Agriculture Reference
In-Depth Information
hydrophobicity,.and.afinity.to.speciic.ligands.and.the.parallel.characteristics.of.
plant.host.proteins.
13,64,141-143
For.mAb-based.PMPs,.Protein.A.or.G.afinity.chromatography.provides.a.superb.
puriication. step
144
. and. has. allowed. successful. puriication. of. many. plant-derived.
mAbs. and. their. derivatives.
21,23,64,145
. Usually,. a. two-column. puriication. scheme.
including. a. protein. A. capture. step. and. an. anion.exchange. polish. step. is.suficient.
to.achieve.high.purity.
58,146
.However,.direct.loading.of.plant.extracts.onto.Protein.A.
(or.G).columns.often.causes.resin.fouling.and.poor.binding.of.target.protein.due.to.
the. fact. that. plants. produce. much. more. solid. debris.than. other.organisms. and. are.
rich. in. phenolics. and. alkaloids.
142,147,148
. For. example,. clariied. tobacco. extract. was.
found. to. foul. Protein. A. Sepharose. and. reduce. the. resin. capacity.
147
. Ion. exchange,.
gel. iltration. chromatography,. and. ultrailtration. methods. have. been. employed. to.
remove. the. interfering. molecules,. but. with. very. little. success.
147
. The. solution. for.
this. problem. came. from. a. nonchromatographic. method. called. aqueous. two-phase.
partitioning.system.(ATPS)..It.was.found.that.ATPS.could.effectively.remove.phe-
nolics,.toxic.alkaloids.and.other.plant.compounds.from.tobacco.extract..This.extract.
could.then.be.directly.loaded.to.a.Protein.A.column.without.causing.resin.fouling,.
which. allowed. eficient. separation. of. target. mAbs. (anti-HIV. mAb. 2F5.and.2G12).
from.tobacco.host.proteins.
149
.ATPS.has.also.been.shown.to.be.effective.in.removing.
interfering.plant.molecules.from.extracts.of.other.plant.species.
150-152
Protein.A.is.an.expensive.resin.and.requires.extreme.low.pH.(2.0-2.5).in.the.elu-
tion.step..In.large-scale.productions,.the.resins.are.routinely.recycled.up.to.50.times.
to.maintain.a.reasonable.cost.
153
.This.practice,.however,.often.leads.to.additional.cost.
in.cleaning.and.revalidation.of.the.resins,.which.is.necessary.to.prevent.leaching.of.
degraded. ligands. into. products.
145,149
. Extensive. researches. have. been. conducted. to.
identify.alternative.resins.with.equivalent.or.better.afinity.to.antibodies.or.cheaper.
ways.to.produce.protein.A.resin.
One.alternative.is.to.create.a.single-use.protein.A-related.resin.with.low.produc-
tion.cost..For.example,.an.attempt.has.been.made.to.produce.transgenic.protein.A.or.
its.fragments.in.plants.to.take.advantage.of.the.inexpensive.nature.of.plant.expression.
systems.
145
.Low-cost.Protein.A.alternatives.have.also.been.developed.in.the.form.of.
fusion.proteins..For.instance,.Protein.A.or.its.afinity.components.were.genetically.
fused. to. molecules. that. are. capable. of. polymerizing. or. binding. to. high. molecular.
weight. structures.. Antibodies. that. bound. to. these. Protein. A-decorated. large. par-
ticles. can. then. be. separated. from. plant. host. proteins. by. simple. centrifugation.
or.ultrailtration.(see.discussion.of.“afinity.precipitation”.on.page.102)..Examples.
of. this. strategy. include. fusion. proteins. of. Protein. A. with. self-polymerizing. bac-
teriophage. capsid. proteins,
154,155
. high. molecular. weight. bacterial. S-layer. proteins,.
cellulose-binding. proteins,. and. starch-binding. domains.
156-160
. Oleosins,. a. class. of.
plant.seed.oil-body-associated.small.proteins,.have.also.been.explored.as.Protein.A.
fusion.partners.
161-163
.This.technology.has.been.developed.and.matured.to.the.com-
mercially.ready.stage.by.a.Canadian.Biotechnology.company.Sembiosys,.in.which.
the.oleosin-Protein.A.fusion-mAbs.complex.can.be.puriied.by.extracting.with.oil.
bodies. (www.sembiosys.com).. The.yield.of. oleosin-Protein. A.fusion. molecules. in.
saflower.plants.is.relatively.low.and.needs.further.improvement.
145
.An.immunoab-
sorbent. nanoparticle. technology. based. on. a. tobamovirus,. displaying. a. 133-amino.
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