Expression and Manufacture of Pharmaceutical Proteins in Genetically Engineered Horticultural Plants - Transgenic Horticultural Crops: Challenges and Opportunities - page 101

One.of.the.most.critical.strategies.for.PMP.puriication.is.to.take.early.measures.
to.prevent.undesirable.host.proteins,.microorganisms,.and.other.contaminants.from.
entering.puriication.feed.streams..For.example,.bioburden,.environmental.contami-
nants,.and.certain.plant.host.proteins.should.be.reduced.as.early.as.possible.prior.to.
the.chromatographic.puriication.steps..These.early.measures.are.not.only.important.
in.simplifying.the.subsequent.puriication.process,.but.are.also.essential.in.ensuring.
the.regulatory.compliance.of.the.inal.product..For.example,.whole.leaf.homogeniza-
tion.should.be.avoided.for.isolating.apoplast-targeted.proteins.with.molecular.weight.
<50.kDa..Instead,.they.should.be.extracted.directly.into.the.extraction.buffer.by.a.
specialized.centrifugation.technique. 203 .Since.this.technique.allows.the.extraction.of.
target.protein.without.breaking.plant.cells,.the.puriication.process.is.greatly.simpli-
ied.as.a.result.of.a.reduction.in.host.protein.contaminants..The.carbon.assimilation.
enzyme. ribulose-1,5-bisphosphate. carboxylase-oxygenase. (RuBisCo). is. the. major.
contaminating. protein. in. plant. leaves,. and. it. should. be. removed. from. the. extract.
by.centrifugation.under.low-pH.buffer.conditions.(∼pH.5.3).prior.to.the.chromato-
graphic.steps. 117 .Phenolics,.alkaloids,.and.other.plant.speciic.molecules.should.also.
be.eliminated.by.nonchromatographic.separation.methods.(see.below).prior.to.col-
umn.chromatography.to.prevent.resin.fouling. 149 .Ongoing.research.by.our.group.has.
established. a. puriication. process. for. vaccine. candidate. NVCP. based. on. low-pH.
precipitation.and.conventional.chromatographic.steps. 130 .SOPs.based.on.this.purii-
cation.scheme.have.been.established.and.used.in.a.cGMP.production.of.NVCP.for.
human.clinical.trials.
n onchromatoGraphIc S eparatIonS
The. promise. of. PMP. production. on. an. agricultural. scale. demands. a. downstream.
processing.platform.with.extraordinarily.large-scale.capabilities..Despite.the.wide-
spread.use.of.chromatographic.separations.in.PMP.puriication,.there.are.growing.
concerns. that. this. approach. cannot. be. scaled. up. suficiently. for. very. large-scale.
production..As.a.result,.alternative.technologies.have.been.explored.for.very.large-
scale. downstream. processing. of. PMPs,. aiming. to. not. only. enhance. manufacture.
scalability,.but.also.to.provide.separation.power.similar.or.better.than.that.of.column.
chromatography,.while.reducing.separation.steps.and.the.manufacture.cost..These.
technologies.include.ATPS,.precipitation,.and.membrane.chromatography. 204
ATPS. has. tremendous. scalability. potential. and. is. able. to. handle. large. biomass.
load.compared.to.other.separation.methods. 205,206 .It.relies.on.the.incompatibility.of.
certain. polymer. mixtures. in. forming. homogeneous. solutions,. but. instead. forming.
two.immiscible.phases.under.speciic.concentrations. 207 .As.discussed.earlier,.ATPS.
is.an.effective.method.in.eliminating.phenolics,.toxic.alkaloids,.pigments,.and.other.
compounds.from.plant.extracts. 149,150 .ATPS.has.been.successfully.used.in.purifying.
tobacco-derived.anti-HIV.mAbs.and.protein.subunit.vaccine.candidates.as.well.as.
nanoparticles.including.VLPs. 149,208-210 .ATPS.offers.high.separation.rates.and,.there-
fore,.minimizes.the.contact.time.between.PMP.protein.and.deleterious.plant.extract..
Inclusion.of.this.system.in.downstream.processing.will.facilitate.plant.extract.clari-
ication,.removal.of.phenolics,.alkaloids.and.other.harmful.compounds,.and.partial.
puriication.of.target.proteins..Overall,.the.advantages.in.low-cost,.scalability,.high.