D2 1 - LCA Tool Adaptation To Pharmaceutical Processes

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D2.1LCATOOLADAPTATIONTOPHARMACEUTICALPROCESSES
INDEX
Summary......................................................................................................................................................3
1 Introduction.........................................................................................................................................4

LCATool
Adaptationto
Pharmaceutical
Processes

ManualtouseintheCluster
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
LCAToolAdaptationtoPharmaceuticalProcesses
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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 ProductConcentrationandChromatographicPurification..................................................16
2.1.4 FilterSterilizationandAPIConditioning...............................................................................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
EcoInnovationofPharmaceuticalBuildingsSupportinginSustainableLCATools(ECO/08/239082),has
among other objectives to identify, describe and present a detailed description of the PRAXIS
Pharmaceutical S.A. production processes. The production processes of this pharmaceutical company
will be used as a case study for the development, within this project, of an opensource LCA tool that
canultimatelybeusedbyeachpharmaceuticalcompanytoanalyzetheirprocesses.ThisLCAtoolcanbe
used to perform an inputoutput analysis of pharmaceutical processes, to evaluate their potential
environmental impacts, to perform a sustainability assessment, and also to identify opportunities for
improvement.
Therefore,thisreportisstructuredintwochapters.Thefirstchapterisasmallintroduction,describing
themotivationforthecreationoftheLCAtoolandasummaryoftheLCAmethodology,accordingtothe
ISO 14040 (2006). The second chapter describes the production processes of lyophilized products via
recombinant biotechnology, obtained from open literature, which are similar to the ones used by
PRAXIS pharmaceutical. This description is an important step in the understanding of the production
processesinvolvedinordertoallowfortheidentificationoftherelevantinputsandoutputsofmaterials
and energy that enter and exit each manufacturing process. For each stage of the primary and
secondary processing, some of the main inputs and outputs are identified, which need to be further
refined and completed in order to be used in the LCAtool development. The inventory data obtained
from PRAXIS can be later used, during the development of the LCA tool, in order to be presented as a
casestudywithquantifieddata.

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1 Introduction
1.1 BACKGROUNDANDMOTIVATION
LifeCycleAssessment(LCA)isamethodologythatcanbeusedtoperformasystematicevaluationofthe
potentialenvironmentalimpactsofamaterial,product,process,activityorserviceacrossallstagesofits
life cycle, from raw material acquisition, product manufacturing, 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
(EnvironmentalmanagementLifecycleassessmentPrinciplesandframework)andISO14044:2006
(EnvironmentalmanagementLifecycleassessmentRequirementsandguidelines).
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
limitsforpollutantemissionsdefinedinexistingenvironmentalregulationsandlaws;
Rapidlyrespondtoanyenvironmentalissue,e.g.setupbyanewenvironmentalstandard;
Obtain precise data that may be used for ecodesign purposes and also, to report reliable
environmentaldata;
Informthepublicabouttheenvironmentalaspectsofanorganizationproductsandprocesses,
inordertobuildaresponsiblecorporateenvironmentalimage.
FewstudiesarepublishedconcerningLCAofpharmaceuticalprocesses.Forexample,JimnezGonzlez
et al. (2004) performed an LCA study for analyzing and identifying the cradletogate environmental
impacts of a typical Active Pharmaceutical Ingredient (API) synthesis, concluding that solvent use
accountsforthemajorityofthepotentialenvironmentalimpacts.
Ponder and Overcash (2010) performed a LCA study of the vancomycin hydrochloride production in a
lowyield fermentation process. Results show that, on a cradletogate perspective, 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
electrical energy. As an environmental measure of the process, these authors determined the process
mass intensity (PMI), calculated as the total gatetogate mass of raw materials per mass of product,
whichmayideallyapproachesthevalueofoneifnowastesaregenerated.
Wernet et al. (2010) performed a LCA of an API production from cradletofactory gate and concluded
that pharmaceutical production is significantly more complex than basic chemical production. Energy
useisdirectlyorindirectly,thecauseofthemajority(andsometimesupto85%)oftheimpacts.These
authors also concluded that the emissions from the chemical processes and transport are minor
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contributors to the overall impacts. Furthermore, this analysis suggests that the most effective way of
increasing these processes sustainability is by optimizing material and energy efficiency. Therefore,
although API are normally produced and used in lower quantities, their lower contribution in mass to
productsshouldnotleadLCApractitionerstoneglecttheirimportance.
Kimetal.(2009)performedanLCAofthreesupportedenzymesforpharmaceuticalapplicationsFLASC
using the life cycle inventory database developed by GlaxoSmithKline. They concluded that production
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)performedalimitedLCAofpharmaceuticalproductsconcludingthatitistheportionofthe
active ingredient in the final product that determines the ecological consequences for activities down
the supply chain. Also, the major energy requirements and environmental impacts of the active
ingredientoverthelifecycleareattributedtotheproductionstage.
Also,somesoftwaretoolsalreadyexisttoperformLCAstudies,evaluatingthepotentialenvironmental
impacts, and others, to assess process potential risks and hazards, or the environmental, health and
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 was developed an LCA tool for pharmaceutical based on a state of the art
aboutavailableenvironmentaltechnologiesforpharmaceuticalproducts(Ecoprofarma,2008)
FLASCFastLifecycleAssessmentofSyntheticChemistry,developedbyGlaxoSmithKline(GSK),is
a webbased tool and methodology designed to evaluate the life cycle environmental impacts
associatedwiththemanufactureofmaterialsusedinatypicalpharmaceuticalprocess(Curzonset
al.,2007).
Sabento Software for the Assessment of Biotechnology Processes. It can be used to
modelbiotechnical production processes and process development. Also, it allows a process
designertoperformeconomicandecologicalassessmentsofprocessalternatives.Thissoftwarecan
beorderedat:http://www.sabento.com/en/
SimaProLCAsoftwaredevelopedbyPRConsultants,includesalargedatabasefortheinventory
andenvironmentalanalysesofseveralprocesses.Itcanbeorderedat:http://www.pre.nl/simapro/
KCLECOLCAsoftware&KCLEcoDatadevelopedbyKCLpulpandpaperResearchCompany,can
beappliedindifferentindustrialsectors.
BEESBuildingforEnvironmentalandEconomicSustainabilitydevelopedbytheBuildingandFire
ResearchLaboratory.
OpenLCA is a modular software for life cycle analysis and sustainability assessments developed by
by 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.
EcosolventLCAtooldevelopedbytheInstituteforChemicalandBioengineeringofETHZurich,for
quantifying the potential environmental impact of wastesolvent treatment. It is available at
http://www.sustchem.ethz.ch/tools/ecosolvent/.
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EHSassessmenttooldevelopedbytheSafetyandEnvironmentalTechnologyGroup,ofETHZurich,
forassessingchemicalsandsolventsbasedonEHScriteria.Thistoolisdemonstratedon26organic
solventsofcommonusewithinthechemicalindustry.Thesubstancesareassessedbasedontheir
performance in nine categories: Release potential, chronic toxicity, fire or explosion, persistency,
reaction or decomposition, air hazard, acute toxicity, water hazard, irritation. It is available at
http://www.sustchem.ethz.ch/tools/ehs/.
HAZOPExpertHAZOPsoftwaredevelopedbytheLaboratoryforIntelligentProcessSystems(LIPS),
School of Chemical Engineering of Purdue University, to analyze different processes for their risks
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, some are not user friendly, the licensing fees may be high and the results
obtainedareoftennotclearforusersinSME(SmallandMediumEnterprises)andforthegeneralpublic.
Also,thesetoolsaremuchtimeconsuming,complicatedandnormallydonotaddressconcernsofsocial
sustainability or of the process economics. Although FLASC is specifically oriented to develop a fast,
streamlined LCA for a wide range of materials commonly used in pharmaceutical products it is only
availabletoGSKscientistsandengineersandaccessibleviatheintranetsiteofthiscompany.
Forthesereasons,thereisastronginterestindevelopinganopensourceLCAtool,easytohandleand
interpret, that can be used by pharmaceutical companies to perform an inputoutput analysis of their
processes, products and materials lifecycle, helping them to meet current environmental legislations
andtheirenvironmentalreportingneeds.
Moreover,theinformationdevelopedinanLCAorLCI(lifecycleinventory)studycanbeusedaspartof
a much more comprehensive decision making process. Furthermore, to achieve the environmental
certificationorecolabelforapharmaceuticalproduct,theirmanufactureandtransformationneedsto
complywithspecialenvironmentalconditionsbasedonalifecycleassessment.
Especially concerning pharmaceutical products, the current European Regulations for Ecologic label do
not take into consideration medicines, health products and others dangerous or toxic products. 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
companies already recognized that one way to grow their bottom lines is by integrating sustainability
performance. Nevertheless, currently no standardized methods exist for integrating, measuring, or
communicatingsustainabilitybypharmaceuticalcompanies.Moreover,Schneideretal.(2010)analyzed
theevolutionofsustainabilityreportinginthepharmaceuticalsector,concludingthatithasincreasedin
breadthanddepth,buthavenowshiftedmoretowardscorporatesocialresponsibility,whichreflectthe
companiesneedtosatisfypublicopinion.Inotherhand,Geibleretal.(2006)inferthatsincethesocial
dimension of sustainability has an intangible and qualitative nature there is a lack of consensus about
whataretherelevantcriteriaforcompaniestoaccountforit.
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Onewayofevaluatingthesustainabilityofpharmaceuticalprocessesisbyusingsustainabilitymetricsor
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 social
sustainabilityinbiotechnologicalprocessesandproductdevelopment.Also,theseauthorsidentified
eightaspectswithsignificantrelevancetothesocialimpactassessmentofthebiotechnologysector:
health and safety, quality of working conditions, impact on employment, education and training,
knowledge management, innovation potential, customer acceptance and societal product benefit,
and social dialogue. For each one of these aspects these authors proposed typical indicators to
assessthem.
ALCHESustainabilityMetrics:TheSustainabilityMetricsworkinggroupofTheAmericanInstituteof
ChemicalEngineers(AICHE)hasdevelopedasetofbaselinesustainabilitymetricsforcompaniesto
measure environmental impacts and has an active project evaluating and testing these metrics in
industry(http://www.aiche.org/cwrt).
IChemESustainabilityMetrics:SustainableDevelopmentProgressMetricsRecommendedforUsein
the Process Industry. IChemE extended sustainability metrics to include measures of the potential
impacts of emissions, effluents and wastes. Thus, this indicators system can be used to evaluate
environmental,economicandsocialconcernsofaprocessunit(IChemE,2002).
Dow Jones Sustainability Index (DJSI) established in 1999 by Sustainable Asset Management
(SAM), this was the first index attempting to assess the ability of businesses to create longterm
shareholder value by embracing opportunities and managing risks deriving from economic,
environmentalandsocialdevelopments(http://www.sustainabilityindex.com/).
SustainableProcessIndex(SPI)developedin1995,SPImeasuresthepotentialimpact(pressure)of
processesormoregenerallyactivitiesontheecosphere.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).
BRIDGESBasicSustainabilityMetricsIntroducedin2002thismetricssystemtoassessproduction
processesbymeasuringthefollowingsetofindicatorsbyunitofoutput(massofproduct,orsales
revenue, or value added): material intensity, water intensity, energy intensity, toxic releases, solid
wastes,pollutanteffects.ExamplesofComplementaryMetricsarealsogiven(Schwarzetal.,2002;
TanzilandBeloff,2006).
ThreeDimensional(3D)SustainabilityMetricsproposedbyMartinsetal.(2007)forevaluatingthe
sustainability of industrial processes and comparing them with alterative production methods. A
three dimensional sustainability framework is also addressed by these authors that proposed 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 and
processes. 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
most frequently used hazard evaluation index. It is used to determine safety risks associated with
fireandexplosion,todeterminetheareasofgreatestlosspotentialinaparticularprocess,andto
predictthephysicaldamagethatwouldoccurintheeventofanincident(Sinnott,2005).
InherentSafetyIndex(ISI)thisindexsystem,suggestedbyHeikkila(1999),addressesthechemical
andprocesssafetyofachemicalplant.Itiscalculatedbasedonsubindicesconcerningthechemical
safety (e.g. chemical reactivity, flammability, explosiveness, toxicity and corrosiveness of chemical
substancespresentintheprocess)andtheprocesssafety(e.g.processtemperatureandpressure,
equipmentsafetyandsafeprocessstructure).
Sustainability Indicators and Indices An indicators system proposed by Tugnoli et al. (2008) to
addressthethreeaspectsofsustainabilityduringearlystagesofprocessdesign.Itcabbeusedfor
comparingdesignalternativesandforanalyzingtheenvironmental,economicandsocialimpactsof
aprocess.
Global Environmental RiskAssessment (GERA) Index proposed byAchour et al.(2005), this index
systemassessestheenvironmentalriskofindustrialprocesses.
Metrics to green chemistry aiming to drive pharmaceutical processes towards more sustainable
practices. Constable et al. (2002) explores several metrics commonly used by chemists, compares
and contrasts these metrics with a new metric known as reaction mass efficiency to assess the
massefficiencyofachemicalsynthesis.
Sustainability Indicators for assessing energy systems an indicators system to assess the
sustainability of energy systems with a focus on resources, environment, societal, and production
efficiency(Afganetal.,2000).
GlobalReportingInitiative(GRI)indicatorstheSustainabilityReportingGuidelineshavebecomea
standardforsustainabilityreportingduetothelackofaformalglobalconsensusonmeasurement
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).
UNCTADenvironmentalandfinancialperformanceindicatorsUNCTADandtheIntergovernmental
WorkingGroupofExpertsonInternationalStandardsofAccountingandReporting(ISAR)prepared
amanualthatpresentsamethodbywhichenvironmentalandfinancialperformanceindicatorscan
be used together to measure an enterprise's progress in attaining ecoefficiency or sustainability
(UNCTAD,2004)
NRTEE ecoefficiency indicators The Canadas National Round Table on the Environment and the
Economy (NRTEE) conducted one of the earliest studies on the development of sustainability
metrics. Its search for a small set of ecoefficiency indicators that is meaningful and applicable by
companies from different industrial sectors. The study (NRTEE, 1999) recommended a set of core
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 Sustainable
Development (WBCSD) for companies pursuing ecoefficient strategies to reduce their impact on
theenvironmentwhileincreasingoratleastnotdecreasing(shareholder)value.TheWBCSDstates
thatecoefficiencyisachievedbythedeliveryofcompetitivelypricedgoodsandservicesthatsatisfy
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humanneedsandbringqualityoflife,whileprogressivelyreducingecologicalimpactsandresource
intensity(Schmidheiny,1992).
BITC social and environmental indicators indicators to help companies report social and
environmental impact effectively, proposed by the Business Impact Review Group (BIRG) of
BusinessintheCommunity(BITC),amovementofover700oftheUKstopcompaniescommitted
toimprovetheirpositiveimpactonsociety(BITC,2003).
PERFORMindicatorsetincludesabout30indicatorsapplicabletoallindustrialsectorsandasmall
number 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
indicators set was developed under the PERFORM project launched by researchers of Science and
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,someofwhichfromthepharmaceuticalindustry.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,
10
EarningsperShare
CashFlow
Wastewater
SolidWaste
EnvironmentalProtection
AccidentsperWorkingHours,
TotalNumberofApprentices,
FrequencyofEHSTraining
Veleva et al. (2003) applied the fivelevel indicator hierarchy developed at the Lowell Center for
SustainableProductiontoanalyzetheenvironmentalreportingofsixpharmaceuticalcompanies.These
authorsconcludedthatthemajorityofindicatorsaddressperformanceorecoefficiency(Level2),some
indicatorslookatenvironmentaleffects(Level3),supplychainandproductlifecycleeffectsarestarting
tobeaddressed(Level4),andnocompaniesareaddressingcarryingcapacityissues(Level5).
Henderson et al. (2008) used a life cycle approach and sustainability metrics to compare the
performance, and the environment, 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.
Ho et al. (2010) analyzed the environmental impacts of protein manufacturing concluding that energy
useandwaterusearethemaincontributorstotheenvironmentalimpactsofsuchoperationsinclean
roomspaces.
BlumKusterer and Hussain (2001) analyzed the main drivers for sustainability improvements in the
Pharmaceuticals Industry concluding that regulation followed by implementation of new technologies
arethemajordriversforprocesschange.
JimnezGonzales and Overcash (2000) evaluated the energy use and related emissions during the
severalstagesofpharmaceuticalsproduction,concludingthatabout70%energyreductionispossibleby
optimizingtheenergyusagesysteminthepilotscalestageduringprocessdevelopments.
JimnezGonzalesetal.(2002)explaintheconceptoftheGreenTechnologyGuide,whichisaseriesof
casescenario comparisons that provide scientists and engineers with comparative environmental and
safety information on technologies for operations commonly found in the pharmaceutical industry.
Technologies are compared using indicators based on unit operation analysis and lifecycle concepts.
These authors propose indicators in four categories: Environment, Safety, Efficiency, and Energy. For
example environmental Indicators are: Mass intensity, Solvent intensity, Waste intensity, Emissions of
compoundsreleased.
ThefollowingindicatorsareproposedintheLCAtooldevelopedunderthisprojectscope,forassessing
pharmaceuticalsprocesses,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(kgCO
2
eq/vialorMJ/API)
abioticdepletion(expressedinantimony
equivalent/vialorMJ/API)
abioticdepletion(expressedinkWh/vialor
MJ/API)
globalwarming(kgCO
2
eq./vialorMJ/API)
ozonelayerdepletion(kgCFC11eq./vialor
MJ/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(kgSO
2
eq./vialorMJ/API)
eutrophication(kgPO
4
eq./vialorMJ/API)
Numberofindirectfull
timejobs
Numberofworkingdays
perpersonperyear
Numberofaccidentsper
workinghours
OperatingProfit
InvestmentinR&D
CashFlow
ExpenditureonEHS
1.2 STUDYOBJECTIVES
TheLCAtooldevelopedforpharmaceuticalcompaniesisbasedonMicrosoftOfficeExcel.Itallowsthe
input of the inventory data and automatically calculates the potential environmental impacts, such as
global warming, acidification, eutrophication, ozone depletion, photochemical oxidation, human
toxicity, aquatic ecotoxicity, and terrestrial ecotoxicity. The LCA tool should facilitate results
interpretation, both for organizational or scientific use, concerning the following three main types of
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 impacts and
sustainability will be evaluated. The technologies to be identified and assessed include the ones listed
below,whichhappenstobetheonescurrentlybeingusedbyPRAXIS:
APIProduction:
o Recombinantbiotechnologytechniques;
o Fermentationtechnologies;
o Formulationandfiltrationsystems;
o Sterilizationsystems(e.g.autoclaving,depyrogenation,radiation,chemical);
MedicineProduction
o 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 ProductionofLyophilizedProductsviaRecombinantBiotechnology
Pharmaceutical production is usually divided into two main production stages: API and Medicine
Production. The former is related to the production of the active pharmaceutical ingredient (API) and
thesecondoneisrelatedtotheconversionoftheAPIintothefinalsdrugpresentationform.Thesetwo
stages are presented and divided into several processing steps that will be described in the following
sections.
2.1 APIPRODUCTION
Microbialcellfermentationhasalonghistoryofuseintheproductionofvariousbiologicalproductsof
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. The rDNA process, sometimes called genetic engineering, is a serial process whereby (i) a
proteinisassociatedwithabiologicactionandisidentified;(ii)aspecifichumangene(aDNAsequence)
is associated with the protein; (iii) the human gene is inserted into bacterial DNA (plasmid); (iv) the
plasmid is placed into the nonhuman host cells (e.g. Escherichia coli bacteria); and (v) the host cells
manufacture their typical variety of proteins and produce a human protein from the human gene. In
fact, over half of all biopharmaceuticals thus far approved are produced by recombinant E. coli or S.
cerevisiae (the most common hosts). As a result, a wealth of technical data and experience has been
accumulatedinthisarea(Walsh,2003,Swarbrick,2005).
FigureshowsatypicalflowdiagramofaprimaryprocessingviarecombinantbiotechnologyfortheAPI
manufacture.
Figure13.Primaryprocessingviarecombinantbiotechnology.
13
2.1.1 PreparationofRawMaterialsforFermentation
Theculturemediumcompositionandthefermentationconditionsrequiredtopromoteanoptimalcell
growth and/or product manufacture needs to be established during the initial product development.
Afterthat,routinebatchproductionisahighlyrepetitiveandahighlyautomatedprocess.
Heatlabileingredientscanbesterilizedbyfiltrationandaddedtothefermenteraftertheheatingstep.
Media composition can vary from a simple defined culture media (usually glucose and some mineral
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 product
production;
Processeconomics(totalmediacost);
Extracellular or intracellularnatureof theproduct. If the biopharmaceutical is an extracellular
product, then the less complex the media composition, the better in order to render
subsequentproductpurificationasstraightforwardaspossible.
Typically, the batch manufacture of a biopharmaceutical product entails filling the production vessel
with the appropriate quantity of waterforinjection (WFI). Heatstablenutrients required for producer
cellgrowtharethenaddedandtheresultantmediaissterilizedinsitu.Thiscanbeachievedbyheatand
manyfermentershaveinbuiltheatingelementsor,alternatively,outerjacketsthroughwhichsteamcan
bepassedinordertoheatthevesselcontents.
Table1showsthemaininputsandoutputsfortheinventoryanalysisrelatedtothepreparationofthe
culturemediumusedforthefermentationprocess(PRAXIS,2009).
Table1Inventoryanalysisofthepreparationoftheculturemediumforthefermentationprocess(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
14
PreparationandMaterialRecuperation
ACSconsumption
AFSconsumption
WFIconsumption
ElectricalEnergy
CompressedAir
2.1.2 InoculationandFermentation
Theupstreamprocessingelementofthebatchmanufactureofabiopharmaceuticalproductstartswith
theremovalofasingleampouleoftheworkingcellbank.Thisvialisusedtoinoculateasmallvolumeof
sterile growth medium. This step entails the use of daughter cells from the new host cell (master
workingcellbank),actuallyremovedfromitsstorageina70Cfreezer.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
enhance cell viability (lifespan) in vitro and functional ability of cells to produce proteins. This starter
culture is in turn used to inoculate a productionscale starter culture, which is used to inoculate the
productionscale bioreactor (Walsh, 2003). It is here that fermentation occurs, with cells from the
inoculumphaseandaddingtheappropriatefortifiedgrowthmedia.Inabatchconfiguration,hostcells
thatcontainanexpressionvectorfortherecombinantproductareaddedtoapredeterminedvolumeof
growth medium. The cells are allowed to grow until the nutrients in the medium are depleted or the
excretedbyproductsreach inhibitorylevels.Meanwhile,theywillproceedtoproduceproteins(inthis
particularcaseextracellularly)intothemedia.Feedingofthehostcellsandremovingofwastefromthe
media need to be done periodically to sustain host viability and productivity. Fermentation follows for
several days subsequent to inoculation with the productionscale starter culture. During this process,
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).
Table 2 shows the main inputs and outputs for the inventory analysis related to the inoculation and
fermentationprocesses(PRAXIS,2009).
Table2Inventorydatafortheinoculationandfermentationprocesses(PRAXIS,2009)
Inoculation
ElectricalEnergy
ACSconsumption
AFSconsumption
WFIconsumption
CompressedAir
O2
CO2
N2
Excipients
15
Timberpackaging,cardboard,
Paper
Plasticpackaging
scrappackstetrabrickand
isothermal
glasspackaging
piecesofsteel
Fermentation
ElectricalEnergy
Containerforbiologicalproducts
ACSconsumption
AFSconsumption
purifiedwaterconsumption
WFIconsumption
Excipients
Compressedair
Auxiliarymaterial
Industrialscale bacterial and yeast fermentation systems are used and share many common features.
Forexample,fermentationorbioreactionvesselsaregenerallymanufacturedfromhighgradestainless
steel, and can vary in size from a few tens of liters to several tens of thousands of liters. An impeller,
driven by an external motor, serves to ensure even distribution of nutrients and cells in the tank. The
baffles(stainlesssteelplatesattachedtothesidewalls)servetoenhanceimpellermixingbypreventing
vortex formation. During fermentation, air (sterilized by 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 than heating is required in some cases. Large scale fermentations, in which cells grow
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 vessel jacket 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 isolation step follows immediately after harvesting of the raw materials. In the isolation step, the
cruderawmaterialisrefinedintoaclarifiedfeedstreamthatisanintermediateprocessfreefromcells
andotherparticulatematter.Anumberofdifferentmethodsmaybeemployedduringtheisolationstep
such as filtration, gravity separation, centrifugation, flocculation, evaporation. The unit operations
employedintheisolationstepdependverymuchontheinitialrawmaterial(JornitzandMeltzer,2007).
In the purification phase, the aim is to prepare a pure biopharmaceutical product from the clarified
feed.Thisisthemostchallengingandexpensivestepindownstreamprocessingbecauseitisnecessary
to separate the desired product from other molecules with similar properties in the fewest possible
stepswiththesimplestpurificationtechnologytoachievetherequiredpurity.
An overview of the steps normally undertaken during downstream processing will be presented in the
followingsections,sincedetailsoftheexactstepsundertakenduringthedownstreamprocessingofany
specific biopharmaceutical product are usually considered highly confidential by the manufacturer and
thusarerarelymadegenerallyavailable(Walsh,2003;RodneyandMilo,2003).
16
2.1.3 ProductConcentrationandChromatographicPurification
The following phase of downstream processing usually entails concentration of the crude protein
product. The reason for this step is because after the removal of the cells and cells debris from the
culturemediumthroughsolidliquidseparation,thefractionofrecombinantproteinintheliquidphase
usually ranges from 2% to 15%. The large volumes associated with these low concentrations make it
impracticaltoproceedtothenextpurificationstep.Indeed,thevolumeofproductcontaminantstobe
purified may far exceed the capacity for chromatographic purification techniques used to isolate the
recombinantproteinsfromothersolublecellularcontaminants.Therefore,aconcentrationstepisused
toreducethevolumeandtherebyincreasetherecombinantproteinconcentration.
The concentration step yields smaller product volumes, which are more convenient to work with and
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
traditionally been used, more specific precipitation reagents are now being tested to improve protein
purification.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
cutoff points. In practice, however, the selection of pore size is more an art than a science, especially
thechoiceofdesignandsizingconfigurationsofthefiltrationsystem.Theselectionofaproperfiltration
systemmayleadtoadditionalbenefitsintermsofanincreaseintheyieldofrecombinantproteinand
its purity. Ultrafiltration is a popular method of concentration since: (Walsh, 2003, Rodney and Milo,
2003)
Highproductrecoveryratesmaybeattained(typicallyoftheorderof99%);
Processingtimesarerapid;
Processscale ultrafiltration equipment is readily available, and running costs are relatively
modest.
After concentration, further purification is needed to increase the purity of the recombinant protein
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).
Detailed description of the theory and practice underlining chromatographic techniques go far beyond
thescopeofthistext,andarefreelyavailableinthescientificliterature.However,asimpledescription
canbegivenhereinordertounderstandthechromatographytechnique.
Chromatography can be defined as a procedure in which proteins bind differentially to solid matrix
supports or media with various functional groups to provide hydrophobic, ionexchange, and affinity
interactions.Theseinteractionstrengthsofeachcomponentwiththestationaryphaseareproportional
toitsretentiontime.
17
Among the many different chromatography formats available, the one that is usually applied in large
scaledownstreamprocessingisthecolumnbasedliquidchromatography,inwhichaliquidfeedstream
is passed over or through a porous, solid matrix or resin held in a column. The components of the
mixture become distributed by virtue of their relative affinity for the solid and liquid phases. So, the
secret here is to introduce the clarified feed stream into the column under conditions where certain
components bind strongly to the resin while others flow through. The composition of the buffer is
chosen to favor the retention or elution of specific components. By changing the composition of this
buffer, molecules that initially bind to the resin can be washed through in subsequent fractions
(SchmidtTraub, 2005).One important reference is that the chromatographic technique should provide
highcapacityandselectivityandfastkinetics.Thematrixmaterialmustwithstandmultiplepurification
cycleswithminimumlossofefficiency.Somematricesusedforindustrialscaleproteinpurificationare
indicatedinthefollowinglist(JornitzandMeltzer,2007;RodneyandMilo,2003).
Agaroseanddextrancomposite
Agaroseandpolyacrylamidecomposite
Agaroseandporouskieselguhrcomposite
Cellulose
Crosslinkedagarose
Crosslinkeddextran
Crosslinkedpolyacrylamide
Ethyleneglycolmethacrylatecopolymer
Hydroxyacrylicpolymer
Hyroxymethacrylatepolymer
Polyacrylamide
Polyacrylamideanddextrancomposite
Polystyrenedivinylbenzene
Poroussilica
Rigidorganicpolymer
Thevariouskindsofphysiochemicalinteractionsthatareusedinchromatographytoproduceselectivity
are called modes of interaction. Examples include electrostatic interactions in ionexchange or ion
chromatography, hydrophobic interactions in reversedphase and hydrophobic interaction
chromatography, and specific interactions in affinity chromatography. However, sometimes this range
of selectivity isnt enough. The reason is related to the different molecular weights of proteins,
hydrophobicity, charge, and structure over a wide range, which renders it impossible to apply a single
chromatographicseparationforcomplexproteinmixtures(JornitzandMeltzer,2007;Gad,2007).
Ingeneral,acombinationoftwotofourdifferentchromatographictechniquesisemployedinatypical
downstream processing procedure, being gel filtration and ion exchange chromatography among the
most common. Affinity chromatography is employed whenever possible, as its high biospecificity
facilitates the achievement of a very high degree of purification. Anyway, the general procedure for
adsorptive chromatography is to introduce the clarified feed stream into the column under conditions
where certain components bind strongly to the resin while others flow through (Jornitz and Meltzer,
2007;RodneyandMilo,2003).
As with most aspects of downstream processing, the operation of chromatographic systems is highly
automated and it is usually computercontrolled. While mediumsize processscale chromatographic
columns(e.g.515litres)aremanufacturedfromtoughenedglassorplastic,largerprocessingcolumns
areavailable,manufacturedfromstainlesssteel(Walsh,2003).
18
Normally, for resins already used, additional analysis is required, which doesnt happen with the new
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 cause
chemicaldegradation(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
performance even at the cost of a lower capacity than the column used for intermediate purification.
High separation performance is needed to minimize contaminant carryover into the recombinant
productforpharmaceuticaluse.(RodneyandMilo,2003)
Table 3 shows the main inputs and outputs for the inventory analysis of the chromatographic
purificationprocess(PRAXIS,2009).
Table3Inventorydataforthechromatographicpurification(PRAXIS,2009)
ChromatographicPurification
ElectricalEnergy
WFIconsumption
Auxiliarymaterial
2.1.4 FilterSterilizationandAPIConditioning
While implementation of good manufacturing practices will ensure that the product carries a low
microbial load, it will not be sterile at this stage. Ideally, sterile filtration removes unwanted particles
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, containing suspended microorganisms would be rendered free of contaminating microbes by
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
the microbiological recovery medium and incubated. Clearly this approach is only suited for products
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).
Table4InventorydataforthefiltersterilizationandconditioningoftheAPI(PRAXIS,2009)
19
FilterSterilization
ElectricalEnergy
inhibitoryagents
plasticpackaging
glasspackaging
AFS
Compressedair
Inspection,controland
quarantine
ElectricalEnergy
plasticpackaging
glasspackaging
Packaging
ElectricalEnergy
plasticpackaging
glasspackaging
AnexampleoftheinventoryanalysisresultsfortheprimaryprocessingisexpressedinTable5(PRAXIS,
2009).
Table5Exampleoftheinventoryanalysisresultsforprimaryprocessing(PRAXIS,2009)
SoilContamination EnergyCostofWasteProductionandManagement
Paper,cardboardandothercellulosic products
Plastic
DangerousWaste
WaterPollution EnergyCostofWaterTreatment
AirPollution
EnergyCostofAirPollution
CO2absorption
CO2emissionsfrom fermentationprocesses
SO2emissionsfromcombustionprocesses
NOXemissionsfromcombustionandmanufacturing
processes
CO2emissionsfromcombustionprocesses
NaturalResourcesDepletion
EnergyCostofNaturalResourcesDepletion
Energyusage(fossilfuelandelectricenergy)
Waterusage
Consumptionofcardboard andothercellulosic
products
20
Plasticinputs consumption
Ferrousmetals consumption
Lubricantagent consumption
BeforethetransformationoftheAPIintothemedicine,theAPIhastotravelbyplainforalongdistance.
Itwasconsideredthattheconsumesduetothattripareimportantfortheoverallimpact,sotheinputs
areshownbelow.
Transportation
QuantityofRawMaterialbeingtransportedbetween
thestorageareatothemanufacturearea
Transportmode
Fuelconsumption
QuatityofRawmaterialpertrip
Kmpertrip
2.2 MEDICINEPRODUCTION
All 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 to
generatesterileproduct,followedbyitsasepticfillingintothefinalproductcontainers;
Freezedrying(orlyophilization)iftheproductistobemarketedinapowderedformat.
Generallyonemayrepresentthesecondaryprocessingforthemanufactureofalyophilizedproductas
inFigure.
Figure14Secondaryprocessingforthemanufactureoflyophilizedproducts.
Asimpledescriptionofthemostimportantstagesofsecondaryprocessingwillbegiveninthefollowing
sections.
21
2.2.1 APIandExcipientsWeightingandProductFormulation
Pharmaceutical dosage forms contain both pharmacologically active compounds and excipients added
tofacilitateformulationandmanufactureofthesubsequentdosageformforadministrationtopatients.
In which concerns freezedrying, excipients are used for various purposes. For example, they act as
bulkingagentstogiveapleasingappearancetothefreezedriedproducts.Buffersarepresenttocontrol
the pH of the products that are stable only within a narrow pH range in solution, both during freezing
andthesubsequentreconstitution(Swarbrick,2007).
Moreover, excipients also play a role in the API protection. This mechanism has not been fully
elucidated,butempiricalobservationshavepointedtothefollowingcontributingfactors:formationofa
glassy state of the proteinexcipient system, crystallinity of the excipients, hydrogen bonding between
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 each
other.Inconclusion,theymustbechosenverycarefully(Rowe,SheskeyandQuinn,2009).
Some excipients that may be present in freezedried powders include solubility enhancers (e.g.,
surfactants or cosolvents), osmotic agents (e.g., saline and sugars), antioxidants (e.g., ascorbic acid),
andpreservativesformultipleinjectioncontainers(e.g.,benzylalcoholandchlorobutanol).Inaddition,
freezedriedbiologicalpowdersmayalsocontainexcipientsthatfunctiontoreduceproteinadsorption
ontothecontainersurface(e.g.,surfactantsandalbumins).Aparticularlyimportantuseofexcipientsfor
therapeuticproteinformulationsisthestabilizationoftheproteinmoleculesinthedrystate.
Examples of some excipients used as stabilizers for proteins 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 (spraydried dextrose) and hydroxypropyl cyclodextrin minimized insulin
aggregation(e.g.usedforbovineandhumaninsulins).
Polysorbate80asprotectorforfreezing;sucroseasprotectorfordrying;histidineaspHbuffer;
glycineforcakeappearance(e.g.usedforrecombinantfactorIX).
Aggregation prevented by amorphous trehalose, sucrose or a combination of sucrose, and
glycineormannitol(e.g.usedforrecombinanthumaninterleukin6).
Sucrose, sorbitol, trehalose and alanine as protectants against aggregation and deamidation;
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)tomodify
theglasstransitiontemperaturesofthefreezedriedpowders(e.g.usedforFK906tripeptide).
RecombinanthumanalbuminOrganicacidexcipientmoleculeswitheitheracarboxylgroupor
anaminogrouppresentatC1positioncompletelystabilizedrHAagainstaggregation
Polyethylene glycol as protectant for freezing; sugars (mannitol, lactose, trehalose) as
lyoprotectants against loss of bioactivity (e.g. used for lactate dehydrogenase
phosphofructokinase).
Lactose and trehalose maintain activity longer at elevated temperatures than mannitol (e.g.
usedforAlkalinephosphatise).
22
Both the excipient type (sucrose, sorbitol, glycerol) and moisture content affected protein
degradation(e.g.usedforrecombinantbovinesomatotropin,lysozyme).
Mannitol protected protein from phase separation induced damage during freeze drying (e.g.
usedforHemoglobin).
Trehalose and sucrose preserved the native dimeric structure of the protein and prevented
aggregatesformation(e.g.usedforRecombinanthumanfactorXIII).
Among others, saccharides are the most widely used excipients for stabilizing freezedried therapeutic
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
ElectricalEnergy
API
ACSconsumption
AFSconsumption
WFIconsumption
plasticpackaging
glasspackaging
Materialreceptionandstorage
conditioning
ElectricalEnergy
ACSconsumption
AFSconsumption
WFIconsumption
plasticpackaging
scrap packstetrabrickandisothermal
glasspackaging
Washingandconditioning
materialpreparation
ElectricalEnergy
ACSconsumption
AFSconsumption
WFIconsumption
Aircompressed
Preparationofthebuffer
solution:Weighting,
ElectricalEnergy
ACSconsumption
23
FormulationandSterile
Filtration
AFSconsumption
pure steamconsumption
WFIconsumption
RawMaterials
API
Aircompressed
2.2.2 FreezeDrying
Freezedrying is a commondrying technique used inthepharmaceutical industry(Swarbrick, 2007).As
manypharmaceuticalscannot beproduced on a commercial scaleby crystallization, a glassy solid may
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.Although relatively expensive, it is employed to convert solutions of labile materials 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 (a
freezedrying process maintains sterility and particle free characteristics of the product much
moreeasilythanotherdryingprocess.Furthermore,theingredientsoftheformulationarenot
stable in the liquid state and other methods of water removal destroy or reduce the active
ingredient);
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
sterilizing after this process cant be considered because the only available techniques require a liquid
producttosterilizeorhightemperatures,whichwoulddenatureproteins(Franks,2007).
24
2.2.2.1 ProductFillingandGlassVialsLoading
Anaqueoussolutioncontainingthedrugandvariousformulationaidsorexcipients,housedtemporarily
inasterileproductholdingtankisasepticallyfilledintopresterilizedfinalproductcontainers(e.g.glass
vials,ampoulesoroccasionallyinsyringes),whichareloadedontothetemperaturecontrolledshelves.
The filling process normally employs highly automated liquid filling systems. All items of equipment,
pipework,etc.withwhichthesterilizedproductcomesintodirectcontactmustobviouslythemselvesbe
sterile.Thisismaintainedbyafilterattheneckofthebottlethatallowsthepassageofwatervaporbut
prevents the ingress of bacteria. Most of such equipment items may also be sterilized by autoclaving
and be aseptically assembled prior to the filling operation. The final product containers must also be
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 its name suggests. Such equipment first moulds plastic into the final product container (the
molding conditions ensure container sterility), followed immediately by automated filling of sterile
product into the container and its subsequent sealing. In this way operator intervention in the filling
processisminimized(Swarbrick,2007,HickeyandGanderton,2001,Franks,2007).
2.2.2.2 TheFreezedryingProcess
Thefreezedryingcycle,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
temperaturecontrolled drying chamber. There, the solution is frozen quickly to prevent concentration
of the solution and to produce fine ice crystals according to physiochemical principles (the energy
transporttotransformiceintowatervaporandthetransportofthewatervaporfromthesublimation
surface through the already dried product into the drying chamber to the condensation or absorbing
systemforthevapor)astheshelftemperatureisloweredtobelowfreezing.
Theshelftemperatureissubsequentlyincreasedbutmaintainedbelowthefreezingpoint.Avacuumis
applied to the chamber to sublimate the solvent. The extent to which the compound is supercooled
depends on the compoundnature, the temperatureprogram of the shelf, theheat transfer properties
ofthecontainer,andthepresenceofparticulatesinthesolution.
Thisphaseofthedryingprocessextractsthemajorityofthesolvent(5080%).Thedrugandexcipients
are typically converted into an amorphous glass also containing large amounts of unfrozen water (15
30%) dissolved in the solid, i.e. glassy amorphous phase. Thus, most of the desiccation actually occurs
duringthefreezingstageofthefreezedryingprocess.
During the secondary drying, the remainder of the solvent is removed at an elevated but still
subfreezing temperature in order to minimize product moisture content. So, heat is supplied to the
dryingsurface.Thepowerdissipatedbytheheatermustbecarefullycontrolledsothatmeltingdoesnot
occur (only drying is accepted) at the icecontainer junction (Franks, 2007, Swarbrick, 2007, Oetjen,
Haseley,2004,HickeyandGanderton,2001).
25
2.2.2.3 TheEquipment
Atypicalproductionscalefreezedryerconsistsofadryingchamberinwhichthesolutioncanbecooled
to the required temperature and be evacuated to a low pressure and several containing temperature
controlled shelves, which can be controlled with a circulating heat exchange fluid. The heat exchange
system is supplied with a pump, which circulates the fluid through the shelves. This circulation system
mustbecapableofmaintainingtheshelftemperatureatthesetdesiredvalues.
Thesystemisalsoconnectedtoacondenserchamberviaalargevalve.Thecondenserchamberhouses
a series of plates or coils capable of being maintained at very low temperature (i.e., less than 50C).
Oneormorevacuumpumpsinseriesareconnectedtothecondenserchambertoachievepressuresin
therangeof0.030.3Torrintheentiresystemduringoperation.
Modern equipment contains computerbased control and monitoring systems by means of which a
desired drying cycle program can be preset. Finally, for pharmaceutical applications, it is essential to
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).
Table 7 shows the main inputs and outputs for the inventory analysis of product filling and glass vials
loading(PRAXIS,2009).
Table7Inventoryanalysisofproductfillingandglassvialsloading(PRAXIS,2009)
FreezeDryingandCapping
ElectricalEnergy
ACSconsumption
AFSconsumption
WFI consumption
FlipOff(capsulesthatcarrythevials)
Sterilevials
Sterilecaps
SterileTop
Compressedair
2.2.3 StopperingandFinalProductStorage
Final conditioning and storage begins with the extraction of the product from the equipment. During
this operation, a great care has to be taken not to lose the refined qualities that have been achieved
duringtheprecedingsteps.Thus,forvials,stopperingundervacuumorneutralgaswithinthechamber
is the current practice. For products in bulk or in ampoules, extraction might be done in a tight gas
chamber by remote operation. Water, oxygen, light, and contaminants are all important threats and
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
26
employed.Bothsmallmoleculesandproteinsshowdegradationduringstorageofthefreezedriedglass.
Insomecases,instabilityisseriousenoughtorequirerefrigeratedstorage.
So, ultimate storage has to be done according to the specific sensitivities of the products. Again,
uncontrolled exposures to water vapor, oxygen (air), light, excess heat, or nonsterile environmentare
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 to analyze the composition and quality of the container itself, i.e.,
glass,elastomersofthestoppers,plasticororganicmembranes(Rey,2004,Swarbrick,2007).
2.2.4 StabilityTests,QualityControlandQuarantine
2.2.4.1 StabilityTests
The stabilityindicating profile for a biotechnological product generally comprises information from a
batteryofassaysandnotfromasinglestabilityindicatingassay.Theexpiry/expirationdateistheactual
dateplacedonthecontainer/labelsofadrugproductdesignatingthetimeduringwhichabatchofthe
drugproduct is expected toremain within the approved shelflife specification if stored under defined
conditionsandafterwhichitmustnotbeused.Toarrive atanexpirationdate,itmustbedetermined
first for how long and under what conditions a pharmaceutical formulation can meet all of its quality
specifications. In general, this issue is answered 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 this case, this step is important in order to minimize degradation of the protein in the
formulationduringstorage.So,theFoodand DrugAdministration(FDA)andotherregulatoryagencies
require that the purity and potency of pharmaceuticals are monitored during the shelf life of the
products. Achieving these requirements involves using a combination of analytical techniques such as
chromatography,electrophoresis,andspectroscopyamongothers.
Because proteins are capable of denaturing via several mechanisms, it is necessary to use more than
onetechniquetodemonstratestability(Walsh,2003).
2.2.4.2 QualityControl
The 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
27
structurally complex than traditional low molecular mass drugs, but also their production in biological
systemsrenderstherangeofpotentialcontaminantsfarbroader.
Recent advances in analytical techniques render practical the routine analysis of complex
biopharmaceuticalproducts(Walsh,2003).Inthefollowingparagraphsadescriptionisprovidedofthe
usualdetectionmethodsusedforthequalitycontrolofproteinbasedfinishedproducts.
Bioassaysrepresentthemostrelevantpotencydeterminingassay,astheydirectlyassessthebiological
activityofthebiopharmaceutical.Itinvolvesapplyingaknownquantityofthesubstancetobeassayed
to a biological system that responds in some way to this applied stimulus. The response is measured
quantitatively,allowinganactivityvaluetobeassignedtothesubstancebeingassayed.
An example of a straightforward bioassay is the traditional assay method for antibiotics. This usually
involved measuring the zone of inhibition of microbial growth around an antibioticcontaining disc,
placed on an agar plate seeded with the test microbe. For modern biopharmaceuticals bioassays are
generallymorecomplex,sincethebiologicalsystemusedcanbewholeanimals,specificorgansortissue
types,orevenindividualmammaliancellsinculture.
Allbioassaysarecomparativeinnature,requiringparallelassayofastandardpreparationagainstwhich
the sample will be compared. Internationally accepted standard preparations of most
biopharmaceuticals are available from organizations such as the World Health Organization (WHO) or
theUnitedStatesPharmacopeia(USP).
Quantificationoftotalproteininthefinalproductrepresentsanotherstandardanalysisundertakenby
qualitycontrol,whereanumberofdifferentproteinassaysmaybepotentiallyemployed.Thesimplest
of such methods is perhaps the detection and quantification of protein by measuring absorbency at
280nm,basedonthefactthatthesidechainsoftheaminoacidstyrosineandtryptophanabsorbatthis
wavelength.Thismethodispopular,asitisfast,easytoperformandisnondestructivetothesample.
However,itisarelativelyinsensitivetechnique,andidenticalconcentrationsofdifferentproteinsyield
different absorbance values if their content of tyrosine and tryptophan vary to any significant extent.
Hence,thismethodisrarelyusedtodeterminetheproteinconcentrationofthefinalproduct,butitis
routinely used during downstream processing to detect protein elution off chromatographic 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
assessment of final product purity. This technique is well established and easy to perform, 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
electrophoresis commonly used to analyze proteins. It is normally run so that mixtures of proteins are
separatedfromeachotheronthebasisofadifferentmolecularpropertyineachdimension,i.e.bytwo
properties in two dimensions on 2D gels. The most commonly used method entails separation of
proteins by isoelectric focusing in the first dimension, with separation in the second dimension being
28
undertaken in the presence of SDS, thus promoting band separation on the basis of protein size.
Applicationofbiopharmaceuticalfinishedproductstosuchsystemsallowsrigorousanalysisofpurity.
Capillaryelectrophoresis(CE),alsoknownascapillaryzoneelectrophoresis(CZE)systems,canbeused
toseparateionicspeciesbytheirchargeandfrictionalforcesandmass.Thesesystemsarealsolikelyto
play an increasingly prominent analytical role in the laboratory quality control. As with other forms of
electrophoresis, separation is based upon different rates of protein migration upon application of an
electricfield.
As the name capillary electrophoresis suggests, this separation occurs within a capillary tube, typically
withadiameterof2050manduptoa1mlong,anditisnormallycoiledtofacilitateeaseofuseand
storage.Thedimensionsofthissystemyieldgreatlyincreasesurfacearea/volumeratio,comparedwith
slabgels,andhencetheefficiencyofheatdissipationfromthesystem.Thisinturn,allowsoperationat
a higher current density, thus speeding upthe rate of migration through the capillary. Sample analysis
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
purityoflowmolecularmasspharmaceuticalsubstances.Italsoplaysanincreasinglyimportantrolein
macromolecules analysis, such as proteins. Most of the chromatographic strategies used to separate
proteinsunderlowpressure(e.g.gelfiltration,ionexchange,etc.)canbeadaptedtooperateunderhigh
pressure. Reversephase, sizeexclusion and, to a lesser extent, ionexchangebased HPLC
chromatographysystemscanbeusedintheanalysisofarangeofbiopharmaceuticalpreparations.
Electrosprayionization(ESI)isatechniqueusedinmassspectrometrytoproduceionsfrombiological
macromolecules, and a very useful technique for their analysis, since it overcomes the propensity of
these molecules to fragment when ionized. ESI allows one to determine the molecular mass of many
proteinstowithinanaccuracyof0.01percent.Aproteinvariantmissingasingleaminoacidresiduecan
easily be distinguished from the native protein in many instances. Although this is a very powerful
technique,analysisoftheresultsobtainedcansometimesbelessthanstraightforward.
Immunologicalapproachestodetectionofcontaminantsorimmunoassaysarebiochemicalteststhat
measure the concentration of a substance in a biological liquid, using the reaction of an antibody or
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, polyclonal antibody preparations capable of binding specifically to these proteins
areproduced.Purificationoftheantibodiesallowstheirincorporationinradioimmunoassayorenzyme
based immunoassay systems, which may subsequently be used to probe the product. Such multi
antigenassaysystemswilldetectthetotalsumofhostcellderivedimpuritiespresentintheproduct.
Immunoassays have found widespread application in detecting andquantifyingproduct 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, as antibodies raised against such impurities would almost certainly cross
react with the product itself. Immunoassays identifying a single potential contaminant can also be
developed.Theseassaysareextremelyspecificandverysensitive,oftendetectingtargetantigendown
29
topartspermillionlevels.Manyimmunoassaysarecommerciallyavailableandtherearecompaniesfor
rapidlydevelopingtailormadeimmunoassaysystemsforbiopharmaceuticalanalysis.
Aminoacidanalysisremainsacharacterizationtechniqueundertakeninmanylaboratories,inparticular
if the product is a peptide or small polypeptide. This is a simple strategy to determine the range and
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.
Full sequencing of a sample of each batch of the protein is the only procedure guaranteed to detect
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,includingoneortwodimensionalelectrophoresis,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
single amino acid residue is likely to alter the peptides physicochemical properties sufficiently to alter
itspositionwithinthepeptidemap.Inthisway,single(ormultiple)aminoacidsubstitutions,deletions,
insertionsormodificationscanusuallybedetected.
Nterminal sequencing (also called Edman sequencing) of the first 2030 amino acid residues of the
protein product became a popular quality control test for finished biopharmaceutical products. The
technique is useful, as it positively identifies the protein, confirms the accuracy of the amino acid
sequenceofatleasttheNterminusoftheprotein,andreadilyidentifiesthepresenceofmodifiedforms
oftheproductinwhichoneormoreaminoacidsaremissingfromtheNterminus.
Analysisofsecondaryandtertiarystructuresuchaspeptidemapping,Nterminalsequencingoramino
acidanalysisyieldinformationrelatingtoapolypeptidesprimarystructure,i.e.itsaminoacidsequence.
Suchtestsyieldnoinformationrelatingtohigherorderstructures,i.e.secondaryandtertiarystructure
of polypeptides, along with quaternary structure of multisubunit proteins. Although a proteins three
dimensional conformation may be studied in great detail by Xray crystallography or nuclear magnetic
resonance (NMR) spectroscopy, routine application of such techniques to biopharmaceutical
manufacture is impractical, both from a technical and an economic standpoint. Limited analysis of
protein secondary and tertiary structure can, however, be more easily undertaken using spectroscopic
methods,particularlyfarUVcirculardichroism.
30
2.2.4.3 Quarantine
Failuretomeetspecificationsornoncompliancewiththeapprovedprocessshouldresultimmediately
inquarantineofthematerialuntildecauseoftheeventisascertained.Thuscomponents,drugproduct
containers and closures shall be stored under quarantine until they have been tested or examined, as
appropriate,andreleased.Sinceproductsareinaquarantinestatusuntilreleasedorrejected,itmaybe
appropriate to retain this quarantine status, particularly if it is expected that the problem can be
resolvedquickly.
Table 8 shows the main inputs and outputs for the inventory analysis of the stability tests, quality
control,quarantineandproductconditioning(PRAXIS,2009).
Table8Inventoryanalysisofqualitycontrol,quarantine,andproductconditioning(PRAXIS,2009)
Reviewquarantineanalysis ElectricalEnergy
ACSconsumption
AFSconsumption
WFIconsumption
Packaging ElectricalEnergy
ACSconsumption
AFSconsumption
WFIconsumption
SterileFilters
plasticpackaging
scrappackstetrabrickand
isothermal
glasspackaging
An example of the inventory analysis results for the secondary processing is presented in Table 9
(PRAXIS,2009).
Table9ExampleoftheresultsforSecondaryProduction
SoilContamination EnergyCostofWasteProductionand
Management
Paper,cardboardandothercellulosicproducts
Organicmaterials
Plastic
Tetrabrickpackages
Glass
FerrousMetals
Non FerrousMetals
DangerousWaste
WaterPollution EnergyCostofWaterTreatment
31
AirPollution
EnergyCostofAirPollution
CO2ofPlasticandorganicwasteincineration
CO2emissionsofsteamgenerationand
transport
SO2emissionsofsteamgenerationand
transport
NOXemissionsofsteamgenerationand
transport
NaturalResources
Depletion
EnergyCostofNaturalResourcesDepletion
Energyusage(fossilfuelandelectricenergy)
Waterusage
Consumptionofcardboardandothercellulosic
products
Plasticinputsconsumption
Tetrabrickinputs
Glassinputs
Ferrousmetalsconsumption
Non FerrousMetalsinputs
Lubricantagentconsumption
2.3 AUXILIARYPROCESSES
Besidesthemainproductionprocessesforthevialsfillingwiththelyophilizedproduct,thereareseveral
auxiliary processes.. The way they connect with each other and the main production processes is
presentedintheschemeofFigure15AsshowninFigure15,therearedifferentoutputsassociatedwith
theauxiliaryprocessesthatentertheproductsmanufactureindifferentstages.Inthefollowingsession
the auxiliary processes (the water treatment, heat sterilization and trigeneration) are described in
detail.
2.3.1 PureWaterTreatmentSystem
Thewatertreatmentaimstorecycleallthewaterusedinthemanufacturingprocessesandtopurifythe
water collected from rain. In the main production processes different types of water are used with
especial purity requirements (from simple tap water to ultrapurified water). The water treatment
processmusthaveseveralstagestofulfilltherequirements,whichareshowninFigure16.
Figure16:StagesoftheWaterTreatmentSystem
The water coming from the rain or recycled from the process is highbiologically contaminated since it
hasbeenincontactwithmicroorganisms.So,ithastobedisinfectedandpassedthroughseveralfilters
untilitreachesthedesiredpurity.Nevertheless,notallthewaterfromtheprocesscanbereused;there
isapurgewhenitreachesthesupplysystem.Thislossiscompensatedwithrainwater.
32
Aftertreatmentwaterisconductedtothetrigenerationsystemwhereitisheatedorboiled.
Table10liststheprocessinputsfromthewatertreatmentsystem.
Table10:InputsoftheWaterTreatmentSystem
Inputs
Electricity
Bisulfitosdico(35%)
NaOH(50%)
NaCl
Polyethylene(PE)Filter
ActivatedCarbonfilters
Effluenttobetreated
2.3.2 HeatSterilizationofWastes
Heatsterilizationistheprocessresponsibleforturningthesolidwasteaninertmatter.Inordertoit,the
biologically contaminated waste, which is essentially the organic matter with yeast gathered in
containers,issubmittedtohightemperatures.Theinertmatteristhentakenoutsidethemanufacturing
plant,whilethewaterusedtoheatthesystemisthentakentothewatertreatmentsystem.
TheinputsandoutputsassociatedwiththisprocessareshowninTable11.
Table11:HeatSterilizationInputsandOutputs
Inputs
Electricity
organicmatter
PureSteam
NaOH(10M)
2.3.3 Trigeneration:PureSteamgenerationandIndustrialSteam
Thetrigenerationprocessisthemostcomplexauxiliaryprocess.Abriefexplanationofitisgiveninthis
sessionsinceamoredetaileddescriptionisoutsidethescopeofthiswork.
The trigeneration process aims to produce heat, cold and electricity. The only outputs are water and
fuel.Thefuelisresponsibleforheatingorboilingthewaterdependingontheneeds.Whileitsheated,
cooled,orboiled,thewaterpassesthroughturbinesthatgenerateelectricity.Thecoldwaterisachieved
bythroughheatexchangersusingdifferentstreamsatdifferenttemperatures.
Thisprocessisthenresponsiblefordistributingthedifferentkindsofwater(withvaryingpurity)tothe
mainprocessstages,asshowninFigure15.
TheinputsfromthetrigenerationprocessarepresentedinTable12.
Table12:TrigenerationInputs
33
Inputs
Electricity
Water
3 LCAToolDescription
Asreferredabove,theobjectiveofanLCAstudyistoevaluatetheenvironmentalburdensofaproduct
or process (e.g. use of resources and environmental consequences of releases) throughout their life
cycle. According to the methodology described by ISO 14040 (2006), an LCA study is divided in the
followingfourmainphases:
Goal and scope definition in which the purpose of the study and the system is described,
including the definition of the system boundaries, the functional unit, and the potential
environmentalimpactscategoriestobeconsideredandhowtheywillbeassessed;
Inventory analysis where the inventory analysis is performed including the gathering of the
systems inputs and outputs. Inventory tables are constructed with, for example, energy and
materialsconsumption,andemissionstoair,waterandsoil;
Impactassessmentinwhichthepotentialenvironmentalimpactsareevaluatedbyrelatingthe
inventory data and the environmental impact categories, using an impact potency factor for
each component emitted to an environmental compartment. This step can be quantitative or
qualitative.
Interpretation where a comparison is made between the inventory analysis and the impact
assessmenttomeettheobjectivesestablishedinthegoalandscopedefinitionphase.Itishere
that some conclusions and recommendations have to be made, as well as an eventual
sensitivityanalysistoevaluatetheresultsrobustness,dependingontheassumptionsthathave
been made to perform the study. Complementary information such as, public opinion or
financial costs can also be considered in order to support decision making. Finally, this step
shouldbeaccompaniedbyseveralalternativestoimproveprocessesandreducethepotential
environmentalimpacts.
3.1 LCATOOLOUTLINE
ForthesystemboundarytobecoveredbytheLCAtool,onemayconsidertheseverallifecyclestagesof
pharmaceuticalmanufacturingprocessesasshowninFigure1(Gainzaetal.,2009).
34
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Figure1.LifecyclestagesofpharmaceuticalmanufacturingprocessestobeincludedintheLCAtool
Depending onthe system boundary definition for the study one may consider all the LCA stages, i.e. a
cradletogate analysis, or just some of them on a gatetogate approach (as shown in Figure 1).
Based on direct inputs and outputs, i.e. an inventory analysis data sheet, and their relation with the
environmental, social and economic aspects, one can get a results sheet, where the most suitable
indicatorsarepresentthatinturnwillbehelpfulforthesustainabilityassessment.
InthisstudyitisconsideredagatetogatetypeofanalysisthatcomprehendstheLCAstagesfromthe
first to the fourth stages, as represented in Figure 1, from the API production to the secondary
processing. A gatetograve analysis would address also the stages five and six as shown in the grey
boxesofFigure1.HowevertheyareoutoftheLCAtoolscopesincetheyarenotdirectlycontrolledby
the pharmaceutical companies because depend on the specific conditions of products used in the
therapeutic applications (e.g. storage time in hospital, hospital waste management). Moreover, a
cradletograve analysis would be more complete, including also the stages of extraction and
processingofrawmaterials,butitisoutoftheLCAtoolscopeandobjective.
3.2 IMPACTEVALUATION
3.2.1 Methodology
Themethodologyappliedinthiswork,basedonLCA,isdividedinseveralsteps.
EmissionFactorsDetermination
Emission factors are related to the quantity of pollutants emitted in manufacturing activities. They
represent average values that can be used as secondary data in LCA studies, whenprimary data is not
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36
oxides,wherethereareonlycharacterizationfactorsforsulfurmonoxideorsulfurdioxide.Thecriterion
hereis,oncemore,istochoosetheonewiththeworsecharacterizationfactor.
Inthedatabase,thereareseveralcharacterizationfactorsthatdependingonthemethodsapplied(CML
method, the critical volumes, the EPS, EcoIndicator 99, etc.) may lead to different impact categories,
suchastheonesfromEcoIndicator.Thusoneneedstochooseonemethodandtherespectiveimpact
categories, for a matter of consistency, to carry on the environmental impacts evaluation. Thus, to be
implementedintheLCAtoolitisdecidedtofollowtheCMLmethod(CentreforEnvironmentalStudies).
Once this task is completed, the environmental impact is calculated, as indicated in Error! No se
encuentraelorigendelareferencia.,basedontheISO14001,withoutthenormalizationstep.
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NormalizationFactors
Normalization factors serve to compare each impact category indicator with a reference value. In the
LCAmethodology,thereferencevaluesconcernaglobezoneorgeographicallocation.Oncemore,the
referencevaluesweretakenfromtheCMLdatabaseandaretheonesregardingtheWestEuropezone
wereselectedtothiswork.
Finally,thereisthelastspreadsheet(Error!Noseencuentraelorigendelareferencia.)concerningthe
potential environmental impact in both steps: the characterization and the normalization. For the
formerone,Equation2isused.
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37
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