Proteins as

Biotechnology Products

ExamplesofProteinProducts?

Medications

Enzymestomakefuelfrom
hardtometabolizebiomass

Textileprocessingenzymes

Decontaminationenzymesfor1stresponders

BIOPROCESSING:
Theuseofbiologicalsystemsto
manufacture(process)aproduct.

AProteinProductisMade
DiscoveryResearch
ProcessDevelopment&Approval
Manufacture
Verification

QualityControl&
QualityAssurance

Packaging

DiscoveryResearch
BasicScienceOngoing
scientificstudy!
Oftengovernmentfunded
Useofmodelorganisms
Communication!
Ideaforabioproduct
Transitiontoindustry:
thestartupcompany

DiscoveryResearch
DiscoveryResearch
Ideaforaproduct,
baseduponDNA
manipulationand
cloning.

ProcessDevelopment
Optimizingthemanufacturingprocess,
butonasmallerscale.
ProcessDevelopmentendswith
productionofamastercellbank
Eventuallytheprocessisscaledupfor
fullscalemanufacturing

ProductManufacturing
Overview
UpstreamProcessing

DownstreamProcessing

UpstreamProcessing
Theactualexpression
oftheprotein
productinthehost
cell.

PhotofromUniv.Hawaii

CommonHostCells
Bacterialcells

Fungalcells

Mammaliancells

E.coli

P.pastoris

ChineseHamsterOvary
CHO

TheBioreactor
Sterilesystem!
NutrientMedium
Processcontrol
dissolvedO2
pressure
pH
temperature
4LiterBioFlow3000,madebyNew
BrunswickScientific

LargeScaleBioreactor

20,000 liter bioreactor in Portsmouth, NH

DownstreamProcessing
Isolationofpure,stable,functional
protein.
Manychallenges!

TheHarvestingChallenge
Cells:ifproductisinsidethecells,harvesting
involvescelllysiswhichisriskytotheprotein
Medium:ifproductissecreted,harvesting
involvesdealingwithhugevolumesofliquid.

HarvestingTechniques
Lysisby:
Freeze/thawing
Mechanicaldisruption(beads,blades,
sonic)
Detergents

StabilizingMethods
ColdTemperature
Proteaseinhibitors
protein

Antifoamingchemicals

protease

Purification
Getridofallunwantedsubstances,but
keepasmuchoftheproteinaspossible.
Dontforget,itmustbeperfect!

TypicalProductPurification
Methods
Filtration
Centrifugation
Chromatography

Filtration
Separationofsubstancesbypassagethroughporousmembrane.

Industrialsizefilters

Centrifugation
Control Panel

Protective enclosure
Door

Separationofparticlesfrom
liquidbyuseofcentrifugal
force.

Cut-away view

Rotor

Motor

Basic components of a centrifuge

Center of rotation

rminimum

Drive
shaft

Centrifugal force

Sedimentation
path of particles
raverage
rmaximum

Pellet
deposited
at an angle

Chromatography
Molecularseparationbasedonphysicalorchemicalcharacteristics

Example:GelFiltrationseparatesbasedonsize.

IndustrialChromatography

2meter
column

Verification
Monitorforthepresence,
quantity,andqualityof
thetargetproteinasitis
beingproducedand
purified.

CommonAnalyticalMethods
HPLCHighPerformanceLiquid
Chromatography
SDSPAGESDSPolyacrylamide
Electrophoresis
IEFIsoelectricFocusing
MassSpectrometry

AnalyticalMethods
HPLC=HighPerformanceliquid
chromatography

FromLCResourcesInc

AnalyticalMethods
SDSPAGE
SodiumDodecylSulfate
PolyacrylamideGel
Electrophoresis

http://fig.cox.miami.edu/~cmallery/255/255hist/ecbxp4x4_SDS.jpg

AnalyticalMethods
IEF
IsoelectricFocusing
SimilartoSDSPAGE,
buthigherresolution

FromAmershamSci.

AnalyticalMethods
MassSpectroscopyamethodofanalyzing
proteinstructure.

LastSteps
1. Formulation:additionof
stabilizingchemicals

2. Filtersterilization
3. Asepticfillingofvials
4. Lyophilizationfreezedry
5. Seal,inspect,label

QualityControltesting
QualityAssuranceoversight
Throughoutallstagestoensure
safetyandcompliancewithFDA
regulations.

DiscoveryResearch

ProcessDevelopmentandDrugApproval

Manufacture

Verification

NEXT!!

QualityControl&
QualityAssurance
Packaging