DCN: 16-000495 v. 3.0 100 Majestic Way, Bangor, PA 18013 / www.biospectra.us Effective Date: 12-May-2020 12-May-2023 : Date of Next Review Initiated By: DeMaio, Jessica 2.0 : Supersedes Reason for Revision: Addition of statement from BLI17-58 Approval: Approvers Date Time Group Name 11-May-2020 03:03:45 PM EDITOR Saam, Amy 11-May-2020 04:55:54 PM QUALITY Miller, Jenna 12-May-2020 04:13:50 PM SNR MGMT Yencho, Amy UREA TESTING METHODS VIEW ONLY Printed On: 19-Oct-2020 10:53:06 AM Ledergerber, Alissa : Printed By Print Expiration: This copy is VIEW ONLY. It is not to be printed or distributed in any manner. Notice: The information contained herein is the property of BioSpectra. The recipient is responsible for its safe-keeping and the prevention of unauthorized appropriation, use, disclosure and copying. PAGE 1 OF 14 Urea Testing Methods DCN: 16-000495 v. 3.0 TABLE OF CONTENTS 1. PURPOSE: ............................................................................................................................................... 3 2. SCOPE: ................................................................................................................................................... 3 3. RESPONSIBILITIES: ................................................................................................................................ 3 4. REFERENCES: ........................................................................................................................................ 3 5. EQUIPMENT: .......................................................................................................................................... 3 6. REAGENTS: ............................................................................................................................................ 4 7. ANALYTICAL PROCEDURES: ................................................................................................................. 4 VIEW ONLY Printed On: 19-Oct-2020 10:53:06 AM Ledergerber, Alissa : Printed By Print Expiration: This copy is VIEW ONLY. It is not to be printed or distributed in any manner. Notice: The information contained herein is the property of BioSpectra. The recipient is responsible for its safe-keeping and the prevention of unauthorized appropriation, use, disclosure and copying. PAGE 2 OF 14 Urea Testing Methods DCN: 16-000495 v. 3.0 1. PURPOSE: 1.1. To provide the Quality Control (QC) Laboratory personnel with a procedure for analyzing Urea Raw Materials, In-Process, Finished Goods, and Stability. 2. SCOPE: 2.1. Applies to the analysis of Urea Raw Materials, In-Process, Finished Goods, and Stability in the QC Laboratory. Methods include testing for all codes of each grade of Urea sold by BioSpectra; only the specific tests required for the desired code must be tested for. This document applies all BioSpectra facilities. 3. RESPONSIBILITIES: 3.1. The Executive Director of Quality Control is responsible for training, maintenance and implementation of this procedure. 3.2. The QC Analysts are responsible for compliance with the terms of this procedure. This includes notifying the appropriate personnel if any analyses fail to meet their respective specifications. 4. REFERENCES: 4.1. ACS, Reagent Chemicals, current edition. 4.2. Balance SOP 4.3. Bangor Portable Turbidimeter Operation and Calibration 4.4. Current USP 4.5. Current EP/BP 4.6. Current JP 4.7. DNase (Endonuclease) Assay 4.8. DNase (Exonuclease) Assay 4.9. Endosafe PTS Endotoxin Reader SOP 4.10. Laboratory Notebooks 4.11. Lambda 25 UV/Vis Operation and Calibration 4.12. Metrohm 914 pH Conductometer Operation and Calibration 4.13. MF-50 Moisture Balance Operation and Calibration 4.14. MP50 Melting Range Operation and Calibration SOP 4.15. Muffle Furnace SOP and Calibration 4.16. NexION 350X ICP-MS SOP 4.17. Portable Turbidimeter SOP and Calibration 4.18. Protease Assay 4.19. RNase (Ribonuclease) Assay 4.20. Spectrum Two UATR SOP 4.21. Standardization of Titrants 4.22. Urea Assay via HPLC 4.23. XL200 pH/mV/Conductivity Meter SOP 4.24. Urea Mother Liquor Testing and Specifications 5. EQUIPMENT: 5.1. Analytical BalanceVIEW ONLY 5.2. Calibrated Oven Printed On: 19-Oct-2020 10:53:06 AM Ledergerber, Alissa : Printed By Print Expiration: This copy is VIEW ONLY. It is not to be printed or distributed in any manner. Notice: The information contained herein is the property of BioSpectra. The recipient is responsible for its safe-keeping and the prevention of unauthorized appropriation, use, disclosure and copying. PAGE 3 OF 14 Urea Testing Methods DCN: 16-000495 v. 3.0 5.3. Hach Portable Turbidimeter, or equivalent 5.4. Lambda 25 UV/Vis Spectrophotometer 5.5. MF-50 Moisture Balance 5.6. MP50 Melting Point Apparatus 5.7. Calibrated Muffle Furnace 5.8. Perkin Elmer NexION 350X ICP-MS 5.9. Perkin Elmer Spectrum Two UATR 5.10. XL200 pH/mV/Conductivity Meter or equivalent 5.11. Endosafe PTS Endotoxin Reader, or equivalent 6. REAGENTS: 6.1. Solution S (EP/BP) -Weigh 10.0g of sample and dissolve in USP Purified Water. Dilute to a total volume of 50 mL with USP Purified Water. 6.2. USP Purified Water is produced by the qualified water system at each BioSpectra facility. 7. ANALYTICAL PROCEDURES: IN PROCESS TESTING 7.1. ML ABSORBANCE 0.2 a.u. max @ 260 nm; Action Limit >0.18 a.u. @ 260 nm: 7.1.1.1. Transfer the entire Mother Liquor sample to a 150 mL beaker and gently heat until all crystals are back in solution, if necessary. 7.1.1.2. Prepare 40 mL of a 1:1 dilution with purified water of the specified Mother Liquor sample. Swirl to dissolve completely. 7.1.1.3. Measure and record the absorbance of the sample solution. Refer to Lambda 25 UV/Vis Operation and Calibration. 7.2. CONDUCTIVITY 0-2000µS/cm; Action Limit 1900-2000µS/cm: 7.2.1.1. Calibrate the conductivity meter prior to sample measurement using the 1413 µS/cm Conductivity standard. 7.2.1.2. Follow the appropriate SOP: 7.2.1.2.1. Stroudsburg: Metrohm 914 pH/Conductometer Operation and Calibration 7.2.1.2.2. Bangor: XL200 pH/mV/Conductivity Meter SOP 7.2.1.3. Rinse the electrode and temperature probe, if necessary, thoroughly with purified water. 7.2.1.4. Use the previously prepared 1:1 dilution sample. 7.2.1.5. Measure the conductivity of the sample solution according to the appropriate SOP. 7.3. BIURET UV Action Limit 0.14 -0.16 a.u.: 7.3.1. To the sample beaker, pipette 1.0 mL of Mother Liquor sample and 4.0 mL of Biuret Test Solution. 7.3.2. To the reference beaker, pipette 1.0 mL of purified water and 4.0 mL oc Biuret Test Solution. 7.3.3. After 15-20 minutes, measure the absorbance of the sample against the reference in a 1.0 cm cell at 540 nm. VIEW ONLY FINISHED GOOD TESTING Printed On: 19-Oct-2020 10:53:06 AM Ledergerber, Alissa : Printed By Print Expiration: This copy is VIEW ONLY. It is not to be printed or distributed in any manner. Notice: The information contained herein is the property of BioSpectra. The recipient is responsible for its safe-keeping and the prevention of unauthorized appropriation, use, disclosure and copying. PAGE 4 OF 14 Urea Testing Methods DCN: 16-000495 v. 3.0 7.4. ABSORBANCE (5M) 0.03 max. @ 280 nm, 0.05 max. @ 260 nm: 7.4.1. Prepare a 5M solution of the specified sample. 7.4.1.1. Accurately weigh 7.5 g of sample. 7.4.1.2. Transfer accurately weighed sample to a graduated cylinder and QS to 25 mL with purified water. Dissolve completely. 7.4.2. Refer to Lambda 25 UV/Vis Operation and Calibration to measure and record the absorbance of the sample. 7.5. ALCOHOL INSOLUBLE MATTER USP & JP (0.04% max.): 7.5.1. Dissolve 5 g of sample in 50mL of warm alcohol, and if any insoluble residue remains, filter the solution on a tared filter, wash the residue and the filter with 20mL of warm alcohol, and dry at 105 ±2°C for 1 hour. The weight of the residue does not exceed 2 mg (0.04%). 7.5.2. Calculate the % Insoluble Matter as follows: Residue Weight(g) % Insoluble Matter = x 100 Sample Weight (g) 7.6. ALCOHOL INSOLUBLE MATTER (0.01% max.): 7.6.1. Dissolve 10 g of sample in 100mL of warm alcohol, and if any insoluble residue remains, filter the solution on a tared filter, wash the residue and the filter with 40mL of warm alcohol, and dry at 105 ±2°C for 1 hour. The weight of the residue does not exceed 1 mg (0.01%). 7.6.2. Calculate the % Insoluble Matter as follows: Residue Weight(g) % Insoluble Matter = x 100 Sample Weight (g) 7.7. ALKALINITY EP/BP (Passes Test): 7.7.1. To 2.5 mL of Solution S, add 7.5 mL of USP Purified Water. 7.7.2. Add 0.1 mL of methyl red solution R and 0.4 mL of 0.01N hydrochloric acid (0.01M hydrochloric acid). 7.7.3. The solution must be red to orange to pass. 7.8. AMMONIUM - METHOD A EP/BP (500 ppm max.): 7.8.1. To 0.1mL of Solution S add 14mL of USP Purified Water in a test tube, make alkaline if necessary by the addition of dilute sodium hydroxide solution R and dilute to 15mL with USP Purified water. To the solution add 0.3mL of alkaline potassium tetraiodomercurate solution R. 7.8.1.1. Dilute sodium hydroxide solution R: Dissolve 8.5g of sodium hydroxide R in USP purified water and dilute to 100mL with USP purified water. 7.8.1.2. Alkaline potassium tetraiodomercurate solution R: Dissolve 11g of potassium iodide R and 15g of mercuric iodided R in USP purified water and dilute to 100mL with USP purified water. 7.8.1.2.1.VIEW Immediately before use,ONLY mix 1 volume of this solution with an equal volume of a 250g/L solution of sodium hydroxide R. Use this solution for analysis. Printed On: 19-Oct-2020 10:53:06 AM Ledergerber, Alissa : Printed By Print Expiration: This copy is VIEW ONLY. It is not to be printed or distributed in any manner.