D2 1_LCA Tool Adaptation to Pharmaceutical Processes

8/2/2019 D2 1_LCA Tool Adaptation to Pharmaceutical Processes

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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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LCAToolAdaptationtoPharmaceutical Processes

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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


plasticpackaging


glasspackaging



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14


Preparationand

Material

Recuperation

ACSconsumption

AFSconsumption




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




WFIconsumption

CompressedAir

O2

CO2

N2Excipients


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15

Timberpackaging,cardboard,

Paper

Plasticpackaging

scrappackstetrabrickand

isothermal

glasspackaging

piecesofsteel


Fermentation

ElectricalEnergy

Containerforbiologicalproducts

ACSconsumption

AFSconsumption



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



WFIconsumption

Auxiliarymaterial


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


plasticpackaging


glasspackaging

packagesorpiecesofsteelpackagesorpiecesofaluminium


AFS

Compressedair


Inspection,controland

quarantine

Electrical Energy


plasticpackaging


glasspackaging



Packaging

Electrical Energy


plasticpackaging


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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20

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




WFIconsumption


plasticpackaging


glasspackaging



Materialreceptionandstorage

conditioning

ElectricalEnergy

ACSconsumption

AFSconsumption




WFIconsumption


plasticpackaging

scrap packstetrabrickandisothermal

glasspackaging



Washingandconditioning

materialpreparation

ElectricalEnergy

ACSconsumption

AFSconsumption




WFIconsumption

Aircompressed

Preparationof

the

buffer

solution:Weighting,ElectricalEnergyACSconsumption


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23

FormulationandSterile

Filtration

AFSconsumption


pure steamconsumption


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



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

Documents

D2 1_LCA Tool Adaptation to Pharmaceutical Processes