Expression and Manufacture of Pharmaceutical Proteins in Genetically Engineered Horticultural Plants - Transgenic Horticultural Crops: Challenges and Opportunities - page 96

plants.do.have.unique.plant-speciic.β-1,2-xylose.and.core.α-1,3-fucose.residues.on.
complex.N-linked.glycans.and.lack.terminal.β1,4-Gal.and. N -acetylneuraminic.acid.
(Neu5Ac).residues. 57 .Extensive.studies.have.shown.that.variations.in. glycosylation.
patterns.do.not.always.lead.to.loss.of.in.vitro.or.in.vivo.function.or.cause.side.effects.
in.humans. 132 .Despite.this.fact,.a.variety.of.strategies.have.been.developed.to.circum-
vent.the.potential.problems.associated.with.the.difference.between.plant.and.human.
glycosylation. patterns.. For. example,. glycoengineering,. involving. sophisticated.
genetic.and.metabolic.engineering.of.plant.with.mammalian.glycosylation.enzymes,.
has.recently.emerged.as.the.most.promising.approach.to.“humanized”.glycosylation.
in. plants. 133 . In. one. of. these. studies,. a. “humanized. knockout”. Arabidopsis thali-
ana .plant.line.has.been.generated.to.express.a.mAb. 134 .Similar.transgenic.tobacco.
( N. benthamiana ). lines. with. humanized. glycans. were. generated. later. 135 . In. these.
plants,. enzymes. for. the. biosynthesis. of. plant-speciic. glycans. are. inactivated,. and.
results.have.shown.that.the.mAb.was.glycosylated.with.a.mammalian-like.pattern.
and.was.structurally.equivalent.to.the.same.mAb.derived.from.mammalian.cells.
In. the. last. few. years,. the. feasibility. of. glycoengineering.at. a. biomanufacturing.
scale. was. demonstrated. 136 . For. example,. an. RNA. interference. (RNAi). strat-
egy. was. employed. to. suppress. the. expression. of. endogenous. enzymes. (α-1,.
3-fucosyltransferase.and.β-1,2-xylosyltransferase).for.plant-speciic.glycans..Results.
showed.that.mAbs. produced. in.plant. lines.with.mutated.α-1,3-fucosyltransferase.
and.β-1,2-xylosyltransferase.had.no.detectable.plant-speciic. N -glycans..More.sig-
niicantly,.the.plant-produced.mAb.contained.only.a.single.major. N -glycan.species.
and.showed.improved.antibody-dependent.cell-mediated.cytotoxicity.(ADCC).and.
effector. cell-binding. activities. in. comparison. with. the. equivalent. mAb. produced.
in. mammalian. cells. 135 . For. certain. class. of. therapeutic. proteins,. the. presence. of.
the. terminal. Neu5Ac. residues. is. required. for. their. biological. activities. and. sta-
bility.. Adding. the. terminal. Neu5Ac. residues. to. plant-derived. proteins. is. the. last.
remaining. challenge. for. the. full. humanization. of. plant. N -glycosylation. pathway..
Recently,.attempts.were.made.to.express.the.three.key.enzymes.of.the.mammalian.
Neu5Ac. biosynthesis. pathway,. namely,. UDP- N -acetylglucosamine. 2-epimerase/.
N -acetylmannosamine. kinase,. N -acetylneuraminic. acid. phosphate. synthase,. and.
CMP- N -acetylneuraminic. acid. synthetase,. in. plants.. Results. showed. that. signii-
cant.amounts. of.Neu5Ac.were.generated. when.the. three.enzymes.are. simultane-
ously.expressed.in.plants. 137,138. Plant.glycoengineering.has.demonstrated.its.ability.
in.producing.mAbs.with.better.glycoform.uniformity.and.enhanced.functionality.
than.mammalian.cell.cultures,.therefore.demonstrating.its.potential.in.producing.
pharmaceutical.proteins.with.fully.humanized.glycans.
DOWNSTREAM PROCESSING OF PLANT-DERIVED
PROTEIN PHARMACEUTICALS
In.the.last.decade,.signiicant.progress.has.been.realized.in.boosting.PMP.expression.
levels.through.the.enhancement.of.genetic.elements.for.stable.transgenic.expression.
and.the.development.of.viral-based.transient.expression.systems..As.a.result,.PMP.
ield. has. increasingly. shifted. its. attention. to. developing. technologies. for. eficient.
PMP.recovery.and.delivery..While.“immunization-by-eating”.still.presents.a.viable.