METHOD FOR THE PRODUCTION OF A LIQUID TROMETHAMINE FORMULATION

Abstract

The present invention relates to a method for the production of a liquid formulation in a glass vial with a rubber stopper, where the formulation is tromethamine in water for injection (WFI), comprising the steps of: a) introducing WFI into a preparation vessel, b) dissolving a quantity of tromethamine in powder form in said WFI, while stirring until the tromethamine in powder form is completely dissolved, c) introducing at least one pH-modifying agent into the tromethamine solution, d) adding further WFI to the tromethamine solution, e) filtering the solution from step d) using a 0.2 µm filter, and f) filling one or more glass vials with the filtered batch solution before the vials are closed with a rubber stopper and the vials are subsequently sealed. The invention also relates to a sterile liquid formulation of tromethamine in WFI and the use thereof.

Description

highlightsthecriticalneedforalternativetherapiesthatdonothavetheseside effects. Accordingly,thereisaclearneedforalternativealkalitreatmentsthatovercomethe limitationsofsodiumbicarbonate.Thepresentinventionaimstoprovidesuchan5 alternative,alongwithmethodsforitsproduction. SUMMARYOFTHEINVENTION Thepresentinventionandembodimentsthereofservetoprovideasolutiontoone ormoreofabove-mentioneddisadvantages.10 Tothisend,thepresentinventionrelatestoamethodfortheproductionofaready- to-useliquidformulationinacontainer,whereintheformulationistromethaminein waterforinjectionaccordingtoclaim1.Saidmethodresultsinastable tromethamineformulationthatservesasanalternativetosodiumbicarbonateused15 forIVadministration,andcanbestoredatroomtemperatureforatleast24months. Preferredembodimentsofthemethodareshowninanyoftheclaims2to13. Inasecondaspect,thepresentinventionrelatestoaready-to-usesterileliquid formulationoftromethamineinwaterforinjectionaccordingtoclaim14.Preferred20 embodimentsofthemethodareshowninclaims15and16. Inathirdaspect,thepresentinventionrelatestoasterileliquidformulationforuse inthetreatment,correctionorpreventionmetabolicacidosisaccordingtoclaim17 or18.Preferredembodimentsareshowninanyoftheclaims19to21.25 Thesolutionorformulationisusedasanalternativetosodiumbicarbonatetoprovide electrolytesintravenouslyandtoincreasebloodpH,inpatientssufferingfromorat risktodevelopmetabolicacidosis.Itfurthercanbeusedtopreventorcorrectthe acidityofACDblood,andcanbeadministeredintravenouslyintotheventricular30 cavityofaheartduringcardiacarrestassociatedwithmetabolicacidosis. DESCRIPTIONOFFIGURES Figure1showstheresultsoftheHPLCAssaydescribedintheExamplesection, Example1,includingthechromatogramoftheblanksolution(A),thestandard35 solution(B),andthesamplesolution(C)regardingthemethodforassayof tromethamineinthedrugproduct.2 BE2025 / 5041 Figure2showstheresultsoftheHPLCAssaydescribedintheExamplesection, Example1,includingthechromatogramoftheblanksolution(A),thestandard solutionat1.08µg / mlwithTromethamineRS(B),andthesamplesolutionat1.08 mg / mlofTromethamine(C)regardingthemethodforassayofimpuritiesof TromethamineintheDrugProduct.5 Figure3showstheHPLCAssaydescribedintheExamplesection,Example1, regardingthestressdegradationstudy,inparticularthechromatogramsofblanco solution(A),mobilephase(B),andtestsolutions:referencesolution(C),solution submittedtolight(D),solutionsubmittedtoheat(E),andsolutionsatalkalinepH 10.8(F),neutralpH7.0(G),andacidicpH5.0(H).10 Figure4showstheHPLCAssaydescribedintheExamplesection,Example1, regardingthestressdegradationstudy,inparticularthechromatogramsofblanco solution(A),solutionofblancoof30mlofH2O2at1%and70mlofwater(B),and solutionsubmittedtooxidation(C).15 DETAILEDDESCRIPTIONOFTHEINVENTION Thepresentinventionconcernsamethodfortheproductionofaready-to-useliquid formulationinaglassvialwitharubberstopperwhereintheformulationis tromethamineinwaterforinjection(WFI).Italsoconcernsaready-to-usesterile liquidformulationoftromethamine,andusesofsaidformulation.Saidformulation20 isanoptimalalternativetosodiumbicarbonatetocorrectorpreventmetabolic acidosis,andcanbestoredforalongtimeatroomtemperature. Definitions Unlessotherwisedefined,alltermsusedindisclosingtheinvention,including25 technicalandscientificterms,havethemeaningascommonlyunderstoodbyoneof ordinaryskillinthearttowhichthisinventionbelongs.Bymeansoffurtherguidance, termdefinitionsareincludedtobetterappreciatetheteachingofthepresent invention. 30 Asusedherein,thefollowingtermshavethefollowingmeanings: tooneormorethanonecompartment.35 amount,atemporalduration,andthelike,ismeanttoencompassvariationsof+ / - 3 BE2025 / 5041 20%orless,preferably+ / -10%orless,morepreferably+ / -5%orless,evenmore preferably+ / -1%orless,andstillmorepreferably+ / -0.1%orlessofandfromthe specifiedvalue,insofarsuchvariationsareappropriatetoperforminthedisclosed invention.However,itistobeunderstoodthatthevaluetowhichthemodifier 5 andareinclusiveoropen-endedtermsthatspecifiesthepresenceofwhat followse.g.componentanddonotexcludeorprecludethepresenceofadditional,10 non-recitedcomponents,features,element,members,steps,knownintheartor disclosedtherein. Furthermore,thetermsfirst,second,thirdandthelikeinthedescriptionandinthe claims,areusedfordistinguishingbetweensimilarelementsandnotnecessarilyfor15 describingasequentialorchronologicalorder,unlessspecified.Itistobeunderstood thatthetermssousedareinterchangeableunderappropriatecircumstancesand thattheembodimentsoftheinventiondescribedhereinarecapableofoperationin othersequencesthandescribedorillustratedherein. 20 Therecitationofnumericalrangesbyendpointsincludesallnumbersandfractions subsumedwithinthatrange,aswellastherecitedendpoints. weightpercent,hereand throughoutthedescriptionunlessotherwisedefined,referstotherelativeweightof25 therespectivecomponentbasedontheoverallweightoftheformulation. onemember(s)ofagroupofmembers,isclearperse,bymeansoffurther exemplification,thetermencompassesinteraliaareferencetoanyoneofsaid30 members,ortoanytwoormoreofsaidmembers,suchas,e.g., Unlessotherwisedefined,alltermsusedindisclosingtheinvention,including technicalandscientificterms,havethemeaningascommonlyunderstoodbyoneof35 ordinaryskillinthearttowhichthisinventionbelongs.Bymeansoffurtherguidance, definitionsforthetermsusedinthedescriptionareincludedtobetterappreciatethe 4 BE2025 / 5041 teachingofthepresentinvention.Thetermsordefinitionsusedhereinareprovided solelytoaidintheunderstandingoftheinvention. Referencethroughoutthisspecificationto"oneembodiment"or"anembodiment" meansthataparticularfeature,structureorcharacteristicdescribedinconnection5 withtheembodimentisincludedinatleastoneembodimentofthepresentinvention. Thus,appearancesofthephrases"inoneembodiment"or"inanembodiment"in variousplacesthroughoutthisspecificationarenotnecessarilyallreferringtothe sameembodiment,butmay.Furthermore,theparticularfeatures,structuresor characteristicsmaybecombinedinanysuitablemanner,aswouldbeapparenttoa10 personskilledintheartfromthisdisclosure,inoneormoreembodiments.Furthermore,whilesomeembodimentsdescribedhereinincludesomebutnotother featuresincludedinotherembodiments,combinationsoffeaturesofdifferent embodimentsaremeanttobewithinthescopeoftheinvention,andformdifferent embodiments,aswouldbeunderstoodbythoseintheart.Forexample,inthe15 followingclaims,anyoftheclaimedembodimentscanbeusedinanycombination. analkalinizingagentwiththechemicalformula thatmybeusedtotreatmetabolicacidosisbybufferingexcesshydrogen ionsintheblood.Itfunctionsbycombiningwithhydrogenionstoformbicarbonate,20 helpingtorestoreacid-basebalance.Tromethamineisparticularlyvaluableinclinical settingsforitsabilitytoincreasebloodpHwithoutproducingcarbondioxide,making itaneffectivealternativetobicarbonateincaseswherecarbondioxideretentionis aconcern. 25 Theterm"waterforinjection"(WFI)referstoahighlypurifiedformofwaterthat meetsspecificqualitystandardssetbyregulatoryagencies,suchastheUnited StatesPharmacopeia(USP)ortheEuropeanPharmacopoeia(Ph.Eur.).WFIisused asasolventordiluentinpharmaceuticalmanufacturingprocesses,particularlyfor thepreparationofinjectablemedications,parenteralsolutions,andothersterile30 productsthatcomeintodirectcontactwiththebloodstreamortissuesofpatients. WFImustundergorigorouspurificationprocessestoremoveimpurities, contaminants,andmicrobialorganismstoensureitssuitabilityforusein pharmaceuticalapplications.Thesepurificationprocessestypicallyincludemultiple stagesoffiltration,distillation,and / orreverseosmosistoachievethedesiredlevel35 ofpurity.WFImustalsomeetstringentmicrobialqualityrequirements,including limitsontotalviablemicroorganisms,endotoxinlevels,andothermicrobial contaminants. 5 BE2025 / 5041 Theterm"pHmodifyingagent"referstoasubstanceorcompoundcapableofaltering thepH(acidityoralkalinity)ofasolutionorenvironment.ThepH-modifyingagent mayeitherincrease(raise)ordecrease(lower)thepHlevelofthesolutionor environment,dependingonitschemicalpropertiesandthedesiredoutcomeofthe5 application.ExamplesofpH-modifyingagentsincludeacids(e.g.,hydrochloricacid, citricacid,sulfuricacid,phosphoricacid,(glacial)aceticacid,nitricacid,lacticacid, formicacid,malicacid,tartaricacid,boricacid)andbases(e.g.,sodiumhydroxide, ammonia,potassiumhydroxide,calciumhydroxide,magnesiumhydroxide,sodium bicarbonate,potassiumcarbonate,sodiumcarbonate,calciumcarbonate,10 triethanolamine),aswellasbufferingagents(e.g.sodiumphosphate,potassium phosphate,sodiumcitrate,potassiumcitrate,sodiumacetate,potassiumacetate, ammoniumchloride,ammoniumhydroxide,sodiumbicarbonate,boricacid)that helpmaintainastablepHlevel. 15 lacialaceticacidconcentrated,anhydrousform inaliquidstate.Knownforitshighpurityandstrongacidity,ithasafreezingpoint of16.6°C,atwhichitsolidifiestoformclear,ice-likecrystals,hencetheterm "glacial."Glacialaceticacidiswidelyusedasasolventandreagentinchemical20 synthesis,pharmaceuticals,andvariousindustrialprocessesduetoitsabilitytoact asapolarproticsolventanditsreactivityasacarboxylicacid. or0.22micrometersrespectively.Thisspecificationindicatesthesizeoftheopenings25 orporeswithinthefiltermembrane,whichdeterminesthetypesandsizesof particlesthatcanpassthroughorberetainedbythefilter.Inpharmaceutical manufacturing,forexample,a0.2µmor0.22µmfiltermaybeemployedtoremove bacteria,fungi,othermicroorganisms,aswellasparticulatematter,fromliquids, ensuringtheproductionofsterilepharmaceuticalproducts.30 Theterm"hydrophilicmembrane"referstoatypeoffiltermembranethathasan affinityforwateroraqueoussolutions.Hydrophilicmembranesaredesignedtoallow thepassageofwatermoleculeswhileblockingthepassageofnon-aqueous substances,suchasoilsororganicsolvents.Hydrophilicmembranesaregenerally35 usedinfiltrationprocesseswherewater-basedsolutionsneedtobeclarified, purified,orsterilized.Non-limitingpreferredexamplesofmembranesthatmaybe hydrophilicarepolyvinylidenefluoride(PVDF)membraneandpolyethersulfone(PES) 6 BE2025 / 5041 membrane.Furtherexamplesarecelluloseacetate(CA)membrane,regenerated cellulose(RC)membrane,nylon(polyamide)membrane,polytetrafluoroethylene (PTFE)membrane(hydrophilictreated),polypropylene(PP)membrane(hydrophilic treated),fluorinatedethylenepropylene(FEP),andpolysulfone(PS)membrane.A "polyvinylidenefluoride(PVDF)membranefilter"maybeatypeofhydrophilic5 membrane.PVDFisasyntheticpolymerthatmaypossessinherenthydrophilic properties,meaningithasanaffinityforwater.WhenPVDFisfabricatedintoa membranefilter,itretainsitshydrophilicnature,allowingittoreadilywetwithwater andpermitthepassageofaqueoussolutionswhileblockingthepassageof nonaqueoussubstances.Theyofferexcellentchemicalresistance,thermalstability,10 andmechanicalstrength,makingthemsuitableforawiderangeoffiltration anothercategoryofhydrophilicmembranes.PES,athermoplasticpolymer,is engineeredwithhydrophilicproperties,enablingittointeractfavorablywithwater. PESmembranes,likePVDF,permitthepassageofaqueoussolutionswhileblocking15 non-aqueoussubstances.Theyalsoboastexcellentchemicalresistance,thermal stability,andmechanicalstrength,makingthemwell-suitedfordiversefiltration anotherspecializedtypeofhydrophilicmembranewithdistinctiveproperties.FEPis afluoropolymerknownforitsexceptionalchemicalresistanceandlowsurface20 energy,whichisenhancedfurtherwhentreatedtoimparthydrophiliccharacteristics. ThistreatmentenablesFEPmembranestointeractfavorablywithwater,allowing themtoefficientlyfilteraqueoussolutionswhilerepellingoilsandorganicsolvents. FEPmembranesareparticularlyvaluedinapplicationsrequiringhighpurityand resistancetoaggressivechemicalsorsolvents.Theirhighthermalstabilityand25 mechanicalstrengthmakethemsuitablefordemandingfiltrationprocesses, includingthoseinthepharmaceutical,biotechnology,andchemicalindustries.The inherentnon-stickpropertiesofFEPmembranesalsofacilitateeasycleaningand maintenance,ensuringprolongedperformanceandreliability. 30 store,andmaintaintheliquidformulationofseleniousacidinwaterforinjection (WFI).Thisincludes,butisnotlimitedto,(glass)vials,ampules,intravenousbags, syringes,bottles,andothersterilepackagingdesignedtoensuretheprotection, preservation,andappropriateadministrationoftheformulation.Thecontaineris35 selectedtomaintaintheintegrityandsterilityoftheformulationduringstorage, handling,andtransportation. 7 BE2025 / 5041 Bytheterm"glassvial"ismeantinthepresentinventionaglasscontainerusedto storeliquidformulations.TheglassvialsmentionedarepreferablyneutralglassType Ivials,whichareknownfortheirchemicalresistanceandsuitabilityforstoring pharmaceuticalpreparations.Inthecontextofthepresentpatentapplication,Type IPlusglassreferstoahigh-quality,borosilicateglassknownforitssuperiorchemical5 resistanceandlowextractablelevels.Thistypeofglassisusedinthemanufacture ofcontainerssuchasvialsandbottlesforpharmaceuticalandmedicalapplications. Itprovidesenhancedprotectionagainstchemicalinteractionsanddegradationofthe contents,ensuringthestabilityandsafetyoftheformulation.TypeIPlusglassis designedtomeetrigorousstandardsforleachablesandextractables,makingit10 particularlysuitableforstoringsensitiveorreactivesubstanceslikepharmaceutical formulations. Inthecontextofthepresentinvention,theterm"rubberstopper"referstoatype ofclosureusedtosealglassvials,ensuringtheircontentsaresecurelyenclosed.15 Non-limitingexamplesincludebromobutylandchlorobutylstoppers.Theserubber stoppersprovideexcellentbarrierstogasesandmoisture,therebypreservingthe stabilityandintegrityoftheformulation.Bromobutylstoppersoffersuperior resistancetopermeationandarecommonlyusedfortheirexcellentsealing propertiesandcompatibilitywithvariousformulations.Chlorobutylstoppersarealso20 utilizedfortheireffectivebarrierpropertiesandchemicalresistance.Bothtypesof stoppersareselectedtomaintainthequalityandeffectivenessoftheformulation throughoutstorageanduse."Aluminum--25 aluminum,preferablycomprisinganaluminumshell,optionallycoatedorcovered withalayercomprisinganothermaterialsuchaspolypropylene,andpreferablyused forsealingvials.Examplesare20mmFlip-Off®CCSseals,whichtypicallycomprises analuminumshellwithapolypropylene(PP)layerontop.However,itisimportant tonotethatthepresenceofaFlip-Off®buttonisnotmandatoryandismentioned30 asanillustrativeexampleonly.Theprimarycomponentofthesealisanaluminum shell,providingstructuralintegrityandservesasthemainprotectivelayer.Ontop ofthealuminumshell,theremaybeanadditionallayer,suchasapolypropylene (PP)layer.Thislayerusuallyincludesa"Flip-Off®"button,whichiscommonlyused foreasyremovaloftheseal,butitsinclusionisnotarequirement.ThePPlayermay35 alsoprovideadditionalsealingandclosurefunctionality.Preferably,thesealsare providedready-to-useandgammasterilized.Thisensuresthattheyarecleanand sterile,makingthemsuitableforpharmaceuticalapplicationswheresterilityis 8 BE2025 / 5041 crucial.Possibly,thealuminumshelliscoatedforlowparticleabrasionandisclear lacquered.Thiscoatinghelpstominimizeparticulategenerationandmaintainthe integrityoftheseal.Overall,thesesealsaredesignedtoprovideareliableandsterile closureforvialscontaininghigh-valuedrugs,makingthemsuitablefor pharmaceuticalapplicationswhereproductqualityandintegrityareparamount.5 Inthecontextofthepresentpatentapplication,"ready-to-use"referstoa formulationorproductthatisfullyprepared,processed,andpackagedsothatitis immediatelysuitableforuseuponreceipt.Thisimpliesthattheformulationhasbeen preparedtotherequiredspecifications,isinitsfinalform,andispackagedinaway10 thatensuresitssafety,stability,andeffectivenesswithouttheneedforfurther preparation,mixing,orhandlingbytheenduser.Thisfeaturemayprovide advantagessuchastimesavings,reducedriskoferrors,andimprovedefficiencyin variousapplications. 15 Theterm"sterile"refersinthepresentinventiontotheabsenceofallliving microorganisms.Inthecontextoftheformulation,sterilitymaybeachievedthrough filtrationandterminalsterilizationmethodssuchasautoclaving. Theterm"compoundingvessel"referstoacontainerorvesselusedin20 pharmaceuticalmanufacturingprocessesforcompounding,mixing,andpreparing liquidformulationsofpharmaceuticalproducts.Thecompoundingvesselplaysa crucialroleintheproductionofsterilesolutions,suspensions,oremulsions, particularlyforinjectablemedicationsorparenteralformulations.Thecompounding vesselistypicallyconstructedfrommaterialsthatarecompatiblewith25 pharmaceuticalmanufacturingrequirements,suchasstainlesssteelorglass,to ensurecleanliness,durability,andchemicalcompatibilitywiththeformulationbeing processed.Thevesselmayhavefeaturessuchasastirringmechanism,temperature controlsystems,portsfortheadditionofingredientsorsampling,andconnections forCO2purgingorfiltration.Duringthecompoundingprocess,thenecessary30 ingredients,suchasactivepharmaceuticalingredients(APIs),excipients,and solventslikewaterforinjection(WFI),areaddedtothecompoundingvessel accordingtoapredeterminedformulationrecipeorbatchformula.Theingredients aremixed,dissolved,ordispersedwithinthevesselundercontrolledconditions,such astemperature,agitation,andinertgasatmosphere,toensureuniformityand35 homogeneityofthefinalproduct. 9 BE2025 / 5041 Theterm"stainlesssteel316Lgradematerial"referstoaspecifictypeofstainless steelalloythatconformstotheASTM(AmericanSocietyforTestingandMaterials) designation316L.Thisdesignationindicatesthecompositionandpropertiesofthe stainlesssteel,with"316"referringtotheseriesofstainlesssteelalloysand"L" indicatingthelowcarboncontentofthealloy.Stainlesssteel316Lisahighly5 corrosion-resistantanddurablematerialcommonlyusedinvariousindustries, includingpharmaceuticalmanufacturing,medicaldevices,andfoodprocessing.It offersexcellentresistancetocorrosioninawiderangeofenvironments,including thosecontainingchloridesandothercorrosiveagents.The"316L"gradedesignation signifiesthatthestainlesssteelalloycontainslowcarboncontent,typicallylessthan10 0.03%,whichimprovesitsweldabilityandresistancetosensitization-induced corrosion.Thismakesitparticularlysuitableforapplicationswhereexposureto corrosiveenvironmentsorhightemperaturesisaconcern. Bytheterm"particlefree"ismeantinthepresentinventionthattheformulation15 doesnotcontainanyvisibleparticulatematter,ensuringitssuitabilityfor intravenousadministration. Apersonofordinaryskillintheartwillappreciatethatelementsoftheaspectsof themethodsreturnintheaspectsoftheready-to-usesterileliquidformulations,and20 theiruses,oftheinvention.Consequently,allaspectsofthepresentinventionare related.Allfeaturesasdescribedinoneoftheaspects,andtheiradvantagesand / or effects,canrelatetoanyoftheseaspects,eveniftheyaredescribedinconjunction withaspecificaspect.25 Description Inafirstaspect,theinventionprovidesamethodfortheproductionofa,preferably ready-to-use,liquidformulationinacontainer,whichpreferablyisaglassvialwith arubberstopper,whereintheformulationistromethamineinwaterforinjection (WFI),comprisingthestepsof:30 a)introducinglessthan100%(suchas80%,85%,90%,95%,or99%)ofa desiredbatchvolumeoftheliquidformulationofWFIinacompoundingvessel, b)addinganddissolvinganamountoftromethamineinpowderedforminsaidWFI toobtainatromethaminesolution,whilestirringuntilcompletedissolutionof thepowderedtromethamine,whereinsaidamountoftromethamineisdivided35 intosmallerportionsofsaidamountoftromethamine,theportionswhichare addedtoanddissolvedinsaidWFIinsequencewhilestirringandwhereina 10 BE2025 / 5041 furtherportionisonlyaddedtosaidWFIuponthecompletedissolutionofa previousportion, c)introducingatleastonepHmodifyingagenttothetromethaminesolutionto obtainatromethaminesolutionhavingapHofbetween8.2to8.5,preferably 8.35to8.45,preferablyabout8.4,5 d)addingfurtherWFItothetromethaminesolutionhavingsaidpHofbetween8, preferably8.35to8.45toobtain100%ofthedesiredbatchvolume, e)filteringthebatchsolutionofstepd)usinga0.2µmfilter,and f)fillingoneormorecontainers,preferablyglassvialswiththefilteredbatch solutionbeforeclosingsuchasstopperingsaidcontainers,preferablyvials,10 preferablywitharubberstopperandsubsequentlysealingthevials.Saidvials arepreferablyTypeIglassvials. Theinventorsfoundthatthepresentmethodresultsinasterileandready-to-use tromethamineformulationthatstaysstableoveraperiodofatleast24monthswhen15 storedat20to25°Candprotectedfromfreezing.Inaddition,theformulationisat apHthatallowsintravenousadministrationoftheformulation. Inanembodiment,saidliquidformulationcomprisestromethamineata concentrationofbetween20and50mg / ml,preferablybetween25and45mg / ml,20 morepreferablybetween30and40mg / ml,morepreferablybetween32.4and39.6 mg / ml,andmostpreferablyabout36mg / ml.Inanotherorfurtherembodiment, saidliquidformulationcomprisestromethamineataconcentrationofbetween15 and40mEq / ml,preferablybetween20and35mEq / ml,morepreferablybetween 25and35mEq / ml,morepreferablybetween27and33mEq / ml,mostpreferably25 about30mEq / ml.Theadvantagethereofisthattheformulationcanbeusedfor intravenousadministrationimmediatelywithoutpriordilution. 36mg / ml30 mEq / ml30 36mg / ml toarangeof32.4to39.6mg / ml(±10%)or34.2to37.8mg / ml 30mEq / ml27to33mEq / ml(±10%)or28.5to31.5 mEq / ml(±5%). 35 1053fortromethamine.Thismeansthat1000mgissolublein1to10mlofwater. Accordingtooneofthetromethaminemanufacturers,thesolubility(innormal 11 BE2025 / 5041 water)is550mg / mlat25°C.Althoughthedesiredconcentrationof36mg / mlis largelybelow550mg / ml,andthepowdereasilyincorporatesinWFI,especiallywhen theWFIisstirredwhenthepowderisadded,itstilltakestimetodissolve.Therefore, theinventorschosetodividetherequiredamountoftromethamineintosmaller portions,andtoonlyaddafurtherportionofpowdertotheWFIuponthecomplete5 dissolutionofapreviousportion.Preferably,thesolutionisstirredforanother10to 15minutesaftervisibledissolutionofthewholeamountoftromethaminepowder. Inanembodiment,theamountoftromethamineisdividedintoatleast2,atleast 3,atleast4,atleast5,atleast6,atleast7,atleast8,atleast9oratleast1010 smallerportions.Inanotherorfurtherembodiment,theamountoftromethamineis dividedintoatleast12,14,16,18,20,25,30,35,40,45,50,60,70,80,90or 100smallerportions. Inanembodiment,avortexiscreatedinsaidWFIbystirringtheWFIinsaid15 compoundingvesselbeforeaddingsaidpowderedtromethamineportionsofstepb). Thisisbecausecontactbetweenthetromethaminepowderandtheinternalwallsof thecompoundingvesselandthestirrershouldbeavoidedasmuchaspossibleduring manufacture.Thiscanbeachievedbymakingavortex,suchaswithastirrer,inthe WFI.Thetromethamineorportionsthereofcanthencarefullybeaddedintothe20 vortex. WFIingeneralhasapHofabout5.7.Whentromethamineisaddedatthepreferred concentrationofabout36mg / ml,thepHrisestoabout10.8.Asindicatedinstepc) ofthemethod,atleastonepHmodifyingagentisaddedtothesolutionof25 tromethamineandWFItoobtainatromethaminesolutionhavingapHofbetween 8.2to8.5.PreferablysaidpHis8.3to8.5,morepreferably8.35to8.45,most preferablyabout8.4.Inanembodiment,saidpHmodifyingagentaddedtothis solutionishydrochloricacidand / orglacialaceticacid,mostpreferablyglacialacetic acidonly.30 Thesolutionisfilteredinstepe),usinga0.2or0.22µmfilter.Thiscanbeanyfilter thatiscompatiblewiththetromethaminesolutioninWFI,withapHofbetween8.4 and8.8,preferablyhaslowadsorptionandabsorptionpropertiesinrelationto tromethamine,andpreferablyresultsinalowamountofextractablesfromthefilter35 tothesolution.Preferablythefilterusedinthisstepisahydrophilicmembrane, preferablyahydrophilicpolyvinylidenefluoride(PVDF)membranefilterora hydrophilicpolyethersulfone(PES)membranefilter.Alternativehydrophilic 12 BE2025 / 5041 membraneshavebeendescribedabove.Intermsofthefilter,a0.2or0.22µmfilter ispreferred.However,filterswithdifferentporesizesmayalsobeused.For example,filterswithporesizesof0.25µmor0.20µmmayalsobeused.More preferably,theporesizeofthefilterisbetween0.20µmand0.25µm,most preferablyaround0.22µm.Thechoiceofthefiltersizeisimportantasitdetermines5 theextenttowhichthesolutionispurified.Asmallerporesizewouldresultina higherdegreeofpurification,butitmayalsoslowdowntherateoffiltration. Therefore,abalancebetweenthedegreeofpurificationandtherateoffiltration needstobestruck.Inamostpreferredembodiment,thefilterisacapsule0.2or 0.22µmPVDFmembranefilter.10 Thefilteredformulationissubsequentlyusedtofillglassvials,whicharethen stopperedandsealed.Althoughinthepresentcontext,focusislaidmainlyonglassvialswhichare15 stopperedandsealed,furthertypesofcontainersmaybeused.Saidcontainermay beanytypeofcontainerknownintheartwhichissuitablevesselorpackagingused toenclose,store,andmaintaintheliquidtromethamineinWFIformulation.This includes,butisnotlimitedto,(glass)vials,ampules,intravenousbags,syringes, bottles,andothersterilepackagingdesignedtoensuretheprotection,preservation,20 andappropriateadministrationoftheformulation.Thecontainerisselectedto maintaintheintegrityandsterilityoftheformulationduringstorage,handling,and transportation.Accordingtoanembodimentoftheinvention,thecontainerisan ampule,vial,orintravenousbag.Accordingtoapreferredembodimentofthe invention,vials,preferablyglassvialsarefilledwiththeliquidtromethamineinWFI25 formulation,andpreferablybeforestopperingsaidvialswitharubberstopperand furtherpreferablysubsequentlysealingthevials.Preferably,thefilledvialsaresubsequently,andpreferablyautomatically,stoppered andcappedorsealed,withthecaporsealcrimpedusingacappingheadpressure30 setto1.0to2.0bar,mostpreferablyabout1.5bar. ItisparticularlypreferredtouseTypeIglassvialsforthispurpose,asthesevials areknownfortheirexcellentchemicalresistanceproperties.Thistypeofglassdoes notdelaminatewhenincontactwiththeformulation,preferablyovertheentire35 foreseenshelflife.Glassdelaminationisaseriousqualityissueasitmayresultin theleachingofglassparticlesintheformulation,whichwouldsubsequentlybe administeredtothepatient.Avoidingdelaminationthusensuresthesafetyand 13 BE2025 / 5041 efficacyoftheproductoveralongperiodoftime.SaidTypeIglassvialsare preferablyneutralglassTypeIvials,preferablymoldedclearTypeIglassvials.Most preferably,preferablyhigh-quality,borosilicateglassisused,knownforitssuperior chemicalresistanceandlowextractablelevels.Itprovidesenhancedprotection againstchemicalinteractionsanddegradationofthecontents,ensuringthestability5 andsafetyoftheformulation.SuchTypeIglassisdesignedtomeetrigorous standardsforleachablesandextractables,makingitparticularlysuitableforstoring sensitiveorreactivesubstanceslikepharmaceuticalformulationsasinthepresent method. 10 Inanembodiment,theusedvialsare500mLvials. Inanembodiment,themethodinvolvestheuseofvialsandstoppersmadeof materialsthatdonotallowadsorptionorabsorptionoftheactiveingredient,the seleniousacidformulation.Thisispreferredasithelpsmaintaintheintegrityand15 efficacyoftheformulation.Inanembodiment,themethodinvolvestheuseof stoppersandcapsthatprovideaneffectivebarrieragainstenvironmentalfactors suchasmoistureandoxygen,whichenhancesthestoragestabilityofthe formulation. 20 Inanembodiment,thestopperusedtostopperthevialsispreferablyarubber stopperand / orthesealingofthevialsispreferablyperformedusingaluminumcaps.Preferredbutnon-limitingexamplesofsuchrubberstopperincludebromobutyland chlorobutylstoppers.Theserubberstoppersprovideexcellentbarrierstogasesand25 moisture,therebypreservingthestabilityandintegrityoftheformulation. Bromobutylstoppersoffersuperiorresistancetopermeationandarecommonlyused fortheirexcellentsealingpropertiesandcompatibilitywithvariousformulations. Chlorobutylstoppersarealsoutilizedfortheireffectivebarrierpropertiesand chemicalresistance.Bothtypesofstoppersaresuitabletomaintainthequalityand30 effectivenessoftheformulationthroughoutstorageanduse.Inaddition,bothtypes ofstoppershavebeenselectedastheinventorsexperimentallydeterminedthatthey donot(orbelowanacceptablepresetthreshold)releaseextractablesintothe presentformulation,maintainingthepurityandintegrityoftheformulation.35 Theuseoftheserubberstoppersinconjunctionwithaluminumcaps,forsealingthe containersorvials,isparticularlypreferred,asthiscombinationeffectivelyprevents 14 BE2025 / 5041 microbialcontamination,whichiscrucialinmaintainingthesterilityofthe formulation,especiallyconsideringitisintendedforintravenousadministration. "aluminum-- capcomprisingaluminum,preferablycomprisinganaluminumshell,optionally5 coatedorcoveredwithalayercomprisinganothermaterialsuchaspolypropylene, andpreferablyusedforsealingvials.Non-limitingexamplesareFlip-Off®CCSseals, whichtypicallycomprisesanaluminumshellwithapolypropylene(PP)layerontop. Overall,thesesealsaredesignedtoprovideareliableandsterileclosureforvials containinghigh-valuedrugs,makingthemsuitableforpharmaceuticalapplications10 whereproductqualityandintegrityareparamount. Theintegrityoftheclosuresystemwasmicrobiologicallychallengedusing Brevundimonasdiminutasuspensions,andvacuum(0.1kPa)conditions(fordetails, seeexamplesection).Theintegrityofthecontainerclosuresystemwas15 demonstratedbytheconformityofthesterilitytestaftersubmissiontoabacteria ingresstestinstress(vacuum)conditionsfilledwithfluidthioglycolatemedium. Inanembodiment,inordertoobtainasterilesolution,thevialscomprisingthe formulationareterminallysterilized,preferablybyautoclavingforaperiodoftime20 sufficienttorendertheformulationsterile.Thepreferredtemperaturefor autoclavingisbetween115°Cand130°C,preferablybetween120and123°C, preferablyforaperiodoftimerangingfrom5to40minutes.Mostpreferably,the liquidformulationisautoclavedatatemperatureofbetween120and123°Csuch as121°C,preferablyforatleast15minutes,preferablyforatleast17minutes,25 preferablyforatleastorabout20minutes.Thisstepensuresthatanyremaining microbialcontaminantsareeffectivelykilled,resultinginahighlysterilesolution. TheinventorsnoticedthatthepHoftheformulationincreasesslightlyupon autoclavingat121°Cfor20minutes. 30 Inanembodiment,theautoclavedformulationhasapHofbetween8.4and8.8, preferablybetween8.4and8.7,preferablybetween8.5and8.7,mostpreferably about8.6. ThepreferredpHofthefinalautoclavedformulationreadytobeadministeredis8.6.35 InordertoobtainsaidpreferredpH,theinventorsshowedthattheformulationprior toautoclavingshouldbeabout8.4.ThepHmodifyingagentusedinstepc)ofthe 15 BE2025 / 5041 method,shouldthereforepreferablybeaddedtothesolutionuntilapHofabout8.4 isobtained. Inanembodiment,thecompoundingvesselisastainlesssteelvessel,preferablya 316Lstainlesssteelvessel.5 Inanotherorfurtherembodiment,alsofurtherpartsofsystemsorsystemscoming incontactwiththesolutionaremadeoutoforcomprisestainlesssteel,preferably 316Lstainlesssteel.Examplesofsaidfurtherpartsofsystemsorsystemsarepumps ofvialfillingmachines,needlesofthefillingmachines,connectiontubingandvalves.10 Inaddition,alsothestirrerusedtostirtheWFI-tromethaminesolutionispreferably astainlesssteelstirrer.Stainlesssteel316Lmaterialhasno(orbelowpresetlimits) adsorptionorabsorptionproperties.Inaddition,theproducedsolution,or componentsusedtopreparesaidsolution,havenoparticularaggressiveproperties againststainlesssteel316L,noristhereachemicalincompatibilityoristhepHof15 theformulationoftheorderthatitcoulddamagethestainlesssteel. Inanembodiment,thefilled,stoppered,sealedand,preferablyautoclaved,glass vialscomprisingtheliquidformulationoftromethamineinWFIaresubsequently labeledand / orpackagedinaboxwithinsert.Suchinsertcomprisesforinstance20 instructionsforuse. Inanembodiment,theglassvialscomprisingtheliquidformulationoftromethamine inWFIaremaintainedinstorageatatemperatureofbetween20°Cand25°C, protectedfromfreezing.25 Inasecondaspect,thepresentinventionrelatestoaready-to-usesterileliquid formulationoftromethamineinwaterforinjection(WFI),whereinthetromethamine ispresentataconcentrationof36mg / mlorataconcentrationof30mEq / ml,further comprisingasexcipientglacialaceticacid,whereintheliquidformulationhasapH30 ofbetween8.5to8.7,andispackagedinTypeIglassvialwitharubberstopper, preferablyabromobutylorchlorobutylrubberstopper,sealedwithanaluminumcap. Inanembodiment,theconcentrationoftromethaminemayalsobebetween20and 50mg / ml,preferablybetween25and45mg / ml,morepreferablybetween30and35 40mg / ml,morepreferablybetween32.4and39.6mg / ml,andmostpreferably about36mg / ml.Inanotherorfurtherembodiment,saidliquidformulation comprisestromethamineataconcentrationofbetween15and40mEq / ml, 16 BE2025 / 5041 preferablybetween20and35mEq / ml,morepreferablybetween25and35mEq / ml, morepreferablybetween27and33mEq / ml,mostpreferablyabout30mEq / ml. Apersonofordinaryskillintheartwillappreciatethatelementsoftheaspectofthe methodasdescribedabovereturnintheaspectoftheformulation,useandmethod5 oftreatmentoftheinvention.Consequently,allaspectsofthepresentinventionare related.Allfeaturesasdescribedinoneoftheaspects,asdescribedaboveaswell asbelow,canrelatetoanyoftheseaspects,eveniftheyaredescribedinconjunction withaspecificaspect.10 Inanembodiment,thepHoftheformulationis8.5to8.7,preferably8.55to8.65, mostpreferably8.6. Inanembodiment,theformulationcomprisesamaximumof2.5endotoxinsperml, preferablyamaximumof1.08endotoxinsperml(=about0.03EU(endotoxinunits)15 permgoftromethamineinaformulationwithatromethamineconcentrationof36 mg / ml). Inanotherorfurtherembodiment,theformulationisvisiblyparticlefree. 20 Inanotherorfurtherembodiment,theformulationisstableforatleast24months whenstoredatatemperatureofbetween20°Cand25°C,protectedfromfreezing. Inanotherorfurtherembodiment,theready-to-usesterileformulationisproduced asdescribedinanyoftheembodimentsdescribedabove.25 Thesterileconditionsunderwhichtheformulationispreparedareimportantfor preventingmicrobialcontamination,whichisparticularlyrelevantforintravenous injections.Thesterilityoftheformulationisachievedthroughtheuseof(sterile) waterforinjection,theuseoffiltration,andpreferablyfollowedbyautoclavingas30 describedabove.Thesemeasuresensurethattheformulationisfreefrombacteria, fungi,andotherpathogensthatcouldposearisktopatients. Inanotherorfurtherembodiment,thestopperedandcapped / sealedglassvial comprisingtheformulationislabeledand / orpackagedinaboxwithinsert.Such35 insertcomprisesforinstanceinstructionsforuse. 17 BE2025 / 5041 Inathirdaspect,theinventionrelatestoauseoftheready-to-usesterileliquid formulationasdescribedinanyofthepreviousembodiments,intheprevention,or correctionortreatmentofmetabolicacidosis,whereintheformulationis administeredintravenously. 5 Metabolicacidosisisaseriouselectrolytedisordercharacterizedbyanimbalancein thebody'sacid-basebalance.Metabolicacidosishasthreemainrootcauses: increasedacidproduction,lossofbicarbonate,andareducedabilityofthekidneys toexcreteexcessacids.Metabolicacidosiscanleadtoacidemia,whichisdefinedas arterialbloodpHthatislowerthan7.35.Theseverityofacidosisisoftenassessed10 usingthebasedeficit(alsoreferredtoas"negativebaseexcess"),whichquantifies theamountofbufferbase,suchasbicarbonate,missingfromtheblood.Thisvalue istypicallyexpressedinmilliequivalentsperliter(mEq / L).Abasedeficitof approximately25mEq / Lisgenerallyindicativeofmildacidosis,whereasmoderate acidosisistypicallycharacterizedbydeficitsintherangeof510mEq / L.Severe15 acidosis,ontheotherhand,isassociatedwithbasedeficitsexceeding10mEq / L. Determiningthebasedeficitisanimportantstepinguidingtreatment. Sodiumbicarbonateisfrequentlyusedasalkalitherapyinpatientswithmetabolic acidosis.However,sincesomepatientscannottoleratethecarbon-dioxide-producing20 effectsofsodiumbicarbonate,thepresentsterileliquidformulationoftromethamine inWFIcanbeadministeredtosaidpatientsasalkalitreatment,andthustorestore acid-basebalanceintheblood.Asmetabolicacidosisisanelectrolytedisorder,theinventionalsorelatesauseof25 thesterileliquidformulationasdescribedinanyofthepreviousembodiments, whereintheformulationisusedaselectrolytereplenisher,whereintheformulation isintravenouslyinjectedinasubjectinneedofelectrolytes. Inanotheraspect,theinventionrelatestoauseoftheready-to-usesterileliquid30 formulationasdescribedinanyofthepreviousembodimentstopreventortreat metabolicacidosisinapatient. Inanotheraspect,theinventionrelatestotheready-to-usesterileliquidformulation asdescribedinanyofthepreviousembodimentsforuseinthetreatmentor35 preventionofmetabolicacidosisinapatient.Inanembodiment,theready-to-use liquidformulationisadministeredintravenouslytosaidpatient. 18 BE2025 / 5041 Inanembodiment,themetabolicacidosisasreferredtoaboveisassociatedwith cardiacbypasssurgeryorcardiacarrest. Inafurtherembodiment,whenmetabolicacidosisisassociatedwithcardiacbypass surgery,theformulationcanbeusedtocorrecttheacidityofACDblood.5 Inanembodiment,theformulationisadministeredbyinjectionorinfusionintothe ventricularcavityofaheartduringcardiacarrest. Itisclearthattheinventionalsorelatestoamethodoftreatingapatientorsubject10 sufferingfromoratrisktodevelopmetabolicacidosis,byadministeringthesterile liquidformulationasdescribedinanyofthepreviousembodiments,whereinthe formulationisintravenouslyinjectedinasubjectorpatient;andtoasterileliquid formulationasdescribedinanyofthepreviousembodimentsforuseinthe preparationofamedicamentforthetreatmentorpreventionofmetabolicacidosis.15 Thepresentinventionwillbenowdescribedinmoredetails,referringtoexamples thatarenotlimitative. EXAMPLES20 Thepresentinventionwillnowbefurtherexemplifiedwithreferencetothefollowing examples.Thepresentinventionisinnowaylimitedtothegivenexamples. Example1:assessmentofimpuritiesandstressdegradationstudy 25 1.Mostimportantcriticalparameters Criticalparametersforaready-to-useliquidformulationoftromethamineinwater forinjection(WFI),suitableforintravenousadministration,oftheinvention,also referredtobelowastheTromethaminedrugproduct.30 Appearanceofthesolution:clear,colorless,particlesfreeliquid pH:8.4-8.8 Assay:95.0-105%oflabelclaim-->preferablyabout36mg / mland / or30 mEq / ml,orthuswithin95.0-105%of36mg / mland / or30mEq / ml Microbialattributes:steriledrugproduct;bacterialendotoxins:<1.08EU / ml35 19 BE2025 / 5041 2.Impurities Inorganicimpurities InorganicimpuritiesthatmaybepresentinTromethaminedrugsubstance(the tromethamineassuch):thefollowingmetalelementsaresearchedin5 Tromethaminedrugsubstancebeforeitsuseinmanufactureofthedrugproduct: MetalelementSpecification Arsenic<5ppm Calcium<5ppm Copper<5ppm Iron<5ppm Lead<5ppm Magnesium<5ppm Organicimpurities10 Oneorganicimpurity,nitromethylidynetri(methanol),couldpossiblybepresent.OneNH2-groupoftromethaminecouldconvertintoanNO2-groupbutonlyinthe presenceofastrongoxidationagentsuchaspotassiumpermanganateand peroxides.Thosecomponentsarenot / shouldnotbepresentinTromethamine drugsubstancenorinTromethaminesolutionofthedrugproduct.15 -->thisimpurityisnotexpected. EstablishmentofspecificationsofimpuritiesofTromethamineintheDrugProduct Asalreadyprovided,noorganicimpuritiesarementionedintheTromethaminedrug20 substanceUSPmonographneitherinTromethamineforInjection. Specificationsofimpuritiesareasfollows: Maximaldailydose(MDD)ofTromethamineencounteredinscientificpapersismore25 than2grams / days,therefore: ReportingThresholdsis0.05%: MDDis>1g / day,sothresholdis0.05%. Onlyimpuritycontentthatisatandabove0.05%isreported. IdentificationsThresholdis0.1%:30 MDDis>2g / day,sothresholdis0.1%. 20 BE2025 / 5041 Limitofconformityofeveryunknownimpurityismaximum:0.1%. Everyunknownimpuritythatismorethan0.1%shouldbeidentified. Qualificationthresholdis0.15%: MDDis>2g / day,sothresholdis0.15%.Limitofconformityofeveryknownimpurityismaximum:0.15%.5 Everyknownimpuritythatismorethan0.15%shouldbequalified. SpecificationsofimpuritiesintheDrugProductTromethamine36mg / mlInjection areestablishedbasedontheaboveasfollows: Eachunknownimpurity:<0.1%.10 Theimpuritynitromethylidynetri(methanol)mentionedinthePhEuris includedinunknownimpuritiesifexist: Totalimpurities:<0.5%.ThisspecificationisbasedonthePhEur specificationof1%foreachunknownimpuritythatistightened50%fortotal impurities.15 AnalyticalDevelopmentinrelationwiththeDrugProductforassayofTromethamine andforcharacterizationandassayofimpurities MethodforassayofTromethamineintheDrugProduct20 ThepHofthepresentproductisadjustedwithglacialaceticacidfromabout10.8, theinitialpHofTromethamine,toabout8.4.Thisdoesnotallowatitrationof Tromethaminebyanacid.Therefore,aHPLCmethodisdevelopedasfortheassay ofTromethamineintheproduct.TromethamineisnotUVabsorbenttherefore,25 themethodisbasedonderivatizationusingareagentthatreactswith Tromethamineandthecomplexisdetectedbyfluorescencedetector. derivatization.Amount ofFluramthatensuresderivatizationofcompleteamountofTromethamineat30 analysisconcentrationisdevelopedandestablished.Thisamountis2mg / ml. ChromatographicconditionsareclassicalofthiskindofHPLCtechniqueusinga derivatizationagentandfluorescencedetector.Theyaredevelopedand establishedasfollows: Column:Lichrospher100RP-18,5µm;250x4mm35 Columntemperature:40°C Detection:excitingwavelength370nm,emissionwavelength450nm Flowrate:10µl 21 BE2025 / 5041 Mobilephase:PICAreagentinacetonitrile / water Elutionmode:isocratic Mobilephase:onevialofPICreagentisdilutedinoneliterflaskwith600ml ofwaterand300mlofacetonitrile.pHisadjustedto7.0withphosphoric acid.Finalvolumeisbroughttooneliterwithwaterandmixed.5 Derivatizationagentsolution:extemporaneouspreparationatmomentof use:Fluraminacetoneat2mg / ml.Blank:invialforchromatography,700µlofpH9bufferaremixedwithequal volumeofderivatizationagentsolution. Standardsolution:establishedconcentrationofTromethaminethatgives10 suitablepeakisof0.0108mg / ml.Itispreparedasfollows: o1mlofsolutionat1.08mg / mlofUSPTromethamineRSinwateris dilutedto100mlwithpH9buffersolution(0.0108mg / ml).700µl aremixedwith700µlofderivatizationagentsolution.Testsolution: 3mlofTromethaminesolution36m / mlaremixedtill100mlofwater.15 1mlisdilutedto100mlwithpH9buffersolution(0.0108mg / ml). 700µlaremixedwith700µlofderivatizationagentsolution. ChromatogramsofassayareprovidedinFigure1,inparticulartheblanksolution isshowninFigure1A,thestandardsolutionisshowninFigure1B,andthesample20 solutioninshowninFigure1C. TheHPLCmethodforassayofTromethamineintheDrugProductTromethamine36 mg / mlisdulyvalidated.SpecificityofthemethodfortheassayofTromethaminein theDrugProductisdemonstratedinthevalidation.25 MethodforassayofimpuritiesofTromethamineintheDrugProduct Aspreviouslyprovided,noorganicimpuritiesarementioned.So,noanalytical methodsforassayofimpuritiesofTromethamineareavailable.Therefore,30 analyticalmethodforcharacterizationandassayofimpuritiesintheDrugProduct isdeveloped.ItisthesameHPLCmethodfortheassay,butatthehighest concentrationpossibleofTromethamineintestsolutioninordertoensurethe detectionofimpurities.Concentrationoftestsolutionisdevelopedandestablished asthemaximumconcentrationinTromethaminethatcanbeanalyzedbythe35 HPLCmethodwithoutgivingasaturatedpeak.Thisconcentrationof Tromethamineisfoundtobe1.08mg / ml. 22 BE2025 / 5041 RequiredamountofFluramthatensuresderivatizationofcompleteamountof Tromethamineat1.08mg / mlanalysisconcentrationisstudiedatdifferent concentrationsofFluramof2mg / ml,4mg / mland6mg / ml.Obtainedresults havebeenthesame,therefore;2mg / mlischosensinceitisthesameusedin theassay.5 Conformityspecificationofeachunknowimpuritypreviouslyestablished accordingtoICHguidelineis0.1%.Therefore,standardsolutionwith TromethamineRSforassayofimpuritiesisestablishedat1.08µg / ml. TromethamineRSisusedforassayofimpuritiesbecausetheonlyconsidered10 theoreticaleventualknownimpuritynitromethylidynetri(methanol)givenbythe PhEur,isnotavailableinthemarket. Descriptionofthemethod: Chromatographicconditionsandsolutionsforderivatization:sameasforassay.15 Concentrationoftestsolution:at1.08mg / ml.Concentrationofstandardsolution: at1.08µg / mlwithTromethamineRS. StabilityindicatingoftheHPLCmethod,capabletodetectandtoquantify Tromethamineimpurities,isstudiedandconfirmedbycomparison.Samesolutions20 ofTromethaminestressdegradationstudyandstabilitystudytill6monthsare analyzedatthesametimebytheHPLCmethodandbythethinlayer chromatographymethodof forimpuritiesdeterminationinTromethamineDrugSubstance.Excipientsofthe DrugProductareaceticacidandWFIthatdonotinterfereinHPLCnorinTLCanalysis25 techniques.ThePhEurThinlayerchromatographymethodforcharacterizationandquantification ofimpuritiesiscompendialofficialmethodofthePhEur.Itisthusvalidatedbythe PhEurfortheassayofimpuritiesincludingspecificity,LOD,LOQandmassbalance.30 Thismethodisthusstabilityindicatingandmassbalanceisthusinconformity.This methodisappropriatefordetectionandquantificationofpossibleimpuritiesof TromethamineasgivenbythePhEur.ofthePhEur forTromethamineimpuritiesisinfactappliedasadditionaltesttothoseoftheUSP fortheassayofimpuritiesinTromethamineDrugSubstanceofthepresent35 application.ThinlayerchromatographymethodofthePhEurmonograph1053for TromethamineDrugSubstanceisasfollows: 23 BE2025 / 5041 Examinebythin-layerchromatography(2.2.27),usingsilicagelGRasthecoating substance.WashtheplatewithmethanolRbeforeapplyingthesolutions.Test solution(a):Dissolve0.20gin1mlofwaterR,withheating,anddiluteto10ml withmethanolR.Testsolution(b):Dilute1mloftestsolution(a)to10mlwith methanolR.Referencesolution(a):Dissolve20mgoftrometamolCRSinmethanol5 Randdiluteto10mlwiththesamesolvent.Referencesolution(b):Dilute1mlof testsolution(a)to100mlwithmethanolR. Applytotheplate10µlofeachsolution.Developoverapathof10cmusinga mixtureof10volumesofdiluteammoniaR1and90volumesof2-propanolR.Dry theplateat100Cto105C.Spraywitha5g / lsolutionofpotassiumpermanganate10 Rina10g / lsolutionofsodiumcarbonateR.Afterabout10minexamineindaylight. Anyspotinthechromatogramobtainedwithtestsolution(a),apartfromthe principalspot,isnotmoreintensethanthespotinthechromatogramobtainedwith referencesolution(b)(1.0percent). 15 Testsolutionisat20mg / mlandstandardsolutionisat0.2mg / ml,whichis1.0% thatisconformityspecificationofeachimpurityfortheDrugSubstanceinthePhEur monograph.Sinceestablishedconformityspecificationofeachimpurityis0.1%in Tromethamine36mg / mlDrugProduct,testsolutionisat36mg / mlwithoutdilution andstandardsolutionisat0.036mg / ml(0.1%oftestsolutionof36mg / ml).Applied20 volumeontheplateis25µl,where0.036mg / mlaredetected.Descriptionofthe TLCmethodisthereforeasfollows: Chromatographicconditions:sameasinthePhEur. Testsolution:36mg / mlTromethaminesolution. Standardsolution:at0.036mg / mlofTromethamineRSinmethanol.Applied25 volumetotheplateofeachsolution:25µl. ChromatogramsoftheHPLCmethodforassayofimpuritiesareprovidedinFigure 2.Inparticular,Figure2Ashowstheblanksolution,Figure2Bshowsthestandard solutionat1.08µg / mlwithTromethamineRS,andFigure2Cshowsthesample30 solutionat1.08mg / mlofTromethamine. TheHPLCmethodforassayofTromethamineimpuritiesintheDrugProduct Tromethamine36mg / mlisdulyvalidated. 35 24 BE2025 / 5041 StabilityofTromethamineinstressconditionsandimpurityprofileinthe DrugProduct StressdegradationstudyofTromethamineinsolution 5 StabilityofTromethamineinsolutionisstudiedinstressconditionsinorderto observethecriticalparametersthatcanaffectdrugproductdevelopmentand performance.Theaimofthestudyis: toknowcriticalparametersoftheDrugSubstancethatcaninfluencetheDrug10 Product, toknowtheoriginofcriticalparameters, toadaptandoptimizethemanufacturingprocessforanoptimalstability conditionsofTromethamineinsolution. toknoweventualcriticalparametersduringthestabilitystudy,15 tooptimizethestorageconditionsofthefinishedproduct. toconfirmthattheHPLCmethodisstabilityindicatingbycomparisonwith theTLCmethodofthePhEurforcharacterizationandquantificationof Tromethamineimpurities. Thestudyisperformedaccordingtotherealandpracticalstressfactorsat20 exaggeratedlevelthatmayaffectTromethamineinsolutionduringmanufacturing processandshelflifeoftheproductofthepresentapplication. Preparationofsolutionsofthestressdegradationstudy: 25 Thestudyisperformedonsolutionsof36mg / mlofTromethamine,asintheDrug Product.Oneliterofextemporaneoussolutionispreparedbydissolving36gramsof TromethamineinoneliterofWFI.pHofthesolution:10.80 Portionsof100mlofeachpreparedsolutionareseparatelysubmittedtothe following:30 Tolight:100mlofthesolutionareintroducedin100mlvolumetricflaskand aresubmittedduring5daystoUVat365nm. Tooxidation:in100mlvolumetricflask,70mlofthesolutionaremixedwith 30mlofH2O2at1%.Mixtureiskeptonehourat20-22°C. Toheat:100mlofthesolutionareintroducedin100mlvolumetricflaskand35 aresubmittedto121°Cduring20minutes. 25 BE2025 / 5041 ToalkalinepHofthesolutionthatis10.80withoutadjustment:100mlofthe solutionareintroducedin100mlvolumetricflaskandarekeptduring5days at20-22°C. ToneutralpH7.0:pHof100mlofthesolutionisadjustedto7.0withglacial aceticacid,introducedin100mlvolumetricflaskandarekeptduring5days5 at20-22°C. ToacidicpH5.0:pHof100mlofthesolutionisadjustedto5.0withglacial aceticacid,introducedin100mlvolumetricflaskandarekeptduring5days at20-22°C. Referencesolution:100mlofthesolutionareintroducedin100mlvolumetric10 flaskandarekeptprotectedfromlightatlaboratorytemperature(20-22°C). Blanco.MobilephaseoftheHPLCmethod. Analysisandanalyticalmethods:15 Stressdegradationsolutionsareanalyzedfor: Appearanceofthesolution:Clearliquid(USP<641>)freefromvisible particle(USP<790). Colorationofthesolution:Colorless(PhEur2.2.2).20 Clarityofthesolution:Clear.