Protein Hydrolysate Injection
»Protein Hydrolysate Injection is a sterile solution of amino acids and short-chain peptides,which represent the approximate nutritive equivalent of the casein,lactalbumin,plasma,fibrin,or other suitable protein from which it is derived by acid,enzymatic,or other methods of hydrolysis.It may be modified by partial removal and restoration or addition of one or more amino acids.It may contain alcohol,dextrose,or other carbohydrate suitable for intravenous infusion.Not less than 50.0percent of the total nitrogen present is in the form of a-amino nitrogen.
Packaging and storage— Preserve in single-dose containers,preferably of Type Ior Type IIglass,and avoid excessive heat.
Labeling— The label of the immediate container bears in a subtitle the name of the protein from which the hydrolysate has been derived and the word “modified”if one or more of the “essential”amino acids has been partially removed,restored,or added.The label bears a statement of the pHrange;the name and percentage of any added other nutritive ingredient;the method of hydrolysis;the nature of the modification,if any,in amino acid content after hydrolysis;the percentage of each essential amino acid or its equivalent;the approximate protein equivalent,in g per liter;the approximate number of calories per liter;the percentage of the total nitrogen in the form of a-amino nitrogen;and the quantity of the sodium and of the potassium ions present in each 100mLof the Injection.Injection that contains not more than 30mg of sodium per 100mLmay be labeled “Protein Hydrolysate Injection,Low Sodium,”or by a similar title the approximate equivalent thereof.
The label states the total osmolar concentration in mOsmol per liter.Where the contents are less than 100mL,or where the label states that the Injection is not for direct injection but is to be diluted before use,the label alternatively may state the total osmolar concentration in mOsmol per mL.
Non-antigenicity—
Sensitizing solution— Select a suitable quantity of the protein identical in nature and quality with that from which the hydrolysate was manufactured,and subject it to the same hydrolytic process used in manufacturing the hydrolysate but reduce the time of hydrolysis to one-third.For purposes of preservation add,if desired,0.5%of chlorobutanol,and package the sensitizing solution in 100-mLmultiple-dose containers.Store in a cold place.
Preparation of animals— Select healthy guinea pigs each weighing between 420and 480g.Inject each animal intraperitoneally with 6mLof the sensitizing solution on the second,fourth,and sixth days of each of two successive weeks.Use the sensitized animals not less than 30days and not more than 37days after the last sensitizing dose.Re-sensitize any animals not used during the 7-day period by injecting intraperitoneally a booster dose of 6mLof the sensitizing solution,and use re-sensitized animals not less than 9days and not more than 16days after the injection of the booster dose.
Procedure— Inject,intravenously,3mLof Injection,at the rate of 2mLper minute,using a 5-mLsyringe fitted with a 27-gauge needle,into each of five guinea pigs prepared as described above.During the injection and during the 15minutes following,observe the animals for any of the following symptoms:(1)licking the nose or rubbing the nose with forefeet;(2)ruffling of the fur;(3)labored breathing;(4)sneezing or coughing (three or more times);(5)retching.The requirements of the test are met if none of the injected animals show more than two of the listed symptoms and none show rales,convulsions,or prostration,or die.If none of the listed symptoms are observed,test the sensitivity of the animals by injecting into one of them 3mLof the sensitizing solution intravenously at the rate of 2mLper minute:the animal shows positive signs of anaphylaxis such as rales,convulsions,prostration,and/or death in addition to one or more of the lesser reaction symptoms.
Bacterial endotoxins á85ñ— It contains not more than 0.5USP Endotoxin Unit per mg of protein hydrolysate.
Biological adequacy— It meets the requirements under Protein—Biological Adequacy Test á141ñ.
pHá791ñ: between 4.0and 7.0,determined potentiometrically,but the variation from the pHrange stated on the label is not greater than ±0.5pHunit.
Content of a-amino nitrogen— Dilute 5.0mLof a 5%Injection,or an appropriate volume of any other concentration,to 25mL.Adjust to a pHof 7,potentiometrically,by the addition of 0.1Nsodium hydroxide or 0.1Nhydrochloric acid.Add 10mLof formaldehyde TS,previously adjusted to a pHof 9.0,potentiometrically,then while stirring the solution,preferably with a mechanical stirrer,and with a suitable glass electrode in the system,add 0.1Nsodium hydroxide VSslowly toward the end,until a pHof 9.0is reached.Continue stirring for 2minutes,check the pH,and adjust if necessary.Record the volume of 0.1Nsodium hydroxide VSadded in the titration.Each mLof 0.10Nsodium hydroxide corresponds to 1.4mg of a-amino nitrogen.
Nitrogen content— Using 0.1mLof Injection,determine the nitrogen content as directed under Method II(see Nitrogen Determination á461ñ).
Potassium content—
Standard solutions— Prepare four standard solutions (numbered 1,2,3,and 4)each containing a suitable wetting agent and 25.0mEq of sodium (1.46g of sodium chloride)per liter,and to the solutions add,respectively,0-,2.0-,3.0-,and 4.0-mg supplements of potassium,in the form of the chloride,per liter.If necessary,because of changes in the sensitivity of the instrument mentioned below,vary the levels of concentration of the potassium,keeping the ratios between solutions approximately as given.
Standard graph— Set a suitable flame photometer to a wavelength of 766nm.Adjust the instrument to zero transmittance with solution 1.Then adjust the instrument to 100%transmittance with solution 4.Read the percent transmittance of solutions 2and 3.Plot the observed transmittance of solutions 2,3,and 4as the ordinate and the concentration as the abscissa on arithmetic coordinate paper.
Procedure— Pipet a portion of the Injection containing approximately 300µg of potassium,or a quantity corresponding to the concentration of the Standard solutions,into a 100-mLvolumetric flask.Add a small amount of wetting agent,and dilute to volume with a sodium solution of such strength that the final sodium concentration is 25.0mEq per liter.Adjust the instrument to zero transmittance with solution 1and to 100%transmittance with solution 4.Read the percent transmittance of the test solution and,by reference to the standard potassium graph,calculate the potassium content of the Injection in mg of potassium per mL.
Sodium content— Proceed as directed under Potassium content,with the following modifications:(1)prepare the Standard solutionsto contain 6.00mEq of potassium (447mg of potassium chloride),and substitute sodium for the stated quantities of potassium;(2)prepare the Standard graphwith the flame photometer set at 589nm instead of 766nm;and (3)under Procedureread “sodium”for “potassium”throughout,but in the second sentence read “a potassium solution of such strength that the final potassium concentration is 6.00mEq per liter”in place of “a sodium solution of such strength that the final sodium concentration is 25.0mEq per liter.”
Other requirements— It meets the requirements under Injections á1ñ.
Auxiliary Information— Staff Liaison:Behnam Davani,Ph.D.,MBA,Senior Scientist
Expert Committee:(PA7)Pharmaceutical Analysis 7
USP28–NF23Page 1668
Phone Number:1-301-816-8394