholic acid in water at pH 6.8 vii. Solution G: Dissolve 11.7 g of sodium chloride, 0.214 g of sodium phosphate dibasic and 1.17 g of sodium phosphate monobasic monohydrate in 800 mL of water. Adjust the pH to 6.5 with 1N HCl and make up the final volume to 1 L with water. viii. Solution H: 0.04 mg/mL of USP Haemophilus influenzae type b PRP RS in Solution G ix. Standard solution 1: 1 g/mL of USP Haemophilus influenzae type b PRP RS from Solution H, in Solution G x. Standard solution 2: 2 g/mL of USP Haemophilus influenzae type b PRP RS from Solution H, in Solution G xi. Standard solution 3: 4 g/mL of USP Haemophilus influenzae type b PRP RS from Solution H, in Solution G xii. Standard solution 4: 8 g/mL of USP Haemophilus influenzae type b PRP RS from Solution H, in Solution G xiii. Standard solution 5: 10 g/mL of USP Haemophilus influenzae type b PRP RS from Solution H, in Solution G xiv. Standard solution 6: 15 g/mL of USP Haemophilus influenzae type b PRP RS from Solution H, in Solution G xv. Hydrolyzed standard solution 1-6: Transfer 1 mL of the Standard solution 1-6 to a 1.5 mL of micro centrifuge tube, add 100 L of Solution D and heat for 3 hr at 55 in dry bath oven. Cool to room temperature and use the solution for analysis. [Note: Standard solution 1-6 will not under go deoxycholate treatment] xvi. Sample solution: Mix 100 g of Haemophilus influenzae type b Tetanus Toxoid Conjugate solution in 900 L of water and 100 L of Solution F and incubate above solution at 2 to 8 for 30 min. Add 50 L of Solution E and centrifuge at 2 to 8 with 7500 rpm for 15 min. Collect the supernatant for analysis. xvii. Hydrolyzed sample solution: Hydrolyze the Sample solution similar to that of the Hydrolyzed standard solution 1-6. [Note: Prepare the Hydrolyzed standard solution and Hydrolyzed sample solution at the same time, using the same stock of reagents and concentrations.] xviii. System suitability solution: Six replicates from the Standard solution 4 xix. Mobile phase: Solution A, Solution B and Solution C (2:1:1) xx. Chromatographic system (See Chromatography<621>, System Suitability.)
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1. Mode: HPIC 2. Detector: Pulsed amperometric detector, (electrochemical), set to the following wave form, See table 2 Table 2 Step 1 2 3 4 5 6 7 8 Time (s) 0.00 0.20 0.40 0.41 0.42 0.43 0.44 0.50 Potential (V) 0.1 0.1 0.1 -2.0 -2.0 0.6 -0.1 -0.1 Integration off Begins off off off off off off

xxi.

xxii.

3. Electrode: Carbohydrate disposable working electrode 4. Reference Electrode: AgCl electrode 5. Column: 4.0 mm 25-cm; packing L53, (similar to Dionex, Carbopac PA10 in series with a 4.0 mm 5-cm; guard column; packing L53, similar to Dionex, Carbopac PA10) 6. Flow rate: 1 mL/min 7. Temperatures Column oven: 30 Auto sampler: 15 8. Injection volume: 100 L System suitability: Sample: System suitability solution Suitability requirements % RSD: NMT 2.0 Analysis: Samples: Hydrolyzed standard solution 1-6 and Hydrolyzed sample solution Calculate the percentage of free PRP content in the Sample solution Result = (MF / MP) 100 MF = Measured free PRP content, g/mL MP = Measured total PRP content, g/mL [Note: proceed as directed in the Assay, Procedure 2 ] MF = (AS I) / S DF AS = Area of the Sample solution I = Intercept S = Slope DF = Dilution factor

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Procedure 3: Free Tetanus Toxoid i. Mobile phase: Dissolve 8.51 g of disodium hydrogen phosphate, 2.74 g of sodium phosphate monobasic monohydrate and 3.5 g of sodium chloride in 1 L of water. ii. Standard solution 1: 1 mg/mL of USP Tetanus Toxoid RS in water iii. Standard solution 2 (1.0%): 10 g/mL of USP Tetanus Toxoid RS in water, from Standard solution 1 iv. Sample solution: 1 mg/mL of Haemophilus influenzae type b Tetanus Toxoid Conjugate solution in water [Note: Based on protein concentration.] v. System suitability solution 1: 10 g/mL of USP Tetanus toxoid RS in water vi. System suitability solution 2: 9.6 g/mL of USP Tetanus toxoid RS from System suitability solution 1, in water vii. Chromatographic system (See Chromatography <621>, System Suitability.) 1. Mode: LC 2. Detector: PDA 214 nm 3. Column: 7.8-mm 30-cm; 1000 ; 10-m packing L59 (similar to TOSOH Biosciences G3000SWXL), two columns connected in series. 4. Flow rate: 0.5 mL/min 5. Temperatures Column oven: 30 Auto sampler: 5 6. Injection volume: 100 L 7. Run time: 60 min viii. System suitability Samples: System suitability solution 1 and System suitability solution 2 Suitability requirements: The area of tetanus toxoid peak in the System suitability solution 2 should be less than that of System suitability solution 1 ix. Analysis Samples: Standard solution 2 and Sample solution The area of free tetanus toxoid in the Sample solution should be less than that of Standard solution 2. SPECIFIC TESTS Molecular size distribution 3 i. Solution A: Agarose for chromatography, cross linked with exclusion limit for polysaccharide 310 to 6 510 (similar to Sepharose CL4 B gel) ii. Solution B: Acetone iii. Solution C: Blue dextran (Make: Amersham biosciences) iv. Mobile phase: 0.2 M Ammonium acetate in water. Adjust with ammonium hydroxide solution to a pH of 7.2. v. System suitability solution: Mix 4.0 mg of Solution C in 1.8 mL of Mobile phase. Add 40 L of Solution B and make up the final volume to 2.0 mL with Mobile phase. vi. Sample solution: 10 mg/mL of dried Haemophilus influenzae type b Tetanus Toxoid Conjugate in Mobile phase vii. Chromatographic system (See Chromatography<621>, System Suitability.) 1. Mode: LC 2. Detector: RI (Make: Shodex, model: 101) 3. RI Detector temperature: 25 4. Column: C 10/40, packing sepharose CL4 B gel (similar to Amersham Biosciences) 5. Flow rate: 0.5 mL/min 6. Temperatures Column oven: 25 Auto sampler: 25 7. Injection volume: 100 L 8. Run time: 100 min
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viii. System suitability: Sample: System suitability solution Suitability requirements Theoretical plate count: NLT 3000 for acetone peak ix. Analysis: Samples: Standard solution and Sample solution Calculate the molecular size distribution of the polysaccharide in the Sample solution. Ve = KD (Vt V0) + V0 Ve = Eluted peak volume of the sample KD = Distribution coefficient [Note: KD value for Haemophilus influenzae type b Tetanus Toxoid Conjugate is 0.2.] Vt = Total volume determined with acetone V0 = Void volume 3. Validation Results 3.1 Identification a. Identification of PRP (by Wellcogen Kit Method) i. Specificity Matrix: Formulation Buffer Negative Control: Tetanus Toxoid Positive Control: Hib PRP Standard solution (NIBSC) Sample solution: Hib Tetanus Toxoid Conjugate Acceptable criteria: Buffer solution should not give any response. Negative Control (Tetanus Toxoid) should give negative response. Positive Control (Hib PRP Standard solution) should give a positive response. Sample (Hib Tetanus Toxoid Conjugate) should give positive response. Observed results: No response obtained form the biuffer solution. Negative Control (Tetanus Toxoid) gives negative response. Positive Control (Hib PRP Standard solution) gives a positive response. Sample solution (Hib Tetanus Toxoid Conjugate) gives positive response. Table 1 Type of Material Positive Control
1 2

Test Latex (Coated with Hib specific Antibody) Positive Negative Positive Negative Negative Positive

Control Latex (Coated with non specific Antibody) Negative Negative Negative Negative Negative Negative

Negative Control

Hib PRP Standard solution (Positive Control) Tetanus Toxoid (Negative Control) Formulation buffer
12

Hib Tetanus Toxoid Conjugate : Provided in the Kit

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b. Identification of Tetanus Toxoid by Ouchterlony double immunodiffusion method i. Specificity Negative Control: 300 g/mL of Hib PRP Positive Control: 300 g/mL of Tetanus Toxoid Sample solution: 300 g/mL of Hib Tetanus Toxoid Conjugate Unrelated Sample solutions: 300 g/mL of Hepatits B Antigen Bulk Acceptable criteria: Negative Control (Hib PRP) should not show precipitin band. Positive Control (Tetanus Toxoid) should show precipitin band. Sample solution should show precipitin band Unrelated sample solutions should not show any precipitin band. Observed results: Negative Control does not show precipitin band. Positive Control shows precipitin band. Sample solution shows precipitin band. Unrelated sample does not show any precipitin band. 3.2 Assay Procedure 1: Total Polysaccharide composition- PRP content by Orcinol assay i. Specificity: Specificity was tested using buffer, PRP, Tetanus Toxoid Conjugate and unrelated molecules Standard solution: D-Ribose-5-phosphate Sample solutions: Buffer (PBS), PRP and Tetanus Toxoid Conjugate Un-related molecules: Tetanus Toxoid, Bovine Serum Albumin (BSA), Glucose & Lipid Acceptable criteria: Buffer solution should not give any response. The response of Sample solutions should fall within the Standard solution response. Calculated and Observed results: No interference observed from the buffer solution. The absorbance of all samples is within the range of Standard solutions The absorbance of unrelated molecules are close to buffer values. Table 2: Raw data of Standard solution and Sample solution Standard solution/ Sample solution Concentration, g/mL 5 10 20 D-Ribose-5-phosphate 30 40 50 Formulation Buffer Pure PRP (2.5 Fold) Bulk Conjugate (4 Fold) Tetanus Toxoid Glucose Lipid BSA 100 100 100 100 100 100 OD -1 0.205 0.383 0.738 1.083 1.423 1.764 0.052 0.702 0.420 0.052 0.046 0.050 0.047 OD-2 0.202 0.38 0.729 1.081 1.416 1.771 0.051 0.673 0.412 0.052 0.050 0.049 0.053 Mean 0.203 0.381 0.734 1.082 1.420 1.767 0.052 0.687 0.416 0.052 0.048 0.050 0.050

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Precision: Repeatibility: Repeatability was performed using 6 independent preparations of Hib Tetanus Toxoid Conjugate by one analyst on a single day. Standard solution: D-Ribose-5-phosphate Sample solution: Tetanus Toxoid Conjugate Acceptable criteria: The mean of all the %RSDs (6 determinants) should bb NMT 2.5% Calculated and Observed results: Repeatability per assay is 0.67 % RSD.

Table 3: Repeatability of measured PRP content, % Replicate 1 2 3 4 5 6 Mean Standard Deviation % RSD Measured PRP Content, mg/mL 0.100 0.099 0.100 0.101 0.100 0.101 0.100 0.001 0.67

Intermediate Precision: Two Analysts performed the assay on 3 different days with six independent sample preparations Standard solution: D-Ribose-5-phosphate Sample solution: 100% Pure PRP Acceptable criteria: The mean of all the %RSDs (36 determinants) should be NMT 5.0% Calculated and Observed results: Overall Intermediate precision: % RSD is 3.05 % Table 4: Inter-day variation Replicate 1 2 3 4 5 6 Measured PRP Content, mg/mL Day-1 0.099 0.101 0.102 0.105 0.103 0.102 Mean Standard Deviation %RSD Day-2 0.105 0.105 0.104 0.104 0.103 0.100 0.103 0.002 1.94

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Table 5: Inter-analyst variation Replicates 1 2 3 4 5 6 Mean Standard Deviation %RSD Measured PRP Content, mg/mL Analyst-1 0.099 0.101 0.102 0.105 0.103 0.102 Analyst-2 0.106 0.107 0.107 0.105 0.105 0.104 0.104 0.002 2.35

Table 6: Overall Variation (between the analysts and days) Measured PRP Content, mg/mL Replicates Day -1 Analyst -1 R-1 R-2 R-3 R-4 R-5 R-6 Mean 0.099 0.101 0.102 0.105 0.103 0.102 0.102 Analyst-2 0.106 0.107 0.107 0.105 0.105 0.104 0.106 Day -2 Analyst - Analyst1 2 0.105 0.105 0.104 0.104 0.103 0.100 0.104 0.107 0.107 0.106 0.107 0.108 0.106 0.107 Mean 0.104 Day -3 Analyst - Analyst1 2 0.108 0.107 0.104 0.098 0.108 0.109 0.106 Standard Deviation 0.003 0.100 0.099 0.099 0.100 0.099 0.094 0.098 %RSD 3.05

Accuracy, Linearity & Range: One Analyst performed the assay with five different simulated concentration (80.0%, 90.0%, 100.0%, 110.0% and 120.0%). Each concentration is tested with six independent sample preparations. Standard Solution: D-Ribose-5-phosphate Sample solution: 80.0%, 90.0%, 100.0%, 110.0% and 120.0% Acceptable criteria: 2 The R value of the linear regression of expected Vs measured % prp content should be 0.98 PPAC (Procedure Performance Acceptance Criteria): Probability of NLT 0.95 at 95.0% to 105.0%. Calculated and observed results: 2 The R value of the linear regression of expected Vs measured % PRP content is 0.994 PPAC: Probability is NLT 0.95 at 95.0% to 105.0%

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Table 7: PPAC calculation for Linearity and Accuracy Expected PRP Content, % Expected PRP Content, mg/mL Measured PRP Content, mg/mL 0.081 0.081 80 0.08 0.081 0.080 0.080 0.078 0.093 0.091 90 0.09 0.090 0.090 0.089 0.089 0.100 0.100 0.10 100 0.100 0.099 0.099 0.099 0.110 0.11 110 0.111 0.110 0.110 0.111 0.111 0.118 0.118 0.12 120 0.118 0.118 0.118 0.117 Mean Recovery, % Standard Deviation

% Recovery 101.42 101.75 101.15 100.60 99.89 97.68 103.10 101.64 100.55 99.96 98.71 99.19 100.49 100.39 99.92 99.12 99.42 98.81 100.26 100.95 99.58 100.41 101.19 101.30 98.06 98.40 98.58 98.36 98.26 97.62

Probability

100.42

1.49

1.00

100.52

1.63

1.00

99.69

0.69

1.00

100.61

0.66

1.00

98.21

0.34

1.00

Range: The range of the method is 80.0 % to 120.0%.

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Procedure 2: Polysaccharide Composition-Ribose content by high performance anion exchange chromatography with pulsed amperometric detection i. Specificity Blank: Sample diluent Formulation buffer System suitability: 6 injections of 8.0 g/mL PRP Standard solution Standard solution: Hib PRP Standard solution (NIBSC) Un-related Sample solution: Tetanus Toxoid and Pullulan. Sample solution: Hib Tetanus Toxoid Conjugate Acceptable criteria: System suitability critera: %RSD between 6 injections of Standard solution of 8 g/mL should be NMT 2.0%. Buffer solution should not show any interference in the integration window. Unrelated samples like Tetanus Toxoid Conjugate and Pullulan should not show any interference in the integration window Specific response should be seen for PRP polysaccharide in the Standard solution (NIBSC) and Sample solution. Calculated and observed results: System suitability criteria met the specification of % RSD, NMT 2.0% between 6 injections of 8 g/mL PRP Standard solution. Buffer solution does not show any interference in the integration window. Unrelated samples Tetanus Toxoid Conjugate and Pullulan do not show any interference in the integration window. Specific response observed for PRP polysaccharide in the Standard solution (NIBSC) and Sample solution. Figure 1: Chromatogram of Sample Diluent (PBS)

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Figure 2: Chromatogram of Formulation buffer

Figure 3: Chromatogram of Un-related molecule (Tetanus Toxoid Conjugate)

Figure 4: Chromatogram of Unrelated molecule (Pullulan)

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Figure 5: Chromatogram of Hib PRP Standard solution

Figure 6: Chromatogram of Hib Tetanus Toxoid Conjugate sample

ii. Precision: Repeatability: Standard solution of 1 mg/mL was analyzed at appropriate dilution for evaluation of repeatability. Repeatibility: Repeatability wa performed using 6 independent preparations of Hib Tetanus Toxoid Conjugate sample solutions by one analyst on a single day. Acceptance criteria: The % RSD between 6 replicates should be NMT 2.0% Calculated and Observed results: The %RSD observed between 6 replicates was 1.03%. Table 8: RSD calculation for Hib PRP Replicates R-1 R-2 R-3 R-4 Conc.of PRP in mg/mL 0.99 0.99 0.98 1.00 0.99 0.01 1.03 Mean Standard Deviation %RSD

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R-5 R-6

1.00 1.01

Intermediate Precision: Parameter performed on 3 different days between two different analysts with six separate sample preparations. Acceptable criteria: The Mean of all the %RSDs (36 determinants) should be NMT 4%. Calculated and Observed results: % RSD of overall Precision is 2.06 % Table 9: Interanalyst precision of day 1 Analyst Replicates R1 R2 Analyst-1 R3 R4 R5 R6 R1 R2 Analyst-2 R3 R4 R5 R6 Mean Standard Deviation %RSD Day 1 PRP conc. in mg/mL 9.92 10.28 10.20 10.06 10.28 10.15 9.77 9.55 9.55 9.56 9.62 9.51 9.9 0.31 3.16

Table 10: Interday Precision of Analyst-1 Day Replicates R1 R2 Day-1 R3 R4 R5 R6 R1 R2 Day-2 R3 R4 R5 R6 Day-3 R1 PRP conc. in mg/mL Analyst 1 9.92 10.28 10.20 10.06 10.28 10.15 10.14 10.01 10.08 9.97 9.97 10.11 10.13

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R2 R3 R4 R5 R6 Mean Standard Deviation %RSD

10.15 10.09 10.21 10.07 10.14 10.1 0.10 1.01

Table 11: Overall variation between the Analysts and Days Replicates R-1 R-2 R-3 R-4 R-5 R-6 Mean Day -1 Analyst -1 9.92 10.28 10.20 10.06 10.28 10.15 10.15 Analyst-2 9.77 9.55 9.55 9.56 9.62 9.51 9.59 10.14 10.01 10.08 9.97 9.97 10.11 10.05 Day -2 Analyst -1 Analyst-2 10.00 10.05 9.93 9.97 10.00 9.88 9.97 Mean 10.0 10.13 10.15 10.09 10.21 10.07 10.14 10.13 Standard Deviation 0.21 Day -3 Analyst -1 Analyst-2 10.06 10.01 9.89 9.95 9.80 9.98 9.95 %RSD 2.06

iii. Linearity, Range & Accuracy: Parameter evaluated at 7 different concentration levels (0.5,1,2,4,8,10,15 g/mL) with six separate sample preparations at each level. Acceptable criteria: 2 R value of the linear curve should be > 0.99 PPAC for the range evaluated should be more than 0.95 at 90% to 110% Calculated and Observed results: 2 R value of the linear curve is 0.999 PPAC for was more thyan 0.95 for 90% to 110% Table 12: PPAC calculation for Linearity and Accuracy Std. conc. in g/mL 0.5 0.5 0.5 0.5 0.5 0.5 1 1 1 1 1 Replicates R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 nC*min 0.917 0.903 0.901 0.896 0.892 0.905 1.828 1.856 1.801 1.856 1.798 Est.conc in g/mL 0.46 0.45 0.45 0.45 0.45 0.45 0.97 0.98 0.95 0.98 0.95 % Recovery 92.1 90.6 90.3 89.8 89.3 90.8 96.8 98.4 95.3 98.4 95.2 96.8 1.42 1.46 1.00 90.5 0.96 1.06 0.69 Mean Recovery Standard Deviation % RSD Probability

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1 2 2 2 2 2 2 4 4 4 4 4 4 8 8 8 8 8 8 10 10 10 10 10 10 15 15 15 15 15 15

R6 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6

1.826 3.716 3.750 3.734 3.698 3.701 3.704 7.348 7.261 7.407 7.318 7.299 7.343 14.781 14.745 14.565 14.860 14.629 14.584 17.882 17.995 17.831 17.730 17.812 17.849 26.847 26.718 27.110 26.720 27.079 27.360

0.97 2.02 2.04 2.03 2.01 2.01 2.01 4.05 4.00 4.08 4.03 4.02 4.04 8.19 8.17 8.07 8.23 8.10 8.08 9.92 9.98 9.89 9.83 9.88 9.90 14.91 14.84 15.06 14.84 15.04 15.20

96.7 101.0 102.0 101.5 100.5 100.6 100.7 101.1 99.9 102.0 100.7 100.5 101.1 102.4 102.1 100.9 102.9 101.3 101.0 99.2 99.8 98.9 98.3 98.8 99.0 99.4 98.9 100.4 99.0 100.3 101.3 99.89 0.95 0.95 1.00 99.0 0.49 0.49 1.00 101.8 0.83 0.81 1.00 100.9 0.69 0.68 1.00 101.1 0.58 0.57 1.00

Procedure 3: Total protein by Lowry method i. Specificity Matrix: Saline solution Negative Control: C4 SPE treated PRP Positive Control: Tetanus Toxoid System suitability : 175 g/mL of BSA Sample solution: Hib Tetanus Toxoid Conjugate Acceptable criteria: Buffer solution should not give any response at 750 nm. Negative Control (C4 SPE treated PRP) should not give response. Positive Control (Tetanus Toxoid) should give a positive response Calculated and observed results: System suitability criteria met. QC Sample gives a recovery within 90 to 100 percent. No interference (absorbance) observed from the buffer solution. Negative Control (C4 SPE treated PRP) does not give response.
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Positive Control (Tetanus Toxoid) gives a positive response Table 9: Specificity Sample conc. Rep 1 Rep 2 (g/mL) 175 NA NA NA NA 0.223 0.228 Back Standard calculated % RSD Deviation value (g/mL) 0.004 NA NA 0.008 0.004 1.57 NA NA 3.52 1.70 182.36 NA NA 199.05 164.05

Sample ID

Mean

% Recovery

System suitability sample (175 g/mL) Saline buffer solution Negative Control (C4 SPE treated PRP) Positive control (Tetanus toxoid) Tetanus Toxoid Conjugate

0.226 -0.002 0.002 0.241 0.209

104.2 Below calibration range Below calibration range NA NA

-0.002 -0.002 0.001 0.235 0.211 0.002 0.247 0.206

ii. Precision: Hib Tetanus Toxoid Conjugate solution of concentration 125 g/mL was used for evaluation of repetability and intermediate precision Repeatibility: Repeatability is performed using 6 independent preparations of Hib Tetanus Toxoid Conjugate sample solutions by one analyst on a single day. Acceptance criteria: The % RSD between 6 replicates should be NMT than 5.0% Calculated and Observed results: The %RSD observed between 6 replicates was 3.8%. Table 10: RSD calculation for Hib Tetanus Toxoid Conjugate Sample 1 2 3 4 5 6 Mean Standard Deviation % RSD Result Conc. 132.1 134.9 133.0 126.5 123.7 123.7 129.0 4.95 3.84

Intermediate Precision: Intermediate Precision performed on three different days between two different analysts with six separate sample preparations. Acceptance criteria: The mean of all the %RSDs (36 determinants) should be NMT 5.0%.

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Calculated and Observed results: Overall Intermediate precision: % RSD is 3.0 % Table 11: Interday/Intranalyst variation of Analyst -1 between three different days Day Replicates R1 R2 Day-1 R3 R4 R5 R6 R1 R2 Day-2 R3 R4 R5 R6 R1 R2 Day-3 R3 R4 R5 R6 Mean Standard Deviation %RSD Analyst 1 Observed Concentration (g/mL) 132.1 134.9 133.0 126.5 123.7 123.7 130.4 130.4 134.4 128.4 121.3 129.4 128.0 130.1 129.1 131.2 127.0 126.0 128.9 3.72 2.89

Table 12: Interanalyst/Intraday variation between two different analysts Analyst Replicates R1 R2 Analyst-1 R3 R4 R5 R6 R1 R2 Analyst-2 R3 R4 R5 R6 Mean Standard Deviation %RSD Day 1 Observed Concentration (g/mL) 132.1 134.9 133.0 126.5 123.7 123.7 122.0 121.2 127.1 122.9 122.0 124.6 126.1 4.71 3.73

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Table 13: Overall variation between the analysts and days Replicates R-1 R-2 R-3 R-4 R-5 R-6 Mean Day -1 Analyst -1 132.1 134.9 133.0 126.5 123.7 123.7 129.0 Analyst-2 122.0 121.2 127.1 122.9 122.0 124.6 123.3 Analyst -1 130.4 130.4 134.4 128.4 121.3 129.4 129.1 Day -2 Analyst-2 121.7 125.2 125.2 122.6 127.0 123.5 124.2 Mean 126.9 Day -3 Analyst -1 128.0 130.1 129.1 131.2 127.0 126.0 128.6 Standard Deviation 3.83 Analyst-2 123.9 131.0 124.7 130.1 128.3 126.5 127.4 %RSD 3.02

iii. Linearity, Range & Accuracy: Parameter evaluated at 6 different concentration levels (50.0, 75.0, 100.0,125.0,150.0 and 200.0 g/mL) with six sample preparations at each level. Acceptable criteria: 2 R value of the linear curve should be > 0.99 PPAC for the range evaluated should be more than 0.95 at 90.0% to 110.0% Calculated and Observed results: 2 R value of the linear curve is 0.999 PPAC was more than 0.95 for 90.0% to 110.0% Table 14: PPAC calculation for Linearity and Accuracy % Mean OD at 750 Est.Conc Standard % Recover Recover nm .% Deviation RSD y y 52.38 104.76 0.073 0.075 0.072 0.068 0.072 0.076 0.102 0.099 0.098 0.096 0.104 0.098 0.123 0.122 0.129 0.132 0.128 0.129 54.14 51.50 47.97 51.50 55.02 77.95 75.30 74.42 72.66 79.71 74.42 96.47 95.58 101.76 104.40 100.87 101.76 108.29 102.99 95.94 102.99 110.05 103.93 100.41 99.23 96.88 106.28 99.23 96.47 95.58 101.76 104.40 100.87 101.76 100.14 3.41 3.41 1.00 100.41 3.53 3.51 1.00 104.17 4.95 4.75 0.88

Replicates R-1 R-2 R-3 R-4 R-5 R-6 R-1 R-2 R-3 R-4 R-5 R-6 R-1 R-2 R-3 R-4 R-5 R-6

Concentratio n (g/ml) 50.00 50.00 50.00 50.00 50.00 50.00 75.00 75.00 75.00 75.00 75.00 75.00 100.00 100.00 100.00 100.00 100.00 100.00

Probabilit y

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R-1 R-2 R-3 R-4 R-5 R-6 R-1 R-2 R-3 R-4 R-5 R-6 R-1 R-2 R-3 R-4 R-5 R-6

125.00 125.00 125.00 125.00 125.00 125.00 150.00 150.00 150.00 150.00 150.00 150.00 200.00 200.00 200.00 200.00 200.00 200.00

0.148 0.157 0.158 0.156 0.146 0.159 0.170 0.185 0.185 0.185 0.184 0.191 0.254 0.234 0.237 0.230 0.249 0.243

118.51 126.44 127.33 125.56 116.74 128.21 137.91 151.13 151.13 151.13 150.25 156.42 211.97 194.34 196.98 190.81 207.56 202.27

94.81 101.15 101.86 100.45 93.40 102.57 91.94 100.75 100.75 100.75 100.17 104.28 105.99 97.17 98.49 95.40 103.78 101.14 100.33 4.05 4.04 0.99 99.77 4.12 4.13 0.98 99.04 3.91 3.95 0.99

IMPURITIES Free PRP A. Orcinol Assay i. Specificity Specificity was tested using formulation buffer, bulk conjugate, vaccine and four unrelated molecules Standard solution: D-Ribose-5-phosphate Sample solutions: Formulation buffer, Tetanus Toxoid Conjugate and Conjugate Vaccine Non-ribose samples: Tetanus Toxoid, Bovine Serum Albumin (BSA), Glucose & Lipid Acceptable criteria: Buffer solution should not give any response. The response of Sample solutions should fall within the Standard solution response. Calculated and Observed results: No interference observed from the buffer solution. The absorbance of PRP Sample solutions is within the range of Standard solutions The absorbance of unrelated molecules are close to buffer values. Table 15: Specificity Standard solution/ Sample solution Conc in g/mL 0.5 1 D-Ribose-5-phosphate 2.5 5 10 20 Formulation Buffer Tetanus Toxoid Conjugate 100 OD -1 0.056 0.076 0.135 0.229 0.431 0.815 0.052 0.118 OD-2 0.058 0.075 0.132 0.228 0.423 0.818 0.053 0.123 Mean 0.057 0.075 0.133 0.228 0.427 0.817 0.053 0.121

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HIB Conjugate Vaccine Tetanus Toxoid Glucose solution Lipid solution BSA solution ii.

100 100 100 100 100

0.138 0.057 0.047 0.05 0.047

0.148 0.057 0.044 0.049 0.053

0.143 0.057 0.046 0.050 0.050

Precision: Repeatibility: Repeatability was performed using 6 independent preparations of HIB Vaccine spiked with PRP sample by one analyst on a single day. Standard solution: D-Ribose-5-phosphate Sample solution: Tetanus Toxoid Conjugate spiked with PRP. Acceptable criteria: The mean of all the %RSDs (6 determinants) should be NMT 10.0%. Calculated and Observed results: Repeatability per assay is 5.39 % RSD.

Table 16: Repeatability of Measured Free PRP content, % Replicate 1 2 3 4 5 6 Mean Standard Deviation % RSD Measured Free PRP Content, % 24.22 24.35 27.7 26.28 25.19 24.54 25.38 1.37 5.39

Intermediate Precision: Two Analysts performed assay on 3 different days with six independent sample preparations Standard solution: D-Ribose-5-phosphate Sample solution: Tetanus Toxoid Conjugate spiked with PRP Acceptable criteria: The mean of all the %RSD (36 determinants) should be NMT 10.0% Calculated and Observed results: Overall Intermediate precision: % RSD is 8.25 % Table 17: Interday variation Measured Free PRP Content, % Day-1 1 2 22.53 22.05 Day-2 24.22 24.35

Replicate

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3 4 5 6 Mean

22.11 22.30 22.27 22.39

27.70 26.28 25.19 24.54 23.83 1.87 7.84

Standard Deviation %RSD Table 18: Interanalyst variation Replicates 1 2 3 4 5 6 Mean Standard Deviation %RSD

Measured Free PRP Content, % Analyst-1 22.53 22.05 22.11 22.30 22.27 22.39 Analyst-2 23.26 21.68 21.41 20.29 19.53 22.61 21.87 1.04 4.75

Table 19: Overall Variation (between the analysts and days) Measured Free PRP Content, % Replicates Day -1 Analyst -1 R-1 R-2 R-3 R-4 R-5 R-6 Mean 22.53 22.05 22.11 22.3 22.27 22.39 22.275 Analyst-2 23.26 21.68 21.41 20.29 19.53 22.61 21.463 Day -2 Analyst - Analyst1 2 24.22 24.35 27.7 26.28 25.19 24.54 25.380 26.82 28.28 24.98 23.58 26.75 23.07 25.580 Mean 23.065 iii. Day -3 Analyst - Analyst1 2 22.47 22.36 22.54 22 21.9 21.97 22.207 Standard Deviation 1.903 22.63 22.22 21.78 21.28 19.77 21.24 21.487 %RSD 8.25

Accuracy, Linearity, Range & LOQ One Analyst performed the assay with five different simulated concentrations (50.0%, 75.0%, 100.0%, 125.0% and 150.0%). Each concentration is tested with six independent sample preparations. Standard solution: D-Ribose-5-phosphate Sample solution: Tetanus Toxoid Conjugate spiked with 50.0%, 75.0%, 100.0%, 125.0% and 150.0% of PRP. Acceptable criteria: 2 The R value of the linear regression of expected Vs measured % prp content should be 0.98

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PPAC (Procedure Performance Acceptance Criteria): Probability of NLT 0.95 at 90% to 110%.

Calculated and observed results: 2 The R value of the linear regression of expected Vs measured % PRP content is 0.997 PPAC: Probability is NLT 0.95 at 90% to 110% Table 20: PPAC calculation for Linearity and Accuracy Expected PRP Content, % Expected Free PRP Content, % Measured Free PRP Content, % 17.99 18.07 50 17.5 18.18 18.28 18.34 17.90 22.34 22.08 75 22.5 22.08 22.54 22.89 22.80 26.66 27.78 100 27.5 27.72 27.79 28.15 28.16 32.58 34.25 125 32.5 33.37 33.20 32.48 33.76 37.37 37.53 150 37.5 37.41 36.92 38.19 37.31 Mean Recovery Standard Deviation

% Recovery 102.81 103.27 103.87 104.48 104.82 102.26 99.28 98.15 98.11 100.17 101.72 101.32 96.94 101.00 100.79 101.04 102.37 102.40 100.23 105.39 102.67 102.15 99.93 103.89 99.64 100.09 99.75 98.45 101.85 99.50

Probability

103.59

0.99

1.00

99.79

1.55

1.00

100.76

1.00

102.37

2.1

1.00

99.88

1.11

1.00

Range: The range of the method is 50 % to 150 %.

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B. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) i. Specificity Blank: Deoxycholate treated water Formulation buffer: Deoxycholate treated formulation buffer Standard solution: Hib PRP Standard solution spiked to deoxycholate treated water Un-related Sample solution: Deoxycholate treated Tetanus Toxoid and Pullulan. Sample solution: Hib PRP Standard solution spiked to deoxycholate treated Hib Tetanus Toxoid Conjugate System suitability: 6 injections of 8 g/mL PRP Standard solution. Acceptable criteria: System suitability critera: %RSD between 6 injections of Standard solution of 8 g/mL should be NMT 2.0%. Matrix should not show any interference in the integration window. Unrelated samples like Tetanus Toxoid Conjugate and Pullulan should not show any interference in the integration window Specific response should be seen for PRP polysaccharide in the Standard solution (NIBSC) and sample solution. Calculated and observed results: System suitability criteria met the specification of % RSD NMT 2.0% between 6 injections of 8 g/ml PRP Standard solution. Matrix does not show any interference in the integration window.. Unrelated samples Tetanus Toxoid Conjugate and Pullulan do not show any interference in the integration window. Specific response observed for PRP in the Standard solution (NIBSC) and Sample solution. Figure 7: Chromatogram for deoxycholate treated water

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Figure 8: Chromatogram for deoxycholate treated formulation buffer

Figure 9: Chromatogram of deoxycholate treated Tetanus toxoid bulk

Figure 10: Chromatogram for deoxycholate treated Pullulan Standard

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Figure 11: Chromatograph for Hib PRP standard in deoxycholate treated Blank

Figure 12: Chromatogram for Hib PRP spiked in deoxycholate treated Hib Tetanus Toxoid

ii. Suitability studies: Sample used for evaluation of repeatability is 5 g/mL Hib PRP spiked to deoxycholate treated Hib Tetanus Toxoid Conjugate Repeatibility: Repeatability was performed using 6 independent preparations of Hib PRP spiked to deoxycholate treated Hib Tetanus Toxoid Conjugate solution by one analyst on a single day. Acceptance criteria: The % RSD between 6 replicates should be NMT 2.0% Results: The %RSD observed between 6 replicates was 1.81%.

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Table 21: RSD calculation for Free PRP Replicates R-1 R-2 R-3 R-4 R-5 R-6 Conc of Free PRP in g/mL 5.82 5.99 6.06 6.03 6.11 6.12 Mean Standard %RSD Deviation

6.02

0.11

1.81

NOTE: A) This method has been validated in the Haemophilus influenza type B PRP (Assay). Therefore, full validation is not required. However, method suitability has been established with suitable matrix solution and precision (repeatability). B) Haemophilus influenza type B PRP (Assay) validation data has been captured below for reference. i. Precision: Standard solution of 10.0 mg/mL was analyzed at appropriate dilution for evaluation of repeatability and intermediate precision. Repeatibility: Repeatability is performed using 6 independent preparations of Haemophilus influenzae type b PRP sample solutions by one analyst on a single day. Acceptable criteria: The % RSD between 6 replicates should be NMT than 2.0% Calculated and Observed results: The %RSD observed between 6 replicates was 1.39%.

Table 22 Repeatibility Replicates R-1 R-2 R-3 R-4 R-5 R-6 Mean Standard Deviation %RSD Conc. of PRP in mg/mL 9.92 10.28 10.20 10.06 10.28 10.15 10.15 0.14 1.39

Intermediate Precision: Parameter performed on 3 different days between two different analysts with six separate sample preparations. Acceptable criteria: The mean of all the %RSDs (36 determinants) should be NMT 4%. Calculated and Observed results: % RSD of overall Precision is 2.06 %

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Table 23: Interanalyst precision of day1 Analyst Replicates R1 R2 Analyst-1 R3 R4 R5 R6 R1 R2 Analyst-2 R3 R4 R5 R6 Mean Standard Deviation %RSD Day 1 PRP conc. in mg/mL 9.92 10.28 10.20 10.06 10.28 10.15 9.77 9.55 9.55 9.56 9.62 9.51 9.9 0.31 3.16

Table 24: Interday Precision of Analyst-1 Day Replicates R1 R2 Day-1 R3 R4 R5 R6 R1 R2 Day-2 R3 R4 R5 R6 R1 R2 Day-3 R3 R4 R5 R6 Mean Standard Deviation %RSD PRP conc. in mg/mL Analyst 1 9.92 10.28 10.20 10.06 10.28 10.15 10.14 10.01 10.08 9.97 9.97 10.11 10.13 10.15 10.09 10.21 10.07 10.14 10.1 0.10 1.01

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Table 25: Overall variation between the analysts and days Day -1 Replicates R-1 R-2 R-3 R-4 R-5 R-6 Mean Analyst -1 9.92 10.28 10.20 10.06 10.28 10.15 10.15 Analyst-2 9.77 9.55 9.55 9.56 9.62 9.51 9.59 Day -2 Analyst - Analyst1 2 10.14 10.00 10.01 10.08 9.97 9.97 10.11 10.05 10.05 9.93 9.97 10.00 9.88 9.97 Mean 10.0 Day -3 Analyst - Analyst1 2 10.13 10.06 10.15 10.09 10.21 10.07 10.14 10.13 Standard Deviation 0.21 10.01 9.89 9.95 9.80 9.98 9.95 %RSD 2.06

iii. Linearity, Range & Accuracy: Parameter evaluated at 7 different concentration levels (0.5,1,2,4,8,10,15 g/mL) with six separate sample preparations at each level. Acceptable criteria: 2 R value of the linear curve should be > 0.99 PPAC for the range evaluated should be more than 0.95 at 90% to 110% Calculated and Observed results: 2 R value of the linear curve is 0.999 PPAC is more than 0.95 for 90% to 110% Table 26: PPAC calculation for Linearity and Accuracy Std. conc. in g/mL 0.5 0.5 0.5 0.5 0.5 0.5 1 1 1 1 1 1 2 2 2 2 2 2 4 4 Replicates R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 R1 R2 nC*min 0.917 0.903 0.901 0.896 0.892 0.905 1.828 1.856 1.801 1.856 1.798 1.826 3.716 3.750 3.734 3.698 3.701 3.704 7.348 7.261 Est.conc in g/mL 0.46 0.45 0.45 0.45 0.45 0.45 0.97 0.98 0.95 0.98 0.95 0.97 2.02 2.04 2.03 2.01 2.01 2.01 4.05 4.00 % Recovery 92.1 90.6 90.3 89.8 89.3 90.8 96.8 98.4 95.3 98.4 95.2 96.7 101.0 102.0 101.5 100.5 100.6 100.7 101.1 99.9 100.9 0.69 0.68 1.00 101.1 0.58 0.57 1.00 96.8 1.42 1.46 1.00 90.5 0.96 1.06 0.69 Mean Recovery Standard Deviation % RSD Probability

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4 4 4 4 8 8 8 8 8 8 10 10 10 10 10 10 15 15 15 15 15 15

R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6

7.407 7.318 7.299 7.343 14.781 14.745 14.565 14.860 14.629 14.584 17.882 17.995 17.831 17.730 17.812 17.849 26.847 26.718 27.110 26.720 27.079 27.360

4.08 4.03 4.02 4.04 8.19 8.17 8.07 8.23 8.10 8.08 9.92 9.98 9.89 9.83 9.88 9.90 14.91 14.84 15.06 14.84 15.04 15.20

102.0 100.7 100.5 101.1 102.4 102.1 100.9 102.9 101.3 101.0 99.2 99.8 98.9 98.3 98.8 99.0 99.4 98.9 100.4 99.0 100.3 101.3 99.89 0.95 0.95 1.00 99.0 0.49 0.49 1.00 101.8 0.83 0.81 1.00

C. Free Tetanus Toxoid by SEC-HPLC i. Specificity System suitability: 1.0% Tetanus Toxoid sample:10.0 g/mL Tetanus Toxoid 0.96% Tetanus Toxoid sample: 9.60 g/mL Tetanus Toxoid Blank: Sample diluent Formulation buffer Standard solution: 1.00%Tetanus Toxoid Un-related Sample solution: Hib Tetanus Toxoid Conjugate Negative control: Hib PRP. Sample solution: Hib Tetanus Toxoid Conjugate ( 1 mg/mL based on protein concentration) spiked with 1.0% Tetanus Toxoid Acceptable criteria: System suitability criteria: The area of 0.96% Tetanus Toxoid sample should be less than area of 1%Tetanus Toxoid sample. Matrix should not show any interference in the integration window. Tetanus toxoid should show specific response. Hib Tetanus Toxoid Conjugate should elute at distinctly different RTs Negative Control like Hib PRP should not show any interference in the integration window. Sample should show distinct peak response for Hib Tetanus Toxoid Conjugate and Tetanus Toxoid bulk Calculated and observed results: System suitability criteria: The area of 0.96% Tetanus Toxoid sample showed less area than that of 1.0% Tetanus Toxoid. Matrix does not show any interference in the integration window. Tetanus Toxoid shows specific response at 31.5 mins. Hib PRP Tetanus Toxoid Conjugate elutes at 21.6 mins Negative Control like Hib PRP does not show any interference in the integration window

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Sample showed distinct peak response for Hib Tetanus Toxoid Conjugate at 21.6 min and Tetanus toxoid bulk at 31.5 min Figure 13: Chromatogram of Mobile phase

0.020 0.018 0.016 0.014 0.012 0.010 0.008

AU

0.006 0.004 0.002 0.000 -0.002 -0.004 -0.006 -0.008 -0.010 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes 35.00 40.00 45.00 50.00 55.00 60.00

Figure 14: Chromatogram of Formulation buffer

0.020 0.018 0.016 0.014 0.012 0.010 0.008
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0.006 0.004 0.002 0.000 -0.002 -0.004 -0.006 -0.008 -0.010 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes 35.00 40.00 45.00 50.00 55.00 60.00

Figure 15: Chromatogram of Hib PRP

0.020 0.018 0.016 0.014 0.012 0.010 0.008
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0.006 0.004 0.002 0.000 -0.002 -0.004 -0.006 -0.008 -0.010 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes 35.00 40.00 45.00 50.00 55.00 60.00

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Figure 16: Chromatogram of 1.0% Tetanus toxoid

0.020 0.018 0.016 0.014 0.012 0.010 0.008
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0.006 0.004 0.002 0.000 -0.002 -0.004 -0.006 -0.008 -0.010 0.00 5.00 10.00 15.00 20.00 25.00

30.00 Minutes

Free TT - 31.514
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40.00

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60.00

Figure 17: Chromatogram of Hib Tetanus Toxoid Conjugate

0.018 0.016 0.014 0.012 0.010 0.008
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0.006 0.004 0.002 0.000 -0.002 -0.004 -0.006 -0.008 -0.010 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes 35.00 40.00 45.00 50.00 55.00 60.00

Figure 18: Chromatogram of Hib Tetanus Toxoid Conjugate spiked with 1.0% Tetanus Toxoid
0.018 0.016 0.014 0.012 0.010 0.008
AU

Hib TT bulk - 21.629

0.020

Hib TT bulk - 21.626

0.020

0.006 0.004 0.002 0.000 -0.002 -0.004 -0.006 -0.008 -0.010 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes

Free TT - 31.585
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40.00

45.00

50.00

55.00

60.00

Suitability study (Precision- Repeatability): Repeatibility: Repeatability was performed using 6 independent preparations of 1% Tetanus Toxoid spiked in Hib Tetanus Toxoid Conjugate (1.0 mg/mL based on protein concentration). Acceptable criteria: The % RSD between the area of spiked 1% Tetanus toxoid for six independent samples should be NMT 2.0%.
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Calculated and Observed results: % RSD between 6 replicates for the area of 1% spiked tetanus toxoid was1.23% Table 27: RSD calculation for Free Tetanus Toxoid Standard Sample Area Mean Deviation 303878 302892 Hib Tetanus Toxoid Conjugate spiked 1.0 % Tetanus Toxoid 305530 294907 303486 301709 Detectability: Contro Sample: 1.00% Tetanus Toxoid spiked in Hib Tetanus Toxoid Conjugate of 1.00 mg/mL based on protein concentration. Test sample 1: Three independent preparations of 1.00 % Tetanus Toxoid spiked in Hib Tetanus Toxoid Conjugate of 1.0 mg/mL based on protein concentration . Test sample 2: Three independent preparations of 0.96% Tetanus Toxoid spiked in Hib Tetanus Toxoid Conjugate of 1.0 mg/mL based on protein concentration Acceptance criteria: Test sample 1 should give the response equivalent to or greater than that of Control sample Each preparation of test sample 2 must provide a response that is less than that of the Control sample Calculated and Observed results: Test sample 1 gave a response equivalent to or greater than that of Control sample Each preparation of test sample 2 gave a response that is less than that of the Control sample Test sample 1 and test sample 2 meets the acceptance criteria for LOD Table 28 Detectability Sample Control sample ((Hib- Tetanus Toxoid (1.0 mg spiked with 1% Tetanus Toxoid (10 g)) Test Sample-1 ((Hib- Tetanus Toxoid (1.0 mg spiked with 1% Tetanus Toxoid (10 g)) Test Sample-2 ((Hib- Tetanus Toxoid (1.0 mg spiked with 0.96% Tetanus Toxoid (9.6 g)) Replicates -R-1 R-2 R-3 R-1 R-2 R-3 Area 303486 303878 302892 305530 289369 288206 285248 287608 2125 0.74 304100 1333 0.44 Mean -Standard %RSD Deviation --302067 3725 1.23

%RSD

SPECIFIC TESTS Molecular size distribution i. Specificity Blank: 1 injection Vo & Vt mix solution (System suitability): 1 injection, (Dextran & Acetone) Un-related Sample solutions: 1 injection each for Tetanus Toxoid, Pullulan Standard solution & Haemophilus influenzae type b PRP-Tetanus Toxoid Conjugate Sample solution: 1 injection, (10.0 mg/mL solution of Haemophilus influenzae type b PRP)

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Acceptable criteria: Buffer solution should not show any interference in the integration window. Haemophilus influenzae type b PRP should show specific response for PRP polysaccharide. Non related samples like Tetanus Toxoid and Haemophilus influenzae type b PRP-Tetanus Toxoid Conjugate should elute at distinctly different RT. System suitability: The therotical plates with respect to acetone peak should be NLT 3000. Calculated and observed results: No interference observed from the buffer solution in the integration window. Haemophilus influenzae type b PRP has shown a specific response for PRP at ~25.9 min Tetanus Toxoid and Haemophilus influenzae type b Tetanus Toxoid Conjugate elutes at different RT other than PRP Figure 19: Chromatogram of Blank solution

Blank

Figure 20: Chromatogram of Vo + Vt mix (Dextran & Acetone)

Dextran + Acetone

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Figure 21: Chromatogram of Haemophilus influenzae type b PRP

PRP (RT ~25.9 min)

Figure 22: Chromatogram of Hib Tetanus Toxoid Conjugate

PRP-Tetanus Toxoid conjugate (RT ~21.7)

Figure 23: Chromatogram of Pullulan Standard solution

Pullulan (RT ~23.3 min)

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Figure 24: Chromatogram of Tetanus Toxoid Conjugate

Tetanus Toxoid Conjugate (RT ~59.3 min)

Precision: Repeatibility: Repeatability is performed using 6 independent preparations of Haemophilus influenzae type b PRP sample solutions by one analyst on a single day. Acceptable criteria: %RSD of molecular size distribution of PRP for 6 replicates should be NMT 2.0%. Calculated and Observed results: The %RSD observed between 6 replicates was 0.27%. Table 29: RSD calculation for Haemophilus influenzae type b PRP Replicates R1 R2 R3 R4 R5 R6 Mean Standard Deviation %RSD % Distribution 51.37 51.66 51.78 51.68 51.57 51.63 51.62 0.14 0.27

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