Trypsin inhibitor, pancreatic basic.
l-Arginyl-l-prolyl-l-aspartyl-l-phenylalanyl-l-cysteinyl-l-leucyl-l-glutamyl-l-prolyl-l-prolyl-l-tyrosyl-l-threonylglycyl-l-prolyl-l-cysteinyl-l-lysyl-l-alanyl-l-arginyl-l-isoleucyl-l-isoleucyl-l-arginyl-l-tyrosyl-l-phenylalanyl-l-tyrosyl-l-asparaginyl-l-alanyl-l-lysyl-l-alanylglycyl-l-leucyl-l-cysteinyl-l-glutaminyl-l-threonyl-l-phenylalanyl-l-valyl-l-tyrosylglycylglycyl-l-cysteinyl-l-arginyl-l-alanyl-l-lysyl-l-arginyl-l-asparaginyl-l-asparaginyl-l-phenylalanyl-l-lysyl-l-seryl-l-alanyl-l-glutamyl-l-aspartyl-l-cysteinyl-l-methionyl-l-arginyl-l-threonyl-l-cysteinylglycylglycyl-l-alanine cyclic (5®55), (14®38), (30®51) tris(disulfide) [9087-70-1].
» Aprotinin is a polypeptide consisting of a chain of 58 amino acid residues, which inhibits stoichiometrically the activity of several proteolytic enzymes such as chymotrypsin, kallikrein, plasmin, and trypsin. Aprotinin is obtained from bovine tissues and purified by a suitable process, and is stored as a bulk solution or lyophilized powder. Its potency calculated on the dried basis is not less than 3 USP Aprotinin Units per mg. In addition, the method of manufacture is validated to result in not more than 0.2 µg of histamine per 3 USP Aprotinin Units using validated methods. The origin and sourcing of bovine material must be specified in compliance with FDA requirements. The manufacturing process is validated to demonstrate the clearance of potential infectious agents (i.e., viruses, TSE agents). One USP Aprotinin Unit is equivalent to 1800 Kallikrein Inhibition Units (K.I.U.).
Packaging and storage For lyophilized powder, preserve in tight containers, and store in a cold place. Protect from light. For bulk solution, preserve in tight containers at a temperature not exceeding 25. Avoid freezing.
Labeling The labeling states the source of material and the number of Kallikrein Inhibition Units per mg or the number of Kallikrein Inhibition Units per mL.
USP Reference standards 11
USP Aprotinin RS.
USP Aprotinin System Suitability RS.
USP Endotoxin RS.
USP Trypsin Crystallized RS.
A: Thin-Layer Chromatographic Identification Test 201
Test solution Prepare a solution of Aprotinin in water having a concentration of about 15 USP Aprotinin Units per mL.
Developing solvent system: a mixture of glacial acetic acid and water (100 : 80) containing 100 g per L of sodium acetate.
Cupric chloride solution Dissolve 1 g of cupric chloride in 100 mL of water.
Spray reagent Dissolve 0.1 g of ninhydrin in a mixture containing 6 mL of Cupric chloride solution, 21 mL of glacial acetic acid, and 70 mL of alcohol.
Procedure Proceed as directed in the chapter, except to spray the plate with the Spray reagent, and heat at 60 to visualize the spots.
B: The retention time of the major peak in the chromatogram of the Test solution corresponds to that in the chromatogram of the Resolution solution, as obtained in the test for Limit of N-pyroglutamyl-aprotinin and related compounds.
Absorbance 851 Prepare a solution containing 3.0 USP Aprotinin Units per mL. The solution shows an absorption maximum at 277 nm. The absorbance at the maximum is not greater than 0.80.
Safety Prepare a solution of Aprotinin that contains 4 USP Aprotinin Units per mL using a sufficient quantity of Water for Injection. It meets the requirements when tested as directed in the section Safety TestsBiologicals under Biological Reactivity Tests, In Vivo 88.
Bacterial endotoxins 85 It contains not more than 0.14 USP Endotoxin Unit per USP Aprotinin Unit. Use a solution that contains 6 USP Aprotinin Units per mL.
Loss on drying 731 [noteThis test should only be performed on the lyophilized powder.] Dry about 100 mg, accurately weighed, in a capillary-stoppered bottle in vacuum at a pressure not exceeding 5 mm of mercury at 60 for 3 hours: it loses not more than 6.0% of its weight.
Specific activity of the dry residue [noteThis test should only be performed when product is a concentrated solution.] Evaporate 25.0 mL of Aprotinin concentrated solution to dryness in a water bath, dry the residue at 110 for 15 hours, and weigh. From the weight of the residue and the activity determined in the Assay, calculate the number of USP Aprotinin Units per mg of dry residue. Not less than 3.0 USP Aprotinin Units per mg of dried residue is found.
Limit of des-Ala-aprotinin and des-Ala-des-Gly-aprotinin
Test solution Dilute a concentrated solution of Aprotinin with water, or weigh out Aprotinin and dissolve in water, to obtain a solution containing about 4 to 7 USP Aprotinin Units per mL.
Standard solution Dilute USP Aprotinin RS with water to obtain a solution having a concentration similar to that of the Test solution.
Capillary zone electrophoresis buffer Dissolve 8.21g of monobasic potassium phosphate in 400 mL of water, adjust with phosphoric acid to a pH of 3.0, and dilute with water to 500 mL.
Capillary zone electrophoresis system (see Capillary Electrophoresis under Biotechnology-Derived ArticlesTest 1047) The capillary electropherograph is equipped with a 214-nm detector and a 45- to 60-cm uncoated fused silica capillary with an internal diameter of 75 µm with the temperature controlled at 25. Apply a field strength of 0.2 kV/cm for 30 minutes, using Capillary zone electrophoresis buffer as the electrolyte in both buffer reservoirs. Electropherograph the Standard solution, and record the peak responses as directed for Procedure: the relative migration times are about 0.98 for des-Ala-des-Gly-aprotinin, 0.99 for des-Ala-aprotinin, and 1 for aprotinin. The resolution, RS, between the des-Ala-des-Gly-aprotinin and des-Ala-aprotinin peaks is not less than 0.8, and the resolution between the des-Ala-aprotinin and aprotinin peaks is not less than 0.5. The migration time for the aprotinin peak is between 19 and 25 minutes. The tailing factor, T, of the aprotinin peak is not more than 3 (see Chromatography 621 for calculation). The baseline is stable and shows little drift. Rinse the capillary for at least 1 minute with at least 10 total capillary volumes of 0.1 N sodium hydroxide, followed by at least 10 total capillary volumes of water, and by at least 20 capillary volumes of Capillary zone electrophoresis buffer between injections.
Procedure Transfer a volume of the Test solution, approximately 15 nL, into the anodic end of the capillary (apply differential pressure of 3.5 kPa for 3 seconds either by vacuum or pressure), record an electropherogram, and measure the peak areas. Calculate the percentage contents of des-Ala-des-Gly-aprotinin and des-Ala-aprotinin by the formula:
100(ri / rs)in which ri is the peak response corresponding to des-Ala-des-Gly-aprotinin or des-Ala-aprotinin; and rs is the sum of the responses of des-Ala-des-Gly-aprotinin, des-Ala-aprotinin, and aprotinin peaks: not more than 8.0% des-Ala-des-Gly-aprotinin and not more than 7.5% des-Ala-aprotinin is found.
Limit of N-pyroglutamyl-aprotinin and related compounds
Solution A Prepare a filtered and degassed solution containing 3.52 g of monobasic potassium phosphate and 7.26 g of dibasic sodium phosphate dissolved in 1000 mL of water.
Solution B Prepare a filtered and degassed solution containing 3.52 g of monobasic potassium phosphate, 7.26 g of dibasic sodium phosphate, and 66.07 g of ammonium sulfate dissolved in 1000 mL of water.
Resolution solution Prepare a solution of USP Aprotinin System Suitability RS containing about 5 USP Aprotinin Units per mL in Solution A.
Test solution Prepare a solution of Aprotinin having a concentration of about 5 USP Aprotinin Units per mL. Dilute with Solution A.
Chromatographic system (see Chromatography 621) The liquid chromatograph is equipped with a 210-nm detector and a 7.5-mm × 7.5-cm column that contains packing L52 and is maintained at a constant temperature of 40. The flow rate is 1 mL per minute. The chromatograph is programmed as follows.
Procedure Inject 40 µL of the Test solution into the chromatograph, record the chromatogram, and measure the peak areas. Calculate the percentage of each impurity peak in the chromatogram by the formula:
100(ri / rs)in which ri is the response of each impurity peak; and rs is the sum of the responses of all peaks in the chromatogram of the Test solution: not more than 1.0% N-pyroglutamyl-aprotinin is found; not more than 0.5% of any other impurity is found; and the sum of all unknown impurities is not more than 1.0%.
Limit of high molecular weight proteins
Mobile phase Prepare a filtered and degassed mixture of water, glacial acetic acid, and acetonitrile (6:2:2).
Resolution solution Prepare an aprotinin solution that contains about 5 USP Aprotinin Units per mL with about 2% aprotinin oligomers. [noteThis solution can be obtained by heating lyophilized aprotinin at 112 for about 2 hours and dissolving the solid at the specified concentration in water.]
Test solution Prepare a solution of Aprotinin in water that contains about 5 USP Aprotinin Units per mL.
Chromatographic system (see Chromatography 621) The liquid chromatograph is equipped with a 280-nm detector and a series of three 7.8-mm × 30-cm columns containing packing L33. The flow rate is about 1.0 mL per minute. Chromatograph the Resolution solution as directed for Procedure: the retention time for aprotinin is between 24.5 and 25.5 minutes; the relative retention times are about 0.9 for the dimer and 1.0 for aprotinin; the resolution, R, between the dimer peak and the aprotinin peak is not less than 1.3; and the tailing factor for the aprotinin peak is not greater than 2.5.
Procedure Inject about 100 µL of the Test solution into the chromatograph, record the chromatogram, and measure the peak areas. Calculate the percentage of each oligomer peak in the chromatogram by the formula:
100ri / rsin which ri is the response of each peak having a retention time less than that of aprotinin monomer; and rs is the sum of the responses of all peaks: the sum of all oligomers is not more than 1.0%.
0.0015 M Borate buffer Transfer about 0.93 g of boric acid into a 1000-mL volumetric flask, dissolve in 900 mL of water, adjust with 5 N sodium hydroxide to a pH of 8.0, dilute with water to volume, and mix. Transfer 100 mL of this solution into a 1000-mL volumetric flask, dilute with water to volume, and mix.
Assay preparation Prepare a solution of Aprotinin with 0.0015 M Borate buffer to obtain a solution having about 1.67 USP Aprotinin Units per mL (about 0.6 mg per mL).
Trypsin solution Prepare a solution of USP Trypsin Crystallized RS containing about 4300 USP Trypsin Units per mL, using 0.001 N hydrochloric acid as the solvent. Use a freshly prepared solution, and keep in ice water.
Trypsin and aprotinin solution To 4.0 mL of the Trypsin solution, add 1.0 mL of the Assay preparation. Dilute immediately with 0.0015 M Borate buffer to 40.0 mL. Allow to stand at room temperature for 10 minutes, and then keep in ice water. [noteUse within 6 hours of preparation.]
Dilute trypsin solution Dilute 0.5 mL of the Trypsin solution with 0.0015 M Borate buffer to 10.0 mL. Allow to stand at room temperature for 10 minutes, and then keep in ice water.
Substrate solution Dissolve 69 mg of N-benzoyl-l-arginine ethyl ester hydrochloride in 10 mL of water. [noteUse within 2 hours.]
Procedure Mix 9.0 mL of 0.0015 M Borate buffer and 1.0 mL of Substrate solution in a jacketed glass vessel with a capacity of about 30 mL and that contains a stirring device. The lid of the reaction vessel should contain five holes to accommodate the electrodes, the tip of a buret, a tube for the admission of nitrogen, and the introduction of reactants. An automated or manual titration apparatus may be used. Adjust to a pH of 8.0 by the addition of 0.1 N sodium hydroxide VS. Maintain an atmosphere of nitrogen within the vessel, and stir continuously. When the temperature has reached equilibrium at 25 ± 0.1, add 1.0 mL of Trypsin and aprotinin solution, and start a timer. Maintain at a pH of 8.0 by the addition of 0.1 N sodium hydroxide VS, and note the volume added every 30 seconds. Continue the reaction for 6 minutes. Determine the volume of 0.1 N sodium hydroxide added per second, in mL (n1). Carry out a similar titration using 1.0 mL of the Dilute trypsin solution. Determine the volume of 0.1 N sodium hydroxide added per second, in mL (n2). For the lyophilized powder, calculate the aprotinin activity in USP Aprotinin Units per mg using the formula:
4000(2n2 n1)/min which m is the quantity, in mg, of Aprotinin used to prepare 1 mL of the Assay preparation. For the concentrated solution, calculate the USP Aprotinin Units per mL using the following formula:
4000(2n2 n1)Din which D is the dilution factor of the concentrated solution used to prepare the Assay preparation.
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USP32NF27 Page 1575Pharmacopeial Forum: Volume No. 31(3) Page 732
Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.