Hyaluronidase for Injection
»Hyaluronidase for Injection is a sterile,dry,soluble enzyme product prepared from mammalian testes and capable of hydrolyzing mucopolysaccharides of the type of hyaluronic acid.Its potency,in USP Hyaluronidase Units,is not less than the labeled potency.Hyaluronidase for Injection contains not more than 0.25µg of tyrosine for each USP Hyaluronidase Unit.It may contain a suitable stabilizer.
Packaging and storage— Preserve in Containers for Sterile Solidsas described under Injections á1ñ,preferably of Type Ior Type IIIglass,and store at controlled room temperature.
Sterility á71ñ: meets the requirements.
Bacterial endotoxins á85ñ— It contains not more than 2.30USP Endotoxin Units per USP Hyaluronidase Unit.
Limit of tyrosine— Dissolve the entire contents of 1or more containers of Hyaluronidase for Injection in sufficient water,accurately measured,to give a concentration of about 60USP Hyaluronidase Units per mL.Transfer 2.0mLof the solution to a 15-mLcentrifuge tube calibrated at 6mL,and evaporate at 105to dryness.Add 200µLof 6Nsodium hydroxide,and heat with steam under pressure at 121for 3hours.Add 300µLof 7Nsulfuric acid,then add 1.5mLof water and 1.5mLof a 15in 100solution of mercuric sulfate in 5Nsulfuric acid.Heat on a steam bath for 10minutes,and cool to room temperature.Add 1mLof 7Nsulfuric acid and 1mLof sodium nitrite solution (1in 500),with shaking.Add water to make 6mL,mix,centrifuge,and decant the supernatant.Twenty minutes after diluting to 6mL,determine the absorbance of the supernatant at 540nm,with a suitable spectrophotometer.
Repeat the preceding test,using the same quantities of the same reagents and in the same manner but omitting the Hyaluronidase for Injection and replacing the 1.5mLof water with 1.5mLof a solution of USPL-Tyrosine RSin 0.4Nsulfuric acid containing 30µg in each mL.
Calculate the quantity,in µg,of tyrosine in the 2-mLaliquot of the solution of Hyaluronidase for Injection taken by the formula:
45(AU/AS),
in which AUand ASare the absorbances of the solution from Hyaluronidase for Injection and the Standard solution,respectively:not more than 0.25µg of tyrosine is found for each USP Hyaluronidase Unit.
Assay—
Acetate buffer solution— Dissolve 14g of potassium acetate and 20.5mLof glacial acetic acid in water to make 1000mL.
Phosphate buffer solution— Dissolve 2.5g of monobasic sodium phosphate,1.0g of anhydrous dibasic sodium phosphate,and 8.2g of sodium chloride in water to make 1000mL.
Hydrolyzed gelatin— Dissolve 50g of bacteriological gelatin in 1000mLof water,heat in an autoclave at 121for 90minutes,and freeze-dry the solution.
Diluent for hyaluronidase solutions— Mix 250mLof Phosphate buffer solutionwith 250mLof water and,within 2hours before use,dissolve 330mg of Hydrolyzed gelatinin the mixture.
Serum stock solution— Constitute dried horse serum with water to its original volume,and dilute it with 9volumes of Acetate buffer solution.Adjust with 4Nhydrochloric acid to a pHof 3.1,and allow the solution to stand at room temperature for 18to 24hours.Store the solution at 0to 4,and use within 30days.
Serum solution— On the day of the assay,dilute 1volume of Serum stock solutionwith 3volumes of Acetate buffer solution,and adjust to room temperature.
Potassium hyaluronate stock solution— Prepare a stock solution to contain,in each mL,500µg of potassium hyaluronate,previously dried in vacuum over phosphorus pentoxide for 48hours.Do not keep the hyaluronate over phosphorus pentoxide indefinitely.The use of a weighing bottle is desirable.Store the solution at a temperature not exceeding 5,and use within 30days.
Hyaluronate solution— On the day of the assay,dilute 1volume of Potassium hyaluronate stock solutionwith 1volume of Phosphate buffer solution.
Standard solution— Dissolve a suitable quantity of USP Hyaluronidase RS,accurately weighed,in cold Diluent for hyaluronidase solutionsto obtain a solution having a known concentration of about 1.5USP Hyaluronidase Units in each mL.Prepare this solution immediately before use in the assay.
Assay solution— Dissolve the contents of 1container of Hyaluronidase for Injection by adding cold Diluent for hyaluronidasedirectly to the container.On the basis of trial or experience,dilute the solution so prepared with cold Diluent for hyaluronidaseso that the observed absorbances with the three dilutions of Hyaluronidase for Injection fall on the upper,linear part of the standard curve prepared as directed below.
Procedure— Prepare a standard concentration-response curve by adding to each of twelve 16-×100-mm test tubes 500µLof Hyaluronate solution.To each of two of the tubes add,respectively,500,400,300,200,100,and 0µLof Diluent for hyaluronidase solutions.If quantities of Standard solutionother than those indicated below are used,change the above-specified quantities of Diluent for hyaluronidase solutionsso that the final volume of solution in each tube,after the addition of the Standard solution,is 1.00mL.
At 30-second intervals,accurately timed,add to each of two tubes 0,100,200,300,400,and 500µL,respectively,of Standard solution.Mix the contents by gentle shaking,and place each tube in a water bath maintained at 37±0.2.After 30±0.25minutes,remove each tube,in order,from the water bath at 30-second intervals,and immediately add 4.0mLof Serum solution.Shake the tube,and allow to stand at room temperature for 30±2minutes.Again shake the tube,and determine the absorbance at 640nm,with a suitable spectrophotometer.Perform a blank determination but omit the hyaluronate,and make any necessary correction.Plot the average absorbance value for each level against the hyaluronidase activity expressed in Units,and draw the smooth curve that best fits the plotted points.
Concurrently,to six test tubes add 500µLof Hyaluronate solutionand sufficient Diluent for hyaluronidase solutionsso that the final volume,after the addition of the Assay solution,is 1.00mL.Add,at 30-second intervals,sufficient Assay solutionso that duplicate tubes contain about 0.30,0.50,and 0.70Unit,respectively.Shake each tube gently,and treat as directed in the preceding paragraph for the Standard solution,beginning with “place each tube in a water bath.”Measure the absorbances in the spectrophotometer,and make any necessary correction for the blank.The potency of Hyaluronidase for Injection is the average of the six activity values read from the standard curve.
Auxiliary Information— Staff Liaison:Ian DeVeau,Ph.D.,Senior Scientist
Expert Committee:(BNT)Biotechnology and Natural Therapeutics/Diagnostics
USP28–NF23Page 950
Pharmacopeial Forum:Volume No.29(5)Page 1505
Phone Number:1-301-816-8178