Expression and Manufacture of Pharmaceutical Proteins in Genetically Engineered Horticultural Plants - Transgenic Horticultural Crops: Challenges and Opportunities - page 102

biocompatibility,.and.ability.for.continuous.processing.qualify.this.system.as.an.ini-
tial.fractionation.strategy.for.large-scale.processing.
Precipitation.is.a.simple.and.robust.method.to.separate.a.precipitable.protein.from.
other. soluble. protein. contaminants.. One. of. the. strategies. to. make. a. target. protein.
precipitable.is.to.fuse.it.to.a.stimulus-responsive.polymer..Since.this.type.of.polymer.
can.transit.reversibly.between.water-soluble.and.water-insoluble.state.depending.on.
temperature,.pH,.or.other.environmental.conditions,.the.target.protein.can.be.easily.
separated.from.host.proteins.by.a.simple.centrifugation.or.iltration.step.while.the.
polymer.is.in.a.water-insoluble.state. 211 .Both.centrifugation.and.iltration.work.well.
in. separating. precipitated. proteins. from. soluble. ones;. the. latter,. however,. has. the.
advantage. of. the. possibility. of. being. operated. in. a. continuous. process. 212 . Several.
PMPs.including.human.interleukin-4,.10, 213 .scFv 214 .and.anti-HIV.mAbs 215 .have.been.
puriied.by.precipitation.using.an.elastinlike.peptide.(ELP)..A.temperature-induced.
“inverse.transition.cycling”.allowed.ELP-fusion.proteins.to.transition.to.an.insoluble.
state..As.a.result,.the.fusion.proteins.were.easily.puriied.by.centrifugation. 216
Afinity. precipitation. is. a. special. case. of. precipitation. methods. and. has. been.
suggested.as.one.of.the.most.promising.nonchromatographic.techniques.for.large-
scale. processing. 146 . The. power. of. afinity. precipitation. is. derived. from. the. unique.
combination. of. the. speciicity. and. selectivity. of. biorecognition. with. the. simplic-
ity,. robustness,. and. high. concentration. factors. associated. with. precipitation.. This.
method. does. not. require. the. fusion. between. target. protein. and. the. precipitation.
carrier.. Instead,. an. afinity. ligand. is. covalently. linked. to. a. stimulus-responsive.
polymer. or. other. molecules. capable. of. forming. high. molecular. weight. particles..
Since. there. is. no. fusion. between. the. PMP. and. the. precipitation. carrier,. structural.
and. functional. integrity. of. the. target. protein. is. better. preserved,. and. the. need. for.
proteases.to.remove.fused.precipitation.carriers.is.eliminated..Afinity.precipitation.
has.been.extensively.tested.with.plant-derived.mAbs..As.discussed.earlier,.protein.A.
or.its.afinity.components.have.been.fused.to.bacteriophage.capsid.proteins,.bacte-
rial.S-layer.proteins,.oleosins,.cellulose,.and.starch-binding.domains,.and.recently.to.
tobamovirus.nanoparticles..Binding.of.mAbs.to.these.Protein.A-precipitation.carrier.
fusions.has.allowed.eficient.puriication.of.a.variety.of.mAbs.from.plants.with.com-
parable.purity.and.recovery.as.mAbs.puriied.by.Protein.A.chromatography,.but.with.
much.superior.cost.effectiveness.and.scalability. 145 .Similarly,.other.afinity.ligands.
have. also. been. successfully. used. to. create. agents. for. afinity. precipitation. of. afin-
ity-tagged. vaccines. and. non-mAb. therapeutics. 162,183 . Another. variation. of. afinity.
precipitation.is.magnetic.separation,.in.which.magnetic.nanoparticles.are.decorated.
with. afinity. ligands,. and. nanoparticle-bound. target-protein. can.be.then. separated.
from. other.soluble.proteins.by. using.magnets. 217,218 .Plant-derived.mAbs.have.been.
successfully.puriied.by.such.method. 24 .In.addition.to.using.magnetic.crystals,.mag-
netic.particles.can.also.be.produced.by.certain.bacteria,.which.can.be.engineered.to.
display.Protein.A.or.other.afinity.ligands.on.their.surface. 219,220
Another. attractive. alternative. to. column. chromatography. is. membrane. separa-
tion or. membrane. chromatography.. The. effectiveness. of. these. membrane-based.
separation. techniques. has. been. successfully. demonstrated. in. purifying. mAbs. and.
other.therapeutic.proteins. 221,222 .For.example,.an.anion-exchange.membrane.eficiently.
removed. impurities. from. the. feed. stream. when. used. in.a.low-through. mode. 223,224 .