Mobile phase— Prepare separate filtered and degassed solutions consisting of a solution (1in 1000)of trifluoroacetic acid in water (Solution A)and a 1in 1000solution of trifluoroacetic acid in acetonitrile (Solution B).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Digestion solution— Dissolve 29.4mg of calcium chloride and 1.8mg of b-alanine in 2mLof water.Adjust with hydrochloric acid to a pHof 4.0.Add 0.4mg of trypsin,and mix.

Standard solution— Dissolve an accurately weighed quantity of USP Sargramostim RSin water,and dilute quantitatively with water to obtain a solution having a known concentration of about 500µg per mL.Transfer 100µLof this solution to a clean test tube,and add 11µLof pH7.6buffer solution (see Buffer Solutionsin the section Reagents,Indicators,and Solutions)containing 0.1Mtris(hydroxymethyl)aminomethane.Add 25µLof Digestion solution,and incubate at 37for 2hours.Quench the reaction by adding 3µLof 20%trifluoroacetic acid.

Test solution— Using an accurately weighed quantity of Sargramostim,proceed as directed for Standard solution.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 220-nm detector and a 25-cm ×4.6-mm column that contains 10-µm packing L1.The column is maintained at ambient temperature,and the flow rate is about 1mLper minute.The system is programmed to provide variable mixtures of Mobile phase.Equilibrate the system with a Mobile phaseconsisting of 100%Solution A.After injection of the solution under test,the composition is changed linearly at a rate of 1%per minute over the next 35minutes so that it then consists of 65%Solution Aand 35%Solution B,and then changed linearly at a rate of 2%per minute over the next 15minutes to a mixture of 35%Solution Aand 65%Solution B.

Procedure— Separately inject equal volumes (about 100µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the eight major peaks as defined in the USP Sargramostim RS Data Sheet.The retention times of the peak responses from the Test solutioncorrespond to those from the Standard solutionif the retention times of corresponding peaks do not differ by more than 0.3minutes,the peak areas ratios for peaks 4,8,and 10are between 0.7and 1.3,and no additional significant peaks or shoulders are found.

Mobile phase— Prepare separate filtered and degassed solutions consisting of a solution (1in 1000)of trifluoroacetic acid in water (Solution A),a 1in 1000solution of trifluoroacetic acid in acetonitrile (Solution B),and a solution prepared by dissolving 116.9g of sodium chloride in 2000mLof water and adding 2mLof trifluoroacetic acid (Solution C).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution— Dissolve an accurately weighed quantity of USP Sargramostim RSin water,and dilute quantitatively with water to obtain a solution having a known concentration of about 1.0mg per mL.

Test solution— Using an accurately weighed quantity of Sargramostim,prepare as directed for Standard solution.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1mLper minute.The system is programmed to provide a Mobile phaseconsisting of a mixture containing variable proportions of Solution Aand Solution B,with a constant 20%of Solution C.Equilibrate the system with a mixture of 55%Solution A,25%Solution B,and 20%Solution C.After injection of the Test solution,the proportion of Solution Ais decreased linearly from 55%to 15%and the proportion of Solution Bis increased linearly from 25%to 65%at a rate of 1%per minute.

Procedure— Separately inject equal volumes (about 50µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The major peaks are from hyperglycosylated sargramostim and from the three glycosylated forms of Sargramostim,as indicated in the USP Sargramostim RS Data Sheet.The peak responses from the Test solutioncorrespond to those from the Standard solution,and no peaks or shoulders are present in the chromatogram of the Test solutionthat are not present in the chromatogram of the Standard solution.Calculate the percentage of hyperglycosylated sargramostim in the Test solutionby the formula: in which rUis the peak response for hyperglycosylated sargramostim,and rsis the sum of the responses of all of the sargramostim peaks:not more than 5.6%is found.Calculate the percentage of each of the three glycosylated forms of Sargramostim in the Test solutionby the formula: in which rnis the peak response for each individual glycosylated form of Sargramostim,and rtis the sum of the peak responses of all three glycosylated forms of Sargramostim.The percentages of each of the three glycosylated forms of Sargramostim,in order of elution,are between 25%and 42%,between 14%and 32%,and between 35%and 53%.

Standard solutions— Dissolve accurately weighed quantities of USP Sargramostim RSin water to obtain solutions having known concentrations of about 100,200,400,600,800,and 1000µg per mL.

Test solution— Dissolve an accurately weighed quantity of Sargramostim in water to obtain a solution having a concentration of between 250and 500µg per mL.

BCAreagent— Dissolve 10g of bicinchoninic acid,20g of sodium carbonate monohydrate,1.6g of sodium tartrate,4g of sodium hydroxide,and 9.5g of sodium bicarbonate in water.Adjust,if necessary,with sodium hydroxide or sodium bicarbonate to a pHof 11.25.Dilute with water to 1liter,and mix.

Copper sulfate reagent— Dissolve 2g of cupric sulfate in water,and dilute with water to 50mL.

Copper-BCAreagent— Mix 1mLof Copper sulfate reagentand 50mLof BCAreagent.[NOTE—If a commercially available kit is used,follow the manufacturer's instructions for preparation of the Copper-BCAreagent.]

Procedure— Mix 0.1mLof each Standard solution,the Test solution,and 0.1mLof water to provide the blank with 2mLof the Copper-BCAreagent.Incubate the solutions at 37for 30minutes,and allow to stand for 5minutes at room temperature.Within 60minutes following incubation,determine the absorbances of the Standard solutionsand the Test solutionin 1-cm cells at 562nm,with a suitable spectrophotometer (see Spectrophotometry and Light-scattering á851ñ),using the blank to set the instrument to zero.Plot the absorbances of the Standard solutionsversus the concentrations,in µg per mL,of USP Sargramostim RS,and draw the straight line best fitting the plotted points.From the graph so obtained,determine the concentration,in µg per mL,of protein in the Test solution.

Iscove's Modified Dulbecco's Medium— Prepare a mixture of the ingredients in the quantities shown in sufficient water to obtain 1liter of medium,and sterilize by filtration.

Medium B— Dissolve an accurately weighed quantity of USP Sargramostim RSin Medium A,and dilute quantitatively with Medium Ato obtain a solution having a known concentration of about 1µg per mL.Prepare aseptically,sterilize by filtration,and store at 2to 8.Use within one month.

Standard preparation— Dissolve an accurately weighed quantity of USP Sargramostim RSin Medium A,and dilute quantitatively with Medium Ato obtain a solution having a known concentration of about 100ng per mL.Dispense aseptically in equal portions,accurately measured,and store at -60or below.Use within 24months.Store thawed portions at a temperature between 2and 8,and use within one month.At the time of use,dilute with Medium Ato obtain a solution having a known concentration of about 2ng per mL.

Assay preparation— Dissolve an accurately weighed quantity of Sargramostim in Medium A,and dilute quantitatively with Medium Ato obtain a solution containing about 2ng per mL.

Cell culture preparation— Prepare cell cultures of [TF-1cells (ATCC CRL-2003)].Passage the cultures every 3to 4days,using a 1:10subculture of the cells for up to 3months.After 3months,initiate a new culture.Use Medium Acontaining 0.5%Medium Bfor passage propagation and storage in the frozen state.

Cell suspension— Wash the cells three times in Medium A,and adjust the cell concentration to 5×104cells per mLin Medium A.

Tritiated thymidine solution— Use a tritiated thymidine stock solution having a concentration of 20Ci per mmol.Add 1.0mLof the tritiated thymidine stock solution to 49mLof Medium A,and store at 2to 8.Use within 2weeks.

Procedure— Use a 96-well,flat-bottom microtitration plate with wells arranged in 8rows (labeled Athrough H)with 12wells (numbered 1to 12)in each row.Place 50µLof Medium Ain wells 2to 12.Place 100µLof the Standard preparation,or each Assay preparationor Medium A(negative control)in well 1.Make serial dilutions by transferring 50µLfrom well 1to well 2,and so on through well 12(serial twofold dilutions).Place 50µLof the Cell suspensionin each well,and incubate the microtitration plate for 72hours at 37in a 10%carbon dioxide incubator.Following incubation,add 25µLof Tritiated thymidine solutionto each well,and return the plate to the same incubator for an additional 4to 5hours.Before harvesting the cells on a filter mat,pre-wet the mat filter,using distilled water.[NOTE—The pre-wetting minimizes background radiation noise.]Using a multiple automated sample harvesting system,place the incubated plate under the harvesting system.Fill the wells to the top with deionized water.Aspirate the water,and pass it through the collecting filter mat.Repeat the procedure at least 5times,or until all the cells have been fully harvested.When all wells have been fully harvested,pour 5to 10mLof alcohol on the plate tray,and aspirate the methanol.Repeat the procedure if further drying of the filter mat is desired.[NOTE—The alcohol helps to dry out the filter mat by carrying away the wash fluid.]Remove the filter mat,and repeat the procedure until all plates under test have been harvested.