d2 1_lca tool adaptation to pharmaceutical processes
TRANSCRIPT
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D2.1LCATOOLADAPTATIONTOPHARMACEUTICAL PROCESSES
INDEX
Summary...................................................................................................................................................... 3
1 Introduction......................................................................................................................................... 4
LCAToolAdaptationto
Pharmaceutical
ProcessesManualtouseintheCluster
January2010
Martins,M.L.;Mata,T.M.; Martins, A.A.; Neto, B.; Costa,C.A.V,Salcedo,R.L.R.
FacultyofEngineeringUniversityofPorto
RuaDr.RobertoFrias,s/n
4200465Porto,Portugal
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1.1 BackgroundandMotivation....................................................................................................... 4
1.2 StudyObjectives....................................................................................................................... 11
2 ProductionofLyophilizedProductsviaRecombinantBiotechnology...............................................12
2.1 APIProduction.......................................................................................................................... 12
2.1.1 PreparationofRawMaterialsforFermentation.................................................................. 13
2.1.2 InoculationandFermentation.............................................................................................. 14
2.1.3 ProductConcentrationandChromatographic Purification.................................................. 16
2.1.4 FilterSterilization andAPIConditioning............................................................................... 18
2.2 MedicineProduction................................................................................................................. 20
2.2.1 APIandExcipientsWeightingandProductFormulation...................................................... 21
2.2.2 FreezeDrying........................................................................................................................ 23
2.2.3 StopperingandFinalProductStorage.................................................................................. 25
2.2.4 StabilityTests,QualityControlandQuarantine................................................................... 26
2.3 AuxiliaryProcesses.................................................................................................................... 31
2.3.1 PureWaterTreatmentSystem............................................................................................. 31
2.3.2 HeatSterilizationofWastes................................................................................................. 32
2.3.3 Trigeneration:PureSteamgenerationandIndustrialSteam..............................................32
3 LCAToolDescription.......................................................................................................................... 33
3.1 LCAToolOutline....................................................................................................................... 33
3.2 ImpactEvaluation..................................................................................................................... 34
3.2.1 Methodology........................................................................................................................ 34
References.................................................................................................................................................. 37
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Summary
Thisreport,donewithinthescopeoftheCIPEIPEcoinnovation2008projectECOPHARMABUILDING
EcoInnovationofPharmaceutical BuildingsSupportinginSustainableLCATools(ECO/08/239082), has
among other objectives to identify, describe and present a detailed description of the PRAXIS
Pharmaceutical S.A.productionprocesses.Theproductionprocessesof thispharmaceutical company
willbeusedasacasestudyforthedevelopment,withinthisproject,ofanopensourceLCAtoolthat
canultimatelybeusedbyeachpharmaceuticalcompanytoanalyzetheirprocesses.ThisLCAtoolcanbe
used to perform an inputoutput analysis of pharmaceutical processes, to evaluate their potential
environmental impacts, toperforma sustainability assessment,andalso to identifyopportunities for
improvement.
Therefore,thisreportisstructuredintwochapters.Thefirstchapterisasmallintroduction,describing
themotivationforthecreationoftheLCAtoolandasummaryoftheLCAmethodology,accordingtothe
ISO14040 (2006).Thesecondchapterdescribes theproductionprocessesof lyophilizedproductsvia
recombinant biotechnology, obtained from open literature, which are similar to the ones used byPRAXISpharmaceutical. Thisdescription isan important step in theunderstandingof theproduction
processesinvolvedinordertoallowfortheidentificationoftherelevantinputsandoutputsofmaterials
and energy that enter and exit each manufacturing process. For each stage of the primary and
secondaryprocessing, someof themain inputsandoutputsare identified,whichneed tobe further
refinedandcompleted inordertobeused intheLCAtooldevelopment.The inventorydataobtained
fromPRAXIScanbe laterused,duringthedevelopmentoftheLCAtool, inordertobepresentedasa
casestudywithquantifieddata.
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1 Introduction
1.1 BACKGROUNDANDMOTIVATIONLifeCycleAssessment(LCA)isamethodologythatcanbeusedtoperformasystematicevaluationofthe
potentialenvironmentalimpactsofamaterial,product,process,activityorserviceacrossallstagesofits
life cycle, from rawmaterialacquisition,productmanufacturing, transportation, sale,use,endoflife
treatment and disposal (i.e. in a cradletograve, cradletogate, gate to gate or gatetograve
perspective). This methodology is described by the international standards ISO 14040:2006
(Environmentalmanagement Lifecycleassessment Principlesandframework)andISO14044:2006
(Environmentalmanagement Lifecycleassessment Requirementsandguidelines).
The first LCA studies (known as ecobalances) emerged in the late 1960s to support corporate
environmentalstrategies,suchastoreducematerialandenergyconsumptionassociatedwithproducts.
By the time of the energy crisis, in the middle 1970s, the use of ecobalance studies by companies
becamemorewidespread,mainlyforthepurposeofperformingenergyassessmentsandevaluatingthe
efficiency of specific energy sources. More recently, new concepts related to other environmental
problems have emerged, including for example, natural resources depletion, atmospheric emissions,
wastewaterandsolidwastesgeneration.
Insummary,thedetailedknowledgeofasystem,providedwhenperforminganLCAstudy,allowsoneto
(Gainzaetal.,2009): Assistanorganizationtoimprovetheirlevelofenvironmentalperformanceevenifthereareno
limitsforpollutantemissionsdefinedinexistingenvironmental regulationsandlaws;
Rapidlyrespondtoanyenvironmentalissue,e.g.setupbyanewenvironmentalstandard; Obtain precise data that may be used for ecodesign purposes and also, to report reliable
environmentaldata;
Informthepublicabouttheenvironmentalaspectsofanorganizationproductsandprocesses,inordertobuildaresponsiblecorporateenvironmental image.
FewstudiesarepublishedconcerningLCAofpharmaceutical processes.Forexample,JimnezGonzlez
et al. (2004) performed an LCA study for analyzing and identifying the cradletogate environmentalimpacts of a typical Active Pharmaceutical Ingredient (API) synthesis, concluding that solvent use
accountsforthemajorityofthepotentialenvironmentalimpacts.
PonderandOvercash(2010)performedaLCAstudyofthevancomycinhydrochlorideproduction ina
lowyield fermentationprocess.Results show that,ona cradletogateperspective, the fermentation
stepconsumesthemostoftherawmaterialsandenergy(47%ofthetotalenergyconsumption).Also,
steam use accounts for more than 75% of the total cradletogate energy consumption, mainly for
sterilization operations. Aeration and agitation in fermentation consume 65% of the cradletogate
electricalenergy.Asanenvironmentalmeasureoftheprocess,theseauthorsdeterminedtheprocess
mass intensity (PMI),calculatedas the totalgatetogatemassof rawmaterialspermassofproduct,
whichmayideallyapproachesthevalueofoneifnowastesaregenerated.
Wernetetal.(2010)performedaLCAofanAPIproductionfromcradletofactorygateandconcludedthatpharmaceutical production is significantlymorecomplex thanbasicchemicalproduction.Energy
useisdirectlyorindirectly,thecauseofthemajority(andsometimesupto85%)oftheimpacts.These
authors also concluded that the emissions from the chemical processes and transport are minor
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contributorstotheoverall impacts.Furthermore,thisanalysissuggeststhatthemosteffectivewayof
increasing these processes sustainability is by optimizing material and energy efficiency. Therefore,
althoughAPIarenormallyproducedandused in lowerquantities,their lowercontribution inmassto
productsshouldnotleadLCApractitionerstoneglecttheirimportance.
Kimetal.(2009)performedanLCAofthreesupportedenzymesforpharmaceutical applicationsFLASCusingthe lifecycle inventorydatabasedevelopedbyGlaxoSmithKline. Theyconcludedthatproduction
of immobilized enzymes is an energy intensive process, being the immobilization and the media
preparation the primary sources of potential environmental impacts such as acidification,
eutrophication, andphotochemicalsmogformation.
Jonge(2003)performedalimitedLCAofpharmaceutical productsconcludingthatitistheportionofthe
active ingredient inthe finalproductthatdeterminestheecologicalconsequences foractivitiesdown
the supply chain. Also, the major energy requirements and environmental impacts of the active
ingredientoverthelifecycleareattributedtotheproductionstage.
Also,somesoftwaretoolsalreadyexisttoperformLCAstudies,evaluatingthepotentialenvironmental
impacts,andothers, toassessprocesspotential risksandhazards,or theenvironmental, healthand
safety(EHS)propertiesofchemicals.Examplesarepresentedbellowandamoreexhaustivelistofthese
softwaretools,servicesanddatacanbefoundathttp://lca.jrc.ec.europa.eu/lcainfohub/toolList.vm
Ecoprofarma Desarrollo de Procesos y Productos Sostenibles para la Industria Farmacutica.Under this project it wasdevelopedan LCA tool forpharmaceuticalbasedon a stateof the art
aboutavailableenvironmentaltechnologiesforpharmaceutical products(Ecoprofarma,2008)
FLASC FastLifecycleAssessmentofSyntheticChemistry,developedbyGlaxoSmithKline (GSK),isa webbased tool and methodology designed to evaluate the life cycle environmental impacts
associatedwiththemanufactureofmaterialsusedinatypicalpharmaceuticalprocess(Curzonsetal.,2007).
Sabento Software for the Assessment of Biotechnology Processes. It can be used tomodelbiotechnical production processes and process development. Also, it allows a process
designertoperformeconomicandecologicalassessmentsofprocessalternatives.Thissoftwarecan
beorderedat:http://www.sabento.com/en/
SimaProLCAsoftwaredevelopedbyPRConsultants, includesalargedatabasefortheinventoryandenvironmental analysesofseveralprocesses.Itcanbeorderedat:http://www.pre.nl/simapro/
KCLECOLCAsoftware&KCLEcoData developedbyKCLpulpandpaperResearchCompany,canbeappliedindifferentindustrialsectors.
BEES BuildingforEnvironmentalandEconomicSustainability developedbytheBuildingandFireResearchLaboratory.
OpenLCA isamodularsoftwarefor lifecycleanalysisandsustainabilityassessmentsdevelopedbyby Green DeltaTC (Ciroth, 2007). This software will be available as open source at
http://www.openlca.org/index.html.
TRACI Tool for Reduction and Assessment of Chemical and Other Environmental Impacts developed by the Environmental Protection Agency (EPA) to assists in making environmental
decisionsandcompletingLCAfordifferentproductionprocesses.
Ecosolvent LCAtooldevelopedbytheInstituteforChemicalandBioengineering ofETHZurich,forquantifying the potential environmental impact of wastesolvent treatment. It is available athttp://www.sustchem.ethz.ch/tools/ecosolvent/.
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EHSassessmenttool developedbytheSafetyandEnvironmentalTechnologyGroup,ofETHZurich,forassessingchemicalsandsolventsbasedonEHScriteria.Thistoolisdemonstratedon26organic
solventsofcommonusewithinthechemicalindustry.Thesubstancesareassessedbasedontheir
performance inninecategories:Releasepotential,chronic toxicity, fireorexplosion,persistency,
reaction or decomposition, air hazard, acute toxicity, water hazard, irritation. It is available at
http://www.sustchem.ethz.ch/tools/ehs/.
HAZOPExpert HAZOPsoftwaredevelopedbytheLaboratoryforIntelligentProcessSystems(LIPS),SchoolofChemicalEngineeringofPurdueUniversity,toanalyzedifferentprocessesfortheirrisks
andhazards(Venkatasubramanianetal.,2000). WAR (WAste Reduction) algorithm software tool developed by the Environmental Protection
Agency(EPA)todescribetheflowandthegenerationofpotentialenvironmentalimpactthrougha
chemicalprocess.Availableathttp://www.epa.gov/nrmrl/std/cppb/war/sim_war.htm.
The problems with some of these software tools is that they are not specifically oriented for
pharmaceutical processes,somearenotuser friendly, the licensing feesmaybehighand the results
obtainedareoftennotclearforusersinSME(SmallandMediumEnterprises) andforthegeneralpublic.
Also,thesetoolsaremuchtimeconsuming,complicatedandnormallydonotaddressconcernsofsocial
sustainabilityoroftheprocesseconomics.AlthoughFLASC isspecificallyorientedtodevelopa fast,
streamlined LCA for a wide range of materials commonly used in pharmaceutical products it is only
availabletoGSKscientistsandengineersandaccessibleviatheintranetsiteofthiscompany.
Forthesereasons,thereisastronginterestindevelopinganopensourceLCAtool,easytohandleand
interpret,thatcanbeusedbypharmaceuticalcompaniestoperforman inputoutputanalysisoftheir
processes,productsandmaterials lifecycle,helping them tomeetcurrentenvironmental legislations
andtheirenvironmentalreportingneeds.
Moreover,theinformationdevelopedinanLCAorLCI(lifecycleinventory)studycanbeusedaspartof
a much more comprehensive decision making process. Furthermore, to achieve the environmentalcertificationorecolabelforapharmaceutical product,theirmanufactureandtransformationneedsto
complywithspecialenvironmentalconditionsbasedonalifecycleassessment.
Especiallyconcerningpharmaceutical products,thecurrentEuropeanRegulationsforEcologic labeldo
not take intoconsiderationmedicines,healthproductsandothersdangerousor toxicproducts.Also,
existing methodologies like LCA and others for Ecodesign are practically not applied in the
pharmaceutical sector or others, like EMAS (EcoManagement and Audit Scheme) are slightly used,
whichmakestheLCAtoolevenmorenecessary.
Concerning pharmaceutical manufacturing companies, there is an increasing pressure to ensure that
information and data about their processes are accurate and reproducible. However, the current
environmental regulations (e.g. for ecoproducts) are not yet specifically oriented to be applied to
pharmaceutical products and processes. Leonard and Schneider (2004) states that pharmaceutical
companiesalreadyrecognizedthatonewaytogrowtheirbottom lines isby integratingsustainability
performance. Nevertheless, currently no standardized methods exist for integrating, measuring, or
communicatingsustainability bypharmaceutical companies.Moreover,Schneideretal.(2010)analyzedtheevolutionofsustainability reportinginthepharmaceutical sector,concludingthatithasincreasedin
breadthanddepth,buthavenowshiftedmoretowardscorporatesocialresponsibility,whichreflectthe
companiesneedtosatisfypublicopinion.Inotherhand,Geibleretal.(2006)inferthatsincethesocialdimensionofsustainabilityhasan intangibleandqualitativenaturethere isa lackofconsensusabout
whataretherelevantcriteriaforcompaniestoaccountforit.
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Onewayofevaluatingthesustainability ofpharmaceutical processesisbyusingsustainabilitymetricsor
indicators.Severalindicatorsandmetricshavebeenproposedbyresearchersovertheyears.Theissue
withsomeofthesemetricsystemsisthattheyonlycovercertaindimensionsofsustainabilityandnotall
the three dimensions. Below is a summary of the key metric systems that have been proposed by
researchersinthisfield.
Key performance indicator (KPI) proposed by Geibler et al. (2006) to account for the socialsustainabilityinbiotechnological processesandproductdevelopment.Also,theseauthorsidentified
eightaspectswithsignificantrelevancetothesocialimpactassessmentofthebiotechnologysector:
healthandsafety,qualityofworkingconditions, impactonemployment,educationand training,
knowledgemanagement, innovationpotential,customeracceptanceandsocietalproductbenefit,
and social dialogue. For each one of these aspects these authors proposed typical indicators to
assessthem.
ALCHESustainabilityMetrics:TheSustainabilityMetricsworkinggroupofTheAmericanInstituteofChemicalEngineers(AICHE)hasdevelopedasetofbaselinesustainability metricsforcompaniesto
measureenvironmental impactsandhasanactiveprojectevaluatingandtestingthesemetrics in
industry(http://www.aiche.org/cwrt).
IChemESustainabilityMetrics:SustainableDevelopmentProgressMetricsRecommendedforUseintheProcess Industry. IChemEextendedsustainability metricsto includemeasuresofthepotential
impactsofemissions,effluentsandwastes.Thus, this indicators systemcanbeused toevaluate
environmental, economicandsocialconcernsofaprocessunit(IChemE,2002).
Dow Jones Sustainability Index (DJSI) established in 1999 by Sustainable Asset Management(SAM), thiswas the first indexattempting toassess theabilityofbusinesses tocreate longterm
shareholder value by embracing opportunities and managing risks deriving from economic,
environmentalandsocialdevelopments(http://www.sustainability index.com/).
SustainableProcessIndex(SPI) developedin1995,SPImeasuresthepotentialimpact(pressure)ofprocessesormoregenerallyactivitiesontheecosphere.ThebasicunitoftheSPIisarea,i.e.itisthe
total surface area required by any activity that exchanges material with the environment to be
sustainably embedded into the ecosphere (or environment). This is based on the principle that
surface area is a limited resource in a sustainable economy because earth has a finite surface
(Narodoslawsky and Krotscheck, 2000). SPIonExcel is developed based on SPI to calculate the
ecologicalfootprintofaprocessonanExcelbasedtool(SandholzerandNarodoslawsky,2007).
BRIDGESBasicSustainabilityMetrics Introducedin2002thismetricssystemtoassessproductionprocessesbymeasuringthefollowingsetofindicatorsbyunitofoutput(massofproduct,orsales
revenue,orvalueadded):material intensity,water intensity,energy intensity,toxicreleases,solid
wastes,pollutanteffects.ExamplesofComplementaryMetricsarealsogiven(Schwarzetal.,2002;TanzilandBeloff,2006).
ThreeDimensional(3D)SustainabilityMetrics proposedbyMartinsetal.(2007)forevaluatingthesustainabilityof industrial processesandcomparing themwithalterativeproductionmethods.A
threedimensionalsustainability framework isalsoaddressedby theseauthors thatproposed the
following sustainability metrics for evaluating chemical processes: Energy intensity, Material
intensity,Potentialchemicalrisk,andPotentialenvironmentalimpact.
BASF ecoefficiency analysis developed by BASF to quantify sustainability of products andprocesses. The environmental impacts are determined on the basis of five main aspects: the
consumptionofrawmaterials,theconsumptionofenergy,resultingemissionstoairwaterandsoil,
thetoxicitypotential,andtheabuseandriskpotential.Also,thetotalcostsarecalculatedoverthe
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life cycle, including the real costs that occur and the subsequent costs that will occur in future
(Salingetal.,2002;Salingetal.,2005;Saling,2005). TheDowfireandexplosionindex(DowF&EI)publishedbyDowChemical,in1964,isprobablythe
mostfrequentlyusedhazardevaluation index.It isusedtodeterminesafetyrisksassociatedwith
fireandexplosion,todeterminetheareasofgreatestlosspotential inaparticularprocess,andto
predictthephysicaldamagethatwouldoccurintheeventofanincident(Sinnott,2005).
InherentSafetyIndex(ISI) thisindexsystem,suggestedbyHeikkila(1999),addressesthechemicalandprocesssafetyofachemicalplant.Itiscalculatedbasedonsubindicesconcerningthechemical
safety(e.g.chemicalreactivity,flammability,explosiveness,toxicityandcorrosivenessofchemical
substancespresent intheprocess)andtheprocesssafety(e.g.processtemperatureandpressure,
equipmentsafetyandsafeprocessstructure).
Sustainability Indicators and Indices An indicators system proposed by Tugnoli et al. (2008) toaddressthethreeaspectsofsustainabilityduringearlystagesofprocessdesign.Itcabbeusedfor
comparingdesignalternativesandforanalyzingtheenvironmental,economicandsocialimpactsof
aprocess.
GlobalEnvironmentalRiskAssessment(GERA)Index proposedbyAchouretal.(2005),this indexsystemassessestheenvironmentalriskofindustrialprocesses.
Metrics togreenchemistry aiming todrivepharmaceuticalprocesses towardsmoresustainablepractices.Constableetal. (2002)exploresseveralmetricscommonlyusedbychemists,comparesandcontraststhesemetricswithanewmetricknownasreactionmassefficiency toassessthe
massefficiencyofachemicalsynthesis.
Sustainability Indicators for assessing energy systems an indicators system to assess thesustainabilityofenergysystemswithafocusonresources,environment,societal,andproduction
efficiency(Afganetal.,2000). GlobalReportingInitiative(GRI)indicatorstheSustainabilityReportingGuidelineshavebecomea
standardforsustainability reportingduetothelackofaformalglobalconsensusonmeasurement
and reporting practices. For reporting on an organizations activities GRI employs quantitative
indicators wherever possible, but in situations where quantitative measures are not effective
qualitativemeasuresarealsopossible(http://www.globalreporting.org).
UNCTADenvironmentalandfinancialperformanceindicatorsUNCTADandtheIntergovernmentalWorkingGroupofExpertsonInternationalStandardsofAccountingandReporting(ISAR)prepared
amanualthatpresentsamethodbywhichenvironmentalandfinancialperformanceindicatorscan
beused together tomeasureanenterprise'sprogress inattainingecoefficiencyor sustainability
(UNCTAD,2004)
NRTEEecoefficiency indicators TheCanadasNationalRoundTableontheEnvironmentandtheEconomy (NRTEE) conducted one of the earliest studies on the development of sustainability
metrics. Itssearchforasmallsetofecoefficiency indicatorsthat ismeaningfulandapplicableby
companies fromdifferent industrialsectors.Thestudy (NRTEE,1999)recommendedasetofcore
metricsincludingmaterialintensity,energyintensity,anddispersionofregulatedtoxicsperunitof
products or services, and also suggested using complementary metrics, such as greenhouse gas
intensity.
WBCSD Ecoefficiency Indicators developed by The World Business Council for SustainableDevelopment (WBCSD) forcompaniespursuingecoefficient strategies to reduce their impacton
theenvironmentwhileincreasingoratleastnotdecreasing(shareholder)value.TheWBCSDstatesthatecoefficiencyisachievedbythedeliveryofcompetitivelypricedgoodsandservicesthatsatisfy
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humanneedsandbringqualityoflife,whileprogressivelyreducingecologicalimpactsandresource
intensity(Schmidheiny,1992).
BITC social and environmental indicators indicators to help companies report social andenvironmental impact effectively, proposed by the Business Impact Review Group (BIRG) of
BusinessintheCommunity(BITC),amovementofover700oftheUKstopcompaniescommitted
toimprovetheirpositiveimpactonsociety(BITC,2003).
PERFORMindicatorsetincludesabout30indicatorsapplicabletoallindustrialsectorsandasmallnumber of additional indicators specific to each sector. It covers the following areas: Economy
(Turnover, Profit, Return on capital, Labor productivity), Environment (Air emissions, Water
emissions, Energy and resource input, Waste, Environmental management), Social responsibility
(Employment, Health and safety, Training and education, Equal opportunities, Community). This
indicatorssetwasdevelopedunderthePERFORMproject launchedbyresearchersofScienceand
TechnologyPolicy(SPRU)attheUniversityofSussex.Tosetobjectivesforimprovement,SPRUhas
implementedafreeserviceallowingcompaniestobenchmarkthemselvesagainsttheircompetitors
using a standard set of economic, environmental and social performance indicators (PERFORM,
2004).
Szkelya and Knirsch M. (2005) lists some of the sustainable performance metrics used by several
companies,someofwhichfromthepharmaceutical industry.Someexamplesarelistedinthefollowing
table:
Economicmetrics Environmentalmetrics Socialmetrics
BASF(CorporateReport,
2003)
Sales,
NetIncome,
EarningsPerShare, CashFlow
GHGEmissions
ReductionofGHGEmissions
EmissionstoWater
ReductionofWaterEmissions
LostTimeAccidents,
WorkforceProfile,
DonationsandSponsoring
BoehringerIngelheim
Pharma(KGESH2000)
Sales
ExpenditureonEHS
TotalEnergyConsumption
GHGEmissions
WaterConsumption
Wastewater
SolidWaste
%ofWasteRecycling
No.ofEmployees
AccidentsperhoursWorked
Henkel(SustainabilityReport
2003)
Sales
OperatingProfit
ProductionVolumes
EnergyConsumption GHGEmissions
DustEmissions
VOCEmissions
WaterConsumption
VolumeofWastewater
COD
HeavyMetalstoWater
WasteRecyclingandDisposal
ComplaintsfromNeighbors No.ofEmployees
AccidentsperHoursWorked
ParticipationinEmployee
TrainingPrograms
No.ofEmployeeProjects
Schering(Environmental
Report2003)
Sales InvestmentR&D
EnergyConsumption
CO2Emissions WaterConsumption
TransportModes(Ship,
Airplane,Truck/Car), TotalNumberEmployee,
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EarningsperShare
CashFlow
Wastewater
SolidWaste
EnvironmentalProtection
AccidentsperWorkingHours,
TotalNumberofApprentices,
FrequencyofEHSTraining
Veleva et al. (2003) applied the fivelevel indicator hierarchy developed at the Lowell Center forSustainableProductiontoanalyzetheenvironmentalreportingofsixpharmaceutical companies.Theseauthorsconcludedthatthemajorityofindicatorsaddressperformanceorecoefficiency(Level2),some
indicatorslookatenvironmentaleffects(Level3),supplychainandproductlifecycleeffectsarestarting
tobeaddressed(Level4),andnocompaniesareaddressingcarryingcapacityissues(Level5).
Henderson et al. (2008) used a life cycle approach and sustainability metrics to compare theperformance,and theenvironment, health, safety, and life cycle impacts of two methods for amino
acidsproduction,concludingthatrawmaterialsproductionhasthelargestenvironmentalimpacts.
Fischer and Hungerbuhler (2000) discussed the use of four different indicators for assessing the
environmental impact of chemical processes: Mass Loss Indices (MLI), Environmental Indices (EI), a
comprehensive EHS (Environment, Health, and Safety) assessment method, and EcoIndicator 95, an
evaluationmethodusedintheLCAframework.
Hoetal.(2010)analyzedtheenvironmental impactsofproteinmanufacturingconcludingthatenergyuseandwaterusearethemaincontributorstotheenvironmental impactsofsuchoperations inclean
roomspaces.
BlumKusterer and Hussain (2001) analyzed the main drivers for sustainability improvements in the
Pharmaceuticals Industryconcluding that regulation followedby implementation ofnewtechnologies
arethemajordriversforprocesschange.
JimnezGonzales and Overcash (2000) evaluated the energy use and related emissions during the
severalstagesofpharmaceuticals production,concludingthatabout70%energyreductionispossibleby
optimizingthe energyusagesysteminthepilotscalestageduringprocessdevelopments.
JimnezGonzalesetal.(2002)explaintheconceptoftheGreenTechnologyGuide,whichisaseriesofcasescenariocomparisons thatprovidescientistsandengineerswithcomparativeenvironmentaland
safety information on technologies for operations commonly found in the pharmaceutical industry.
Technologiesarecomparedusing indicatorsbasedonunitoperationanalysisand lifecycleconcepts.
Theseauthorspropose indicators in four categories:Environment, Safety,Efficiency,andEnergy. For
exampleenvironmental Indicatorsare:Mass intensity,Solvent intensity,Waste intensity,Emissionsof
compoundsreleased.
ThefollowingindicatorsareproposedintheLCAtooldevelopedunderthisprojectscope,forassessing
pharmaceuticals processes,basedonthePRAXIScurrentprocesses:
Environmentalmetrics Socialmetrics Economicmetrics
Energyintensity(MJ/vialorMJ/API)
Materialintensity(gram/vialorMJ/API)
WaterConsumption(liter/vialorMJ/API)
SolidWaste(gram/vialorMJ/API)
Wastewater(liter/vialorMJ/API)
GHGEmissions(kgCO2eq/vialorMJ/API)
abioticdepletion(expressedinantimony
equivalent/vialorMJ/API)
abioticdepletion(expressedinkWh/vialor
MJ/API)
globalwarming(kgCO2eq./vialorMJ/API)
ozonelayerdepletion(kgCFC11eq./vialorMJ/API)
humantoxicity(kg1,4dichlorobenzeneeq./vial
Deathsorpermanent
disabilitiesinthepast5
years
Partialdisability(number
ofaffectedinthelast5
years)
Partialdisabilities(%
average)
Annualabsences(days/yr)
Absencecosts(euros/day)
Numberofdirectfulltime
jobs Numberofhoursper
personperyear
Electricitycost(EUR/g
vial)
Watercost(EUR/vial)
Costofwoodpacking
material,paperand
cardboard(kg/grAPI)
Costoffuel(EUR/
year)
Costofrawmaterial
forAPImanufacture
(EUR/year)
Sales NetIncome
ProductionVolumes
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orMJ/API)
Freshwateraquaticecotoxicity(kg1,4
dichlorobenzeneeq./vialorMJ/API)
Marineaquaticecotoxicity(kg1,4
dichlorobenzeneeq./vialorMJ/API)
Terrestrialecotoxicity(kg1,4dichlorobenzene
eq./vialorMJ/API)
photochemicaloxidation(kgethyleneeq./vialor
MJ/API)
acidification(kgSO2eq./vialorMJ/API)
eutrophication(kgPO4eq./vialorMJ/API)
Numberofindirectfull
timejobs
Numberofworkingdays
perpersonperyear
Numberofaccidentsper
workinghours
OperatingProfit
InvestmentinR&D
CashFlow
ExpenditureonEHS
1.2 STUDYOBJECTIVESTheLCAtooldevelopedforpharmaceutical companiesisbasedonMicrosoftOfficeExcel.Itallowsthe
inputofthe inventorydataandautomaticallycalculatesthepotentialenvironmental impacts,suchas
global warming, acidification, eutrophication, ozone depletion, photochemical oxidation, human
toxicity, aquatic ecotoxicity, and terrestrial ecotoxicity. The LCA tool should facilitate results
interpretation,both fororganizationalor scientificuse,concerning the following threemain typesof
contents(Gainzaetal.,2009): Ecologicalcontent EcoSocialcontent Economiccontent
During the development of the LCA tool, the best available technologies for specific production
processes in the pharmaceutical industry will be analyzed and also their environmental impactsand
sustainabilitywillbeevaluated.Thetechnologiestobe identifiedandassessed includetheones listed
below,whichhappenstobetheonescurrentlybeingusedbyPRAXIS:
APIProduction:o Recombinantbiotechnologytechniques;o Fermentationtechnologies; o Formulationandfiltrationsystems;o Sterilizationsystems(e.g.autoclaving,depyrogenation,radiation,chemical);
MedicineProductiono Materialwashingandpreparationsystemsthat:
Avoidcrosscontamination; Saveenergy,e.g.choosingasteamheatingsourceoverelectricalsystems; Reducewaterconsumption,speciallyofWFI(WaterforInjection); Combinewashingandsterilizationsystemsinonesingleequipment;
o Dosingandfillingsystemsforliquidsandsolids;o Freezedrying;o Microandnanostopperingtechnologies;o Sterileconditioningtechnologies; o Chromatographicseparation;o Filtersterilizationandproductconservation;o Preparationandconditioningtechnologies.
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2 ProductionofLyophilizedProductsviaRecombinantBiotechnologyPharmaceutical production is usually divided into two main production stages: API and Medicine
Production.The former isrelatedtotheproductionoftheactivepharmaceutical ingredient (API)and
thesecondoneisrelatedtotheconversionoftheAPIintothefinalsdrugpresentationform.Thesetwo
stagesarepresentedanddivided intoseveralprocessingsteps thatwillbedescribed inthe following
sections.
2.1 APIPRODUCTIONMicrobialcellfermentationhasalonghistoryofuseintheproductionofvariousbiologicalproductsof
commercial significance. It started in the early 1970s when there was the development of two core
biotechnologies (rDNA and Mabs), accounting in 2006, for 80 of the 140 commercially available
products.TherDNAprocess,sometimescalled geneticengineering, isaserialprocesswhereby (i)a
proteinisassociatedwithabiologicactionandisidentified;(ii)aspecifichumangene(aDNAsequence)
is associatedwith theprotein; (iii) the humangene is inserted intobacterialDNA (plasmid); (iv) the
plasmid isplaced intothenonhumanhostcells (e.g.Escherichia colibacteria);and (v)thehostcells
manufacture their typicalvarietyofproteinsandproduceahumanprotein from thehumangene. In
fact,overhalfofallbiopharmaceuticals thus farapprovedareproducedby recombinantE. colior S.cerevisiae (themostcommonhosts).Asa result,awealthof technicaldataandexperiencehasbeenaccumulatedinthisarea(Walsh,2003,Swarbrick,2005).
Figure showsatypicalflowdiagramofaprimaryprocessingviarecombinantbiotechnologyfortheAPI
manufacture.
Figure13.Primaryprocessingviarecombinantbiotechnology.
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2.1.1 PreparationofRawMaterialsforFermentationTheculturemediumcompositionandthefermentationconditionsrequiredtopromoteanoptimalcell
growthand/orproductmanufactureneeds tobeestablishedduring the initialproductdevelopment.
Afterthat,routinebatchproductionisahighlyrepetitiveandahighlyautomatedprocess.
Heatlabileingredientscanbesterilizedbyfiltrationandaddedtothefermenteraftertheheatingstep.
Mediacompositioncan vary froma simpledefinedculturemedia (usuallyglucoseand somemineral
salts) to a more complex media, using yeast extract and peptone. The choice of the culture media
dependsuponseveralfactorssuchas(Walsh,2003):
Exact nutrient requirements of producer cell line to maximize cell growth and productproduction;
Processeconomics(totalmediacost); Extracellularor intracellularnatureoftheproduct.Ifthebiopharmaceutical isanextracellular
product, then the less complex the media composition, the better in order to render
subsequentproductpurificationasstraightforwardaspossible.
Typically, the batch manufacture of a biopharmaceutical productentails filling theproduction vessel
withtheappropriatequantityofwaterforinjection(WFI).Heatstablenutrientsrequiredforproducer
cellgrowtharethenaddedandtheresultantmediaissterilizedinsitu.Thiscanbeachievedbyheatand
manyfermentershaveinbuiltheatingelementsor,alternatively, outerjacketsthroughwhichsteamcan
bepassedinordertoheatthevesselcontents.
Table1showsthemaininputsandoutputsfortheinventoryanalysisrelatedtothepreparationofthe
culturemediumusedforthefermentationprocess(PRAXIS,2009).
Table1 Inventoryanalysisofthepreparationoftheculturemediumforthefermentationprocess(PRAXIS,2009)
ReceptionandcontrolofRawmaterial
RawMaterials
ElectricalEnergy
ACSconsumption
AFSconsumption
industrialsteamconsumption
puresteamconsumption
WFIconsumption
timberpackaging,cardboard,paper
plasticpackaging
scrappackstetrabrickandisothermal
glasspackaging
packagesorpiecesofsteel
packagesorpiecesofaluminium
PreparationofRawmaterial
ElectricalEnergy
ACSconsumption
AFSconsumption
Industrialsteamconsumption
Puresteamconsumption
PurifiedWaterconsumption
WFIconsumption
timberpackaging,cardboard,paper
plasticpackaging
scrappackstetrabrickandisothermal
glasspackaging
packagesorpiecesofsteel
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packagesorpiecesofaluminium
Preparationand
Material
Recuperation
ACSconsumption
AFSconsumption
Industrialsteamconsumption
Puresteamconsumption
PurifiedWaterconsumption
WFIconsumption
ElectricalEnergy
CompressedAir
2.1.2 InoculationandFermentationTheupstreamprocessingelementofthebatchmanufactureofabiopharmaceutical productstartswith
theremovalofasingleampouleoftheworkingcellbank.Thisvialisusedtoinoculateasmallvolumeof
sterile growth medium. This step entails the use of daughter cells from the new host cell (master
workingcellbank),actuallyremovedfromitsstorageina 70Cfreezer.Thedaughtercellsaregrownin
specific media in serially larger flasks and assessed for normal growth characteristics. The growth
medium (liquid and air) is a unique and specific mixture of minerals, compounds, and nutrients to
enhancecellviability (lifespan) invitroand functionalabilityofcells toproduceproteins.Thisstarter
culture is in turnused to inoculateaproductionscale starter culture,which isused to inoculate the
productionscale bioreactor (Walsh, 2003). It is here that fermentation occurs, with cells from the
inoculumphaseandaddingtheappropriatefortifiedgrowthmedia.Inabatchconfiguration,hostcells
thatcontainanexpressionvectorfortherecombinantproductareaddedtoapredeterminedvolumeof
growthmedium.Thecellsareallowedtogrowuntilthenutrients inthemediumaredepletedorthe
excretedbyproductsreach inhibitorylevels.Meanwhile,theywillproceedtoproduceproteins(inthisparticularcaseextracellularly)intothemedia.Feedingofthehostcellsandremovingofwastefromthe
medianeedtobedoneperiodicallytosustainhostviabilityandproductivity.Fermentationfollowsfor
severaldays subsequent to inoculationwith theproductionscale starterculture.During thisprocess,
the biomass (i.e. cell mass) accumulates. From each master frozen culture, a subculture stock is
established for use in largescale production. The subculture stock becomes the inoculum for every
batch of product. In this way each batch of cultured cells is initiated with a common lineage of
recombinanthostcells(Swarbrick,2005;Walsh,2003;RodneyandMilo,2003).
Table2 shows themain inputsandoutputs for the inventoryanalysis related to the inoculationand
fermentationprocesses(PRAXIS,2009).
Table2 Inventorydatafortheinoculationandfermentationprocesses(PRAXIS,2009)
Inoculation
ElectricalEnergy
ACSconsumption
AFSconsumption
Industrialsteamconsumption
Puresteamconsumption
PurifiedWaterconsumption
WFIconsumption
CompressedAir
O2
CO2
N2Excipients
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Timberpackaging,cardboard,
Paper
Plasticpackaging
scrappackstetrabrickand
isothermal
glasspackaging
piecesofsteel
packagesorpiecesofaluminium
Fermentation
ElectricalEnergy
Containerforbiologicalproducts
ACSconsumption
AFSconsumption
industrialsteamconsumption
puresteamconsumption
purifiedwaterconsumption
WFIconsumption
Excipients
Compressedair
Auxiliarymaterial
Industrialscalebacterialandyeast fermentationsystemsareusedandsharemanycommon features.
Forexample,fermentationorbioreactionvesselsaregenerallymanufacturedfromhighgradestainless
steel,andcanvary insizefromafewtensof literstoseveraltensofthousandsof liters.An impeller,
drivenbyanexternalmotor,servestoensureevendistributionofnutrientsandcells inthetank.The
baffles(stainlesssteelplatesattachedtothesidewalls)servetoenhanceimpellermixingbypreventing
vortex formation. During fermentation, air (sterilizedby filtration) is sparged into the tank to supply
oxygentothefermenterthatisoperatedatanappropriatetemperaturetooptimalcellgrowth(usually
between 2537C) depending upon the producer cell type. In order to maintain this temperature,
cooling rather thanheating is required insomecases.Large scale fermentations, inwhichcellsgrow
rapidlyandtoahighcelldensity,cangenerateconsiderableheatduetomicrobialmetabolismandalso
mechanical activity, e.g. stirring. Cooling is achieved by passing the coolant (cold water or glycol)
through a circulating system associated with the vesseljacket or sometimes via internal vessel coils
(Walsh, 2003). Various ports are also available through which probes are inserted to monitor pH,
temperatureandsometimestheconcentrationofacriticalmetabolite(e.g.thecarbonsource).
The isolationstepfollows immediatelyafterharvestingoftherawmaterials. Inthe isolationstep,the
cruderawmaterialisrefinedintoaclarifiedfeedstreamthatisanintermediateprocessfreefromcells
andotherparticulatematter.Anumberofdifferentmethodsmaybeemployedduringtheisolationstep
such as filtration, gravity separation, centrifugation, flocculation, evaporation. The unit operations
employedintheisolationstepdependverymuchontheinitialrawmaterial(JornitzandMeltzer,2007).
In the purificationphase, the aim is toprepare apure biopharmaceutical product from the clarified
feed.Thisisthemostchallengingandexpensivestepindownstreamprocessingbecauseitisnecessary
to separate the desiredproduct from othermoleculeswith similar properties in the fewest possible
stepswiththesimplestpurificationtechnologytoachievetherequiredpurity.
Anoverviewofthestepsnormallyundertakenduringdownstreamprocessingwillbepresented inthe
followingsections,sincedetailsoftheexactstepsundertakenduringthedownstreamprocessingofany
specificbiopharmaceutical productareusuallyconsideredhighlyconfidentialbythemanufacturerand
thusarerarelymadegenerallyavailable(Walsh,2003;RodneyandMilo,2003).
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2.1.3 ProductConcentrationandChromatographicPurificationThe following phase of downstream processing usually entails concentration of the crude protein
product.The reason for this step isbecauseafter the removalof the cellsandcellsdebris from the
culturemediumthroughsolidliquidseparation,thefractionofrecombinantproteinintheliquidphase
usually ranges from2% to15%.The largevolumesassociatedwith these lowconcentrationsmake itimpracticaltoproceedtothenextpurificationstep.Indeed,thevolumeofproductcontaminantstobe
purifiedmay farexceed the capacity for chromatographic purification techniquesused to isolate the
recombinantproteinsfromothersolublecellularcontaminants.Therefore,aconcentrationstepisused
toreducethevolumeandtherebyincreasetherecombinantproteinconcentration.
Theconcentrationstepyieldssmallerproductvolumes,whicharemoreconvenienttoworkwithand
cansubsequentlybeprocessedwithgreaterspeed.Concentrationmaybeachievedbyinducingproduct
precipitation using, for example, salts such as ammonium sulphate or solvents such as ethanol.
Moreover,proteinprecipitation, usingagentsthatdecreasethesolubilityoftherecombinantproduct,is
a well established technique in the pharmaceutical industry. While salts and organic solvents have
traditionallybeenused,morespecificprecipitationreagentsarenowbeingtestedto improveproteinpurification.However,ultrafiltrationisthemorecommonlyemployedmethod.
Ultrafiltration is a lessdestructive approach to protein concentration. In most cases, pore size is
selectedtoretaintherecombinantmacromoleculewhileallowingthepassageofwaterandothersmall
molecules. Ultrafiltration membranes are usually manufactured from tough plasticbased polymers,
suchaspolyvinylchlorideorpolycarbonate.Arangeofmembranesareavailablewhichdisplaydifferent
cutoffpoints.Inpractice,however,theselectionofporesize ismoreanartthanascience,especially
thechoiceofdesignandsizingconfigurationsofthefiltrationsystem.Theselectionofaproperfiltration
systemmay leadtoadditionalbenefitsintermsofanincreaseintheyieldofrecombinantproteinand
itspurity.Ultrafiltration isapopularmethodofconcentration since: (Walsh,2003,RodneyandMilo,
2003)
Highproductrecoveryratesmaybeattained(typicallyoftheorderof99%); Processingtimesarerapid; Processscale ultrafiltration equipment is readily available, and running costs are relatively
modest.
After concentration, furtherpurification isneeded to increase the purity of the recombinantprotein
foundintheconcentratedsolution.Inthisstage,increasedpurityisachievedthroughremovalofmost
contaminant proteins, nucleic acids, endotoxins, and viruses, by means of chromatography. High
resolution chromatographic purification is usually undertaken for which, a variety of different
chromatographic techniques are available to separate proteins from each other on the basis of
differences in various physiochemical characteristics (Jornitz and Meltzer, 2007, Rodney and Milo,
2003).
Detaileddescriptionofthetheoryandpracticeunderliningchromatographictechniquesgofarbeyond
thescopeofthistext,andarefreelyavailableinthescientificliterature.However,asimpledescription
canbegivenhereinordertounderstandthechromatographytechnique.
Chromatography can be defined as a procedure in which proteins bind differentially to solid matrix
supports ormediawith various functionalgroups to providehydrophobic, ionexchange,andaffinity
interactions.Theseinteractionstrengthsofeachcomponentwiththestationaryphaseareproportional
toitsretentiontime.
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Amongthemanydifferentchromatography formatsavailable,theonethat isusuallyapplied in large
scaledownstreamprocessingisthecolumnbasedliquidchromatography,inwhichaliquidfeedstream
is passed over or through a porous, solid matrix or resin held in a column. The components of the
mixturebecomedistributedbyvirtueof their relativeaffinity for the solidand liquidphases.So, the
secrethere is to introduce theclarified feed stream into thecolumnunderconditionswherecertain
components bind strongly to the resin while others flow through. The composition of the buffer ischosen to favor the retentionorelutionof specificcomponents.Bychanging thecompositionof this
buffer, molecules that initially bind to the resin can be washed through in subsequent fractions
(SchmidtTraub,2005).One importantreference isthatthechromatographictechniqueshouldprovide
highcapacityandselectivityandfastkinetics.Thematrixmaterialmustwithstandmultiplepurification
cycleswithminimumlossofefficiency.Somematricesusedfor industrialscaleproteinpurificationare
indicatedinthefollowinglist(JornitzandMeltzer,2007;RodneyandMilo,2003).
Agaroseanddextrancomposite Agaroseandpolyacrylamidecomposite Agaroseandporouskieselguhrcomposite Cellulose Crosslinkedagarose Crosslinkeddextran Crosslinkedpolyacrylamide Ethyleneglycolmethacrylatecopolymer Hydroxyacrylicpolymer Hyroxymethacrylate polymer Polyacrylamide Polyacrylamideanddextrancomposite Polystyrenedivinylbenzene Poroussilica Rigidorganicpolymer
Thevariouskindsofphysiochemical interactionsthatareusedinchromatographytoproduceselectivity
are called modes of interaction. Examples include electrostatic interactions in ionexchange or ion
chromatography, hydrophobic interactions in reversedphase and hydrophobic interaction
chromatography,andspecific interactions inaffinitychromatography. However,sometimesthisrange
of selectivity isnt enough. The reason is related to the different molecular weights of proteins,
hydrophobicity,charge,andstructureoverawiderange,whichrenders it impossibletoapplyasingle
chromatographicseparationforcomplexproteinmixtures(JornitzandMeltzer,2007;Gad,2007).
Ingeneral,acombinationoftwotofourdifferentchromatographic techniquesisemployedinatypicaldownstreamprocessingprocedure,beinggel filtrationand ionexchange chromatographyamong the
most common. Affinity chromatography is employed whenever possible, as its high biospecificity
facilitates theachievementofa very high degree ofpurification. Anyway, thegeneralprocedure for
adsorptivechromatography isto introducetheclarifiedfeedstream intothecolumnunderconditions
wherecertaincomponentsbind strongly to the resinwhileothers flow through (JornitzandMeltzer,
2007;RodneyandMilo,2003).
Aswithmostaspectsofdownstreamprocessing, theoperationofchromatographic systems ishighly
automated and it is usually computercontrolled. While mediumsize processscale chromatographic
columns(e.g.515litres)aremanufacturedfromtoughenedglassorplastic,largerprocessingcolumns
areavailable,manufacturedfromstainlesssteel(Walsh,2003).
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Normally, forresinsalreadyused,additionalanalysis isrequired,whichdoesnthappenwiththenew
resins.Theseanalysisincludeforexample,titrationofsmallionbindingcapacity,measurementoftotal
protein capacity, comparison of flow versus pressure plots to indicate particle size (and attrition),
particle size distribution, total organic carbon (TOC) removal by cleaning solutions, and microbial
contamination/ endotoxin (LAL) analysis. Exposure to cleaning/regeneration solutions, rather than
contact with mild buffers and protein solutions during normal processing, is most likely to causechemicaldegradation(Gad,2007).
A final purification, known as the polishing step, is designed to remove trace contaminants and
impurities so that a biologically active recombinant protein with a safety profile suitable for
pharmaceutical application is obtained. Chromatography systems for final purifications demand high
performanceevenatthecostofa lowercapacitythanthecolumnusedfor intermediatepurification.
High separation performance is needed to minimize contaminant carryover into the recombinant
productforpharmaceutical use.(RodneyandMilo,2003)
Table 3 shows the main inputs and outputs for the inventory analysis of the chromatographic
purificationprocess(PRAXIS,2009).
Table3 Inventorydataforthechromatographic purification(PRAXIS,2009)
ChromatographicPurification
ElectricalEnergy
puresteamconsumption
purifiedwaterconsumption
WFIconsumption
Auxiliarymaterial
Containerforbiologicalproducts
2.1.4 FilterSterilizationandAPIConditioningWhile implementation of good manufacturing practices will ensure that the product carries a low
microbial load, itwillnotbesterileat thisstage. Ideally,sterile filtration removesunwantedparticles
andbacteriawhileallowingtheformulationtoremainunadulterated.
In the membrane filtration method, the product samples are put aseptically into a volume of non
inhibitorydiluentandthenpassedthroughasterilemembranefilterwithaporeof0.22to0.45mm.This
cancompletelyeliminateviableorganismsofanyspeciesfromafluid(JornitzandMeltzer,2007).Thus,
a liquid,containingsuspendedmicroorganismswouldberendered freeofcontaminatingmicrobesby
separationofthemfromtheliquid.Theadvantageofthisstepis,aswithmanyoftheotherassessment
techniques, that it must be conducted offline in a timeconsuming manner. As a result, it is not
determinedwhetherbatchesaresatisfactoryuntilprocessingiscompleted(Gad,2007).
Thetestforsterilitymaybeperformedinoneoftwoways,bydirectinoculation(directtransfer)orby
membranefiltration(Swarbrick,2007).Indirectinoculation,theproductsamplesareputasepticallyinto
themicrobiological recoverymediumand incubated.Clearly thisapproach isonlysuited forproducts
that are not likely to be inhibitory to the growth of microorganisms in the recovery medium. An
incubationperiodof14daysisspecified(JornitzandMeltzer,2007).
Table 4 shows the main inputs and outputs for the inventory analysis of filter sterilization and
conditioningoftheAPIsteps(PRAXIS,2009).
Table4 InventorydataforthefiltersterilizationandconditioningoftheAPI(PRAXIS,2009)
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FilterSterilization
Electrical Energy
inhibitoryagents
timberpackaging,cardboard,paper
plasticpackaging
scrappackstetrabrickandisothermal
glasspackaging
packagesorpiecesofsteelpackagesorpiecesofaluminium
Containerforbiologicalproducts
AFS
Compressedair
Industrialsteamconsumption
Inspection,controland
quarantine
Electrical Energy
timberpackaging,cardboard,paper
plasticpackaging
scrappackstetrabrickandisothermal
glasspackaging
packagesorpiecesofsteel
packagesorpiecesofaluminium
Packaging
Electrical Energy
timberpackaging,cardboard,paper
plasticpackaging
scrappackstetrabrickandisothermal
glasspackaging
packagesorpiecesofsteelpackagesorpiecesofaluminium
AnexampleoftheinventoryanalysisresultsfortheprimaryprocessingisexpressedinTable5(PRAXIS,
2009).
Table5Exampleoftheinventoryanalysisresultsforprimaryprocessing(PRAXIS,2009)
SoilContamination EnergyCostofWasteProductionandManagement
Paper,cardboardandothercellulosic products
Plastic
DangerousWasteWaterPollution EnergyCostofWaterTreatment
AirPollution
EnergyCostofAirPollution
CO2absorption
CO2emissionsfrom fermentationprocesses
SO2emissionsfromcombustionprocesses
NOXemissionsfromcombustionandmanufacturing
processes
CO2emissionsfromcombustionprocesses
NaturalResourcesDepletion
EnergyCostofNaturalResourcesDepletion
Energyusage(fossilfuelandelectricenergy)
Waterusage
Consumptionofcardboard andothercellulosicproducts
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Plasticinputs consumption
Ferrousmetals consumption
Lubricantagent consumption
BeforethetransformationoftheAPIintothemedicine,theAPIhastotravelbyplainforalongdistance.
Itwasconsideredthattheconsumesduetothattripareimportantfortheoverallimpact,sotheinputs
areshownbelow.
Transportation
QuantityofRawMaterialbeingtransportedbetween
thestorageareatothemanufacturearea
Transportmode
Fuelconsumption
QuatityofRawmaterialpertrip
Kmpertrip
2.2 MEDICINEPRODUCTIONAll the steps after purification (except in some cases milling) are usually included in the Medicine
Production.itsgoalistomaketheformulationintothefinalproduct(BennettandCole,2003).
Thisgenerallyinvolvesthefollowingsteps:
Addition of the various excipients, which are substances other than the active ingredient(s)which, for example, stabilize the final product or enhance the characteristics of the final
productinsomeotherway;
Filter sterilization of the final product (e.g. through a 0.22mm absolute filter) in order togeneratesterileproduct,followedbyitsasepticfillingintothefinalproductcontainers;
Freezedrying(orlyophilization) iftheproductistobemarketedinapowderedformat.Generallyonemayrepresentthesecondaryprocessingforthemanufactureofalyophilizedproductas
inFigure.
Figure14 Secondaryprocessingforthemanufactureoflyophilizedproducts.
Asimpledescriptionofthemostimportantstagesofsecondaryprocessingwillbegiveninthefollowing
sections.
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2.2.1 APIandExcipientsWeightingandProductFormulationPharmaceutical dosage formscontainbothpharmacologically activecompoundsandexcipientsadded
tofacilitateformulationandmanufactureofthesubsequentdosageformforadministration topatients.
In which concerns freezedrying, excipients are used for various purposes. For example, they act as
bulkingagentstogiveapleasingappearancetothefreezedriedproducts.BuffersarepresenttocontrolthepHoftheproductsthatarestableonlywithinanarrowpHrange insolution,bothduringfreezing
andthesubsequentreconstitution(Swarbrick,2007).
Moreover, excipients also play a role in the API protection. This mechanism has not been fully
elucidated,butempiricalobservationshavepointedtothefollowingcontributingfactors:formationofa
glassystateoftheproteinexcipientsystem,crystallinityoftheexcipients,hydrogenbondingbetween
the excipient and protein molecules, and residual water content. In particularly, water affects the
stabilityofproteinsbyenhancingthemobilityoftheproteinmolecules.Ithasbeenestablishedthatan
optimallevelofwaterisrequiredtomaintainstabilityofproteinsduringstorage.Indeed,theproperties
ofthefinaldosageform(i.e.itsbioavailabilityandstability)are,forthemostpart,highlydependenton
the excipients chosen, their concentration and interaction with both the active compound and eachother.Inconclusion,theymustbechosenverycarefully(Rowe,SheskeyandQuinn,2009).
Some excipients that may be present in freezedried powders include solubility enhancers (e.g.,
surfactantsor cosolvents),osmoticagents (e.g., salineand sugars),antioxidants (e.g.,ascorbicacid),
andpreservativesformultipleinjectioncontainers(e.g.,benzylalcoholandchlorobutanol).Inaddition,
freezedriedbiologicalpowdersmayalsocontainexcipientsthatfunctiontoreduceproteinadsorption
ontothecontainersurface(e.g.,surfactantsandalbumins).Aparticularlyimportantuseofexcipientsfor
therapeuticproteinformulationsisthestabilizationoftheproteinmoleculesinthedrystate.
Examplesof some excipientsusedas stabilizers forproteins in freezedried formulations include the
following(Swarbrick,2007):
Mannitol and glycine as amorphous excipients to prevent human growth hormone (hGH)aggregation.Trehaloseasalyoprotectant, preservesthesecondarystructureofrhGH(e.g.used
forrecombinanthumangrowthhormone(rhGH)).
Dextrin,EmdexTM (spraydrieddextrose)andhydroxypropyl cyclodextrinminimized insulinaggregation(e.g.usedforbovineandhumaninsulins).
Polysorbate80asprotectorforfreezing;sucroseasprotectorfordrying;histidineaspHbuffer;glycineforcakeappearance(e.g.usedforrecombinantfactorIX).
Aggregation prevented by amorphous trehalose, sucrose or a combination of sucrose, andglycineormannitol(e.g.usedforrecombinanthumaninterleukin6).
Sucrose, sorbitol, trehaloseandalanineasprotectantsagainstaggregationanddeamidation;mannitol and glycine as bulking agent; sodium citrate as buffer (e.g. used for recombinant
humaninterleukin1receptorantagonist)
Sugars(sucrose,lactose,trehalose,maltose),polymer(dextran)andsalts(NaCl,KCl)tomodifytheglasstransitiontemperaturesofthefreezedriedpowders(e.g.usedforFK906tripeptide).
Recombinanthumanalbumin OrganicacidexcipientmoleculeswitheitheracarboxylgrouporanaminogrouppresentatC1positioncompletelystabilizedrHAagainstaggregation
Polyethylene glycol as protectant for freezing; sugars (mannitol, lactose, trehalose) aslyoprotectants against loss of bioactivity (e.g. used for lactate dehydrogenase
phosphofructokinase).
Lactoseand trehalose maintain activity longeratelevated temperatures than mannitol (e.g.usedforAlkalinephosphatise).
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Both the excipient type (sucrose, sorbitol, glycerol) and moisture content affected proteindegradation(e.g.usedforrecombinantbovinesomatotropin,lysozyme).
Mannitolprotectedproteinfromphaseseparation induceddamageduringfreezedrying(e.g.usedforHemoglobin).
Trehalose and sucrose preserved the native dimeric structure of the protein and preventedaggregatesformation(e.g.usedforRecombinanthumanfactorXIII).
Amongothers,saccharidesarethemostwidelyusedexcipientsforstabilizingfreezedriedtherapeutic
proteins.
Table 6 presents the main inputs and outputs for the inventory analysis for the API and excipients
weightingandproductformulation.ThisdatawasprovidedbythepharmaceuticalcompanyPRAXIS.
Table6InventoryanalysisoftheAPIandexcipientsweightingandproductformulation(PRAXIS,2009)
Receptionandcontrolofraw
materialsandexcipients
ElectricalEnergyAPI
ACSconsumption
AFSconsumption
industrialsteamconsumption
puresteamconsumption
purifiedwaterconsumption
WFIconsumption
timberpackaging,cardboard,paper
plasticpackaging
scrappackstetrabrickandisothermal
glasspackaging
packagesorpiecesofsteel
packagesorpiecesofaluminium
Materialreceptionandstorage
conditioning
ElectricalEnergy
ACSconsumption
AFSconsumption
industrialsteamconsumption
puresteamconsumption
purifiedwaterconsumption
WFIconsumption
timberpackaging,cardboard,paper
plasticpackaging
scrap packstetrabrickandisothermal
glasspackaging
packagesorpiecesofsteel
packagesorpiecesofaluminium
Washingandconditioning
materialpreparation
ElectricalEnergy
ACSconsumption
AFSconsumption
industrialsteamconsumption
puresteamconsumption
purifiedwaterconsumption
WFIconsumption
Aircompressed
Preparationof
the
buffer
solution:Weighting,ElectricalEnergyACSconsumption
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FormulationandSterile
Filtration
AFSconsumption
industrialsteamconsumption
pure steamconsumption
purifiedwaterconsumption
WFIconsumption
RawMaterials
API
Aircompressed
2.2.2 FreezeDryingFreezedrying isacommondryingtechniqueused inthepharmaceutical industry(Swarbrick,2007).As
manypharmaceuticals cannotbeproducedonacommercialscalebycrystallization, aglassysolidmay
betheonlysolidstateoption.FreezeDryingisanextremeformofvacuumdrying,whereaformulation
isdriedto1%waterorless,withoutanyoftheproductexceeding30C.Theprocessinvolvesfreezingof
an aqueousbased drug solution in a glass vial followed by sublimation of the ice in a vacuum
environment.Althoughrelativelyexpensive, it isemployedtoconvertsolutionsof labilematerials into
highlyporous,amorphoussolidcakesofsufficientstabilityfordistributionandstorage(Swarbrick,2007;
HickeyandGanderton,2001).
Someadvantagesoffreezedryingoverotherdryingtechniquesarethefollowing(KudraandMujundar,
2009;OetjenandHaseley,2004):
The use of low temperatures with the purpose of protecting the API during processing (afreezedryingprocessmaintainssterilityandparticle freecharacteristicsoftheproductmuch
moreeasilythanotherdryingprocess.Furthermore,theingredientsoftheformulationarenot
stable in the liquid stateandothermethodsofwater removaldestroyor reduce the activeingredient);
Thisprocessisapprovedbyregulatoryauthorities; Itcanbeperformedundersterileconditions(thesolutionissterilefilteredimmediatelybefore
fillingintothefinalcontainer,andfurtherprocessing);
Thedriedproductcanberapidlyrehydratedwhennecessary; Theamountoftheactiveingredientisverysmall Thefreezedryingprocesshasthereputationofbeingsimpletoperform; Ithasbeensuccessfullyusedbyseveralcompaniesand/orforseveralproducts; Moisture and headspace gas can be easily controlled, an important advantage for products
whosestoragestabilityisadverselyaffectedbyresidualmoistureand/oroxygen; Development of freezedried products requires less material for formulation and process
development.
The only requirement is the product sterility and it is of overriding importance that the whole
procedure, from vial filling to the final sealing stages, is performed under strictly controlled
environmental conditions, so that the final product is viable for consumption. The hypothesis of
sterilizingafterthisprocesscantbeconsideredbecausetheonlyavailabletechniquesrequirea liquid
producttosterilizeorhightemperatures,whichwoulddenatureproteins(Franks,2007).
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2.2.2.1 ProductFillingandGlassVialsLoadingAnaqueoussolutioncontainingthedrugandvariousformulationaidsorexcipients,housedtemporarily
inasterileproductholdingtankisasepticallyfilledintopresterilizedfinalproductcontainers(e.g.glass
vials,ampoulesoroccasionallyinsyringes),whichareloadedontothetemperaturecontrolledshelves.
The filling processnormallyemployshighly automated liquid filling systems. All items of equipment,
pipework,etc.withwhichthesterilizedproductcomesintodirectcontactmustobviouslythemselvesbe
sterile.Thisismaintainedbyafilterattheneckofthebottlethatallowsthepassageofwatervaporbut
prevents the ingressofbacteria.Mostofsuchequipment itemsmayalsobesterilizedbyautoclaving
andbeasepticallyassembledprior to the fillingoperation.The finalproductcontainersmustalsobe
presterilized. This may be achieved by autoclaving, or passage through special equipment which
subjectsthevialstoahotWFIrinse,followedbysterilizingdryheatandUVtreatment.
If the product can be filled into plasticbased containers, alternative blowfillseal systems may be
used,as itsname suggests.Suchequipment firstmouldsplastic into the finalproductcontainer (the
molding conditions ensure container sterility), followed immediately by automated filling of sterile
product into thecontainerand its subsequent sealing. In thiswayoperator intervention in the filling
processisminimized(Swarbrick,2007,HickeyandGanderton,2001,Franks,2007).
2.2.2.2 TheFreezedryingProcessThefreezedryingcycle,asappliedtoasolution,consistsofasequenceoftwodistinctprocesses
(1)Primarydrying(coolingtobelowthefreezingtemperatureinordertomaximizetheicecontentand
sublimatetheiceatsomesubfreezingtemperature,usuallyperformedunderreducedpressure);
(2)Secondarydrying(removalofresidualunfrozenwaterfromthesolidifiedsolution).
During the primary drying, the drug solution is filled into glass vials and then placed within a
temperaturecontrolleddryingchamber.There,thesolution isfrozenquicklytopreventconcentration
of the solution and to produce fine ice crystals according to physiochemical principles (the energy
transporttotransformiceintowatervaporandthetransportofthewatervaporfromthesublimation
surface through thealreadydriedproduct into thedryingchamber to thecondensationorabsorbing
systemforthevapor)astheshelftemperatureisloweredtobelowfreezing.
Theshelftemperatureissubsequentlyincreasedbutmaintainedbelowthefreezingpoint.Avacuumis
applied to the chamber to sublimate the solvent.Theextent towhich the compound is supercooled
dependsonthecompoundnature,thetemperatureprogramoftheshelf,theheattransferproperties
ofthecontainer,andthepresenceofparticulatesinthesolution.
Thisphaseofthedryingprocessextractsthemajorityofthesolvent(5080%).Thedrugandexcipients
aretypicallyconverted intoanamorphousglassalsocontaining largeamountsofunfrozenwater(15
30%)dissolved inthesolid, i.e.glassyamorphousphase.Thus,mostofthedesiccationactuallyoccurs
duringthefreezingstageofthefreezedryingprocess.
During the secondary drying, the remainder of the solvent is removed at an elevated but still
subfreezing temperature inorder to minimize product moisture content. So, heat is supplied to the
dryingsurface.Thepowerdissipatedbytheheatermustbecarefullycontrolledsothatmeltingdoesnot
occur (only drying is accepted) at the icecontainerjunction (Franks, 2007, Swarbrick, 2007, Oetjen,
Haseley,2004,HickeyandGanderton,2001).
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2.2.2.3 TheEquipmentAtypicalproductionscalefreezedryerconsistsofadryingchamberinwhichthesolutioncanbecooled
totherequiredtemperatureandbeevacuatedtoa lowpressureandseveralcontainingtemperature
controlledshelves,whichcanbecontrolledwithacirculatingheatexchange fluid.Theheatexchange
system issuppliedwithapump,whichcirculatesthefluidthroughtheshelves.Thiscirculationsystemmustbecapableofmaintainingtheshelftemperatureatthesetdesiredvalues.
Thesystemisalsoconnectedtoacondenserchamberviaalargevalve.Thecondenserchamberhouses
aseriesofplatesorcoilscapableofbeingmaintainedatvery lowtemperature (i.e., lessthan 50C).
Oneormorevacuumpumpsinseriesareconnectedtothecondenserchambertoachievepressuresin
therangeof0.030.3Torrintheentiresystemduringoperation.
Modern equipment contains computerbased control and monitoring systems by means of which a
desireddryingcycleprogramcanbepreset.Finally, forpharmaceutical applications, it isessentialto
prevent crosscontamination between consecutive batches. A cleaninplace (CIP) system and a
steriliseinplace system are therefore provided by means of which the chamber can be cleaned
betweensuccessiveproductioncycles(Swarbrick,2007;Franks,2007).
Table7showsthemain inputsandoutputs forthe inventoryanalysisofproductfillingandglassvials
loading(PRAXIS,2009).
Table7 Inventoryanalysisofproductfillingandglassvialsloading(PRAXIS,2009)
FreezeDryingandCapping
ElectricalEnergy
ACSconsumption
AFSconsumption
industrialsteamconsumption
puresteamconsumption
purifiedwaterconsumptionWFI consumption
FlipOff(capsulesthatcarrythevials)
Sterilevials
Sterilecaps
SterileTop
Compressedair
2.2.3 StopperingandFinalProductStorageFinalconditioningand storagebeginswith theextractionof theproduct from theequipment.During
thisoperation,agreatcarehastobetakennotto losetherefinedqualitiesthathavebeenachieved
duringtheprecedingsteps.Thus,forvials,stopperingundervacuumorneutralgaswithinthechamber
is the currentpractice. Forproducts inbulk or in ampoules, extractionmight bedone in a tightgas
chamberby remoteoperation.Water,oxygen, light,and contaminantsareall important threatsand
mustbemonitoredandcontrolled.
Stabilizationisamatterofimportanceandmustbecontrolledparticularlyinfreezedryingandstorage
steps. While freezedrying has a long history in the pharmaceutical industry as a technique for
stabilizationoflabiledrugs,includingproteins,manyproteinssufferirreversiblechange,ordegradation,
duringthefreezedryingprocess.Evenwhenthelabiledrugsurvivesthefreezedryingprocesswithout
degradation,theresultingproductisrarelyfoundperfectlystableduringlongtermstorage,particularly
when analytical techniques with a sensitivity to detect low levels of degradation (around 0.1%) are
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employed.Bothsmallmoleculesandproteinsshowdegradationduringstorageofthefreezedriedglass.
Insomecases,instabilityisseriousenoughtorequirerefrigeratedstorage.
So, ultimate storage has to be done according to the specific sensitivities of the products. Again,
uncontrolledexposurestowatervapor,oxygen(air),light,excessheat,ornonsterileenvironmentare
major factors to be considered. Stability problems are most often addressed by a combination of
formulationoptimizationandattentiontoprocesscontrol.Lyoprotectantsareaddedforstabilityduring
the freezedrying process as well as to provide storage stability, and the level and type of buffer is
optimized. Finally, it is important toanalyze the compositionand quality of the container itself, i.e.,
glass,elastomersofthestoppers,plasticororganicmembranes(Rey,2004,Swarbrick,2007).
2.2.4 StabilityTests,QualityControlandQuarantine
2.2.4.1 StabilityTestsThe stabilityindicatingprofile for abiotechnological productgenerally comprises information from a
batteryofassaysandnotfromasinglestabilityindicatingassay.Theexpiry/expirationdateistheactual
dateplacedonthecontainer/labels ofadrugproductdesignatingthetimeduringwhichabatchofthe
drugproduct isexpectedtoremainwithintheapprovedshelflifespecification ifstoredunderdefined
conditionsandafterwhichitmustnotbeused.Toarriveatanexpirationdate, itmustbedetermined
first forhow longandunderwhatconditionsapharmaceutical formulationcanmeetallof itsquality
specifications. In general, this issue isanswered through stability testing that monitors chemical and
physical product attributes as a function of time, temperature, and other environmental factors. To
supporttheexpirationdating,thestabilityofthedrugproductanddrugsubstancemustbeassessedby
methodsthathavebeenvalidatedandaredetailedinaprotocolspecifictothatproduct.Thisprotocol
includesthetestingintervalsandthespecificationsthattheproductmustmeet.
Particularly in thiscase, thisstep is important inorder tominimizedegradationof theprotein in the
formulationduringstorage.So,theFoodandDrugAdministration (FDA)andotherregulatoryagencies
require that the purity and potency of pharmaceuticals are monitored during the shelf life of the
products.Achieving theserequirements involvesusingacombinationofanalytical techniquessuchas
chromatography,electrophoresis, andspectroscopyamongothers.
Becauseproteinsarecapableofdenaturingviaseveralmechanisms, it isnecessarytousemore than
onetechniquetodemonstratestability(Walsh,2003).
2.2.4.2 QualityControlThe final product must undergo a quality control testing in order to confirm their conformance to
predeterminedspecifications. Forexample,potencytestingisofobviousimportance,ensuringthatthe
drugwillbeefficaciouswhenadministeredtothepatient.Otherprominentaspectissafetytestingthat
entailsanalysisofproductforthepresenceofvariouspotentialcontaminants(Walsh,2003).
The range and complexity of analytical testing undertaken for recombinant biopharmaceuticals far
outweighs that undertaken with regard to traditional pharmaceuticals manufactured by organic
synthesis.Notonlyareproteinsoradditionalbiopharmaceuticals, suchasnucleicacids,muchlargerand
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structurallycomplexthantraditional lowmolecularmassdrugs,butalsotheirproduction inbiological
systemsrenderstherangeofpotentialcontaminantsfarbroader.
Recent advances in analytical techniques render practical the routine analysis of complex
biopharmaceuticalproducts(Walsh,2003).Inthefollowingparagraphsadescriptionisprovidedofthe
usualdetectionmethodsusedforthequalitycontrolofproteinbasedfinishedproducts.
Bioassaysrepresentthemostrelevantpotencydeterminingassay,astheydirectlyassessthebiological
activityofthebiopharmaceutical. Itinvolvesapplyingaknownquantityofthesubstancetobeassayed
toabiologicalsystem that responds insomeway to thisappliedstimulus.The response ismeasured
quantitatively,allowinganactivityvaluetobeassignedtothesubstancebeingassayed.
Anexampleofa straightforward bioassay is the traditionalassaymethod forantibiotics.Thisusually
involved measuring the zone of inhibition of microbial growth around an antibioticcontaining disc,
placedonanagarplate seededwith the testmicrobe.Formodernbiopharmaceuticals bioassaysare
generallymorecomplex,sincethebiologicalsystemusedcanbewholeanimals,specificorgansortissue
types,orevenindividualmammaliancellsinculture.
Allbioassaysarecomparativeinnature,requiringparallelassayofastandardpreparationagainstwhich
the sample will be compared. Internationally accepted standard preparations of most
biopharmaceuticals areavailable fromorganizationssuchastheWorldHealthOrganization (WHO)or
theUnitedStatesPharmacopeia(USP).
Quantificationoftotalproteininthefinalproductrepresentsanotherstandardanalysisundertakenby
qualitycontrol,whereanumberofdifferentproteinassaysmaybepotentiallyemployed.Thesimplest
of suchmethods isperhaps the detectionandquantification ofprotein bymeasuringabsorbency at
280nm,basedonthefactthatthesidechainsoftheaminoacidstyrosineandtryptophanabsorbatthis
wavelength.Thismethodispopular,asitisfast,easytoperformandisnondestructivetothesample.
However,itisarelativelyinsensitivetechnique,andidenticalconcentrationsofdifferentproteinsyield
differentabsorbancevalues if theircontentoftyrosineandtryptophanvary toanysignificantextent.
Hence,thismethodisrarelyusedtodeterminetheproteinconcentrationofthefinalproduct,butitis
routinelyusedduringdownstreamprocessing todetectproteinelutionoffchromatographic columns,
andhencetrackthepurificationprocess.
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDSPAGE) represents the most
widely used analytical technique in biochemistry, forensics, genetics and molecular biology for the
assessmentof finalproductpurity.This technique iswellestablishedandeasy toperform,providing
highresolution separation of polypeptides on the basis of their molecular mass or for separating
proteins according to their electrophoretic mobility, a function of length of polypeptide chain or
molecularweight.
SDSPAGEisnormallyrununderreducingconditions,wheretheadditionofareducingagentsuchas2
mercaptoethanol or dithiothreitol (DTT) disrupts interchain and/or intrachain disulfide linkages.
Individualpolypeptidesheldtogetherviadisulfidelinkagesinoligomericproteinswillthusseparatefrom
eachotheronthebasisoftheirmolecularmass.
Twodimensional gel electrophoresis, abbreviated as 2DE or 2D electrophoresis, is a form of gel
electrophoresiscommonlyusedtoanalyzeproteins.It isnormallyrunsothatmixturesofproteinsare
separatedfromeachotheronthebasisofadifferentmolecularpropertyineachdimension,i.e.bytwo
properties in two dimensions on 2D gels. The most commonly used method entails separation of
proteinsby isoelectric focusing in the firstdimension,withseparation in theseconddimensionbeing
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undertaken in the presence of SDS, thus promoting band separation on the basis of protein size.
Applicationofbiopharmaceutical finishedproductstosuchsystemsallowsrigorousanalysisofpurity.
Capillaryelectrophoresis(CE),alsoknownascapillaryzoneelectrophoresis(CZE)systems,canbeused
toseparateionicspeciesbytheirchargeandfrictionalforcesandmass.Thesesystemsarealsolikelyto
playan increasinglyprominentanalyticalrole inthe laboratoryqualitycontrol.Aswithotherformsof
electrophoresis, separation isbasedupondifferent ratesofproteinmigrationuponapplicationofan
electricfield.
Asthenamecapillaryelectrophoresissuggests,thisseparationoccurswithinacapillarytube,typically
withadiameterof2050manduptoa1mlong,anditisnormallycoiledtofacilitateeaseofuseand
storage.Thedimensionsofthissystemyieldgreatlyincreasesurfacearea/volumeratio,comparedwith
slabgels,andhencetheefficiencyofheatdissipationfromthesystem.Thisinturn,allowsoperationat
ahighercurrentdensity,thusspeedinguptherateofmigrationthroughthecapillary.Sampleanalysis
can be undertaken in 1530 min and online detection at the end of the column allows automatic
detectionandquantificationofelutingbands.
Highperformance liquid chromatography (HPLC) occupies a central analytical role in assessing the
purityoflowmolecularmasspharmaceutical substances.Italsoplaysanincreasinglyimportantrolein
macromoleculesanalysis, suchasproteins. Mostof thechromatographic strategiesused to separate
proteinsunderlowpressure(e.g.gelfiltration,ionexchange,etc.)canbeadaptedtooperateunderhigh
pressure. Reversephase, sizeexclusion and, to a lesser extent, ionexchangebased HPLC
chromatography systemscanbeusedintheanalysisofarangeofbiopharmaceuticalpreparations.
Electrosprayionization(ESI)isatechniqueusedinmassspectrometrytoproduceionsfrombiological
macromolecules, anda veryuseful technique for theiranalysis, since itovercomes thepropensityof
thesemolecules to fragmentwhen ionized.ESIallowsone todetermine themolecularmassofmany
proteinstowithinanaccuracyof0.01percent.Aproteinvariantmissingasingleaminoacidresiduecan
easily be distinguished from the native protein in many instances. Although this is a very powerful
technique,analysisoftheresultsobtainedcansometimesbelessthanstraightforward.
Immunologicalapproachestodetectionofcontaminantsorimmunoassaysarebiochemicalteststhat
measure theconcentrationofa substance inabiological liquid,using the reactionofanantibodyor
antibodiestoitsantigen.
Thestrategyusuallyemployedtodevelopsuchimmunoassaysistermedtheblankrunapproach.This
entailsconstructingahostcellidenticalinallaspectstothenaturalproducercell,exceptthatitlacksthe
genecodingforthedesiredproduct.Thisblankproducercellisthensubjectedtoupstreamprocessing
procedures identical to those undertaken with the normal producer cell. Cellular extracts are
subsequently subjected to the normal product purification process, but only to a stage immediately
priortothefinalpurificationsteps.Thisproducesanarrayofproteinsthatcouldcopurifywiththefinal
product.Therefore,polyclonalantibodypreparationscapableofbinding specifically to theseproteins
areproduced.Purificationoftheantibodiesallowstheirincorporationinradioimmunoassay orenzyme
based immunoassay systems, which may subsequently be used to probe the product. Such multi
antigenassaysystemswilldetectthetotalsumofhostcellderivedimpuritiespresentintheproduct.
Immunoassayshavefoundwidespreadapplication indetectingandquantifyingproduct impurities.For
example, an immunoassay may be conveniently used to detect and quantify nonproductrelated
impurities in a final preparation. Generally immunoassays may not be used to determine levels of
productrelated impurities,asantibodies raisedagainst such impuritieswouldalmost certainly cross
react with the product itself. Immunoassays identifying a single potential contaminant can also be
developed.Theseassaysareextremelyspecificandverysensitive,oftendetectingtargetantigendown
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topartspermillionlevels.Manyimmunoassaysarecommerciallyavailableandtherearecompaniesfor
rapidlydevelopingtailormadeimmunoassaysystemsforbiopharmaceuticalanalysis.
Aminoacidanalysisremainsacharacterization techniqueundertakeninmanylaboratories,inparticular
iftheproduct isapeptideorsmallpolypeptide.This isasimplestrategytodetermine therangeand
quantity of aminoacids present in the final product and to compare the results obtained with the
expectedtheoreticalvalues.Thustheresultsshouldbecomparable.
Peptidemapping(orfingerprint)isapowerfulidentitytestforproteins,especiallythoseobtainedbyr
DNA technology, capable of detecting whether alterations in protein structure have occurred, and
demonstratesprocessconsistencyandgeneticstability.
Fullsequencingofa sampleofeachbatchof theprotein is theonlyprocedureguaranteed todetect
alterations in gene transcription or translation. This potential occurrence of point mutations in the
products gene is a major concern relating to biopharmaceuticals produced in highexpression
recombinantsystems,sinceitleadstoanalteredprimarystructurei.e.aminoacidsequence.
Peptidemappinginvolvesthechemicalorenzymatictreatmentofaproteinresultingintheformationof
peptidefragments,forexampleexposureoftheproteinproducttoareagentthatpromoteshydrolysis
of peptide bonds at specific points along the protein backbone. This generates a series of peptide
fragmentsthatcanbeseparatedandidentifiedfromeachotherinareproduciblemannerbyavarietyof
techniques,includingone ortwodimensionalelectrophoresis, andRPHPLCinparticular.
Astandardizedsampleoftheproteinproductwhensubjectedtothisprocedurewillyieldcharacteristic
peptide fingerprint, or map, with which the peptide maps obtained with each batch of product can
subsequently be compared. Thus, the information obtained in this test is compared to a Reference
StandardorReferenceMaterialsimilarlytreatedthatconfirmstheprimarystructureoftheprotein.This
comparison is possible since each protein presents unique characteristics which must be well
understoodsothatthescientificandanalyticalapproachespermitvalidateddevelopmentofapeptide
mapthatprovidessufficientspecificity.Ifthepeptidesgeneratedarerelativelyshort,thenachangeina
singleaminoacidresidue is likelytoalterthepeptidesphysicochemical propertiessufficientlytoalter
itspositionwithinthepeptidemap.Inthisway,single(ormultiple)aminoacidsubstitutions,deletions,
insertionsormodificationscanusuallybedetected.
Nterminal sequencing (also called Edman sequencing) of the first 2030aminoacid residuesof the
protein product became a popular quality control test for finished bi