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By Dr. Megha Agrawal, Dr. Shyamasri Biswas, Dr.

Kim Van Vliet, Contributing Editors

Vacuum Infiltration
in Plant Biotechnology:
A Promising Approach for Gene Delivery
for Production of Pharmaceutical Proteins

uman biological or medical


products have always been intended to treat diseases and various medical conditions. They are most
commonly produced by mammalian cell
culture-based fermentation technologies.
However, such technologies have limitations due to their restricted scalability
and high cost production which prevent
fermentation based processes from meeting the ever increasing global demand of
biological products. To overcome such
technological challenges, biotechnologists have explored plants that have been
considered as alternatives to mammalian,
insect and bacterial cell cultures for the
expression and production of recombinant pharmaceutical proteins.
Plants are considered to be a novel
alternative system for the production of
pharmaceutical proteins that are more
scalable, cost-effective and safer than
current expression paradigms as they
offer multiple advantages over current
bioreactor-based platforms for protein
production. This is due to the fact that the
production cost of plant-based biological
products is significantly less than the
current mammalian cell culture-based
systems that require considerable startup

investments and expensive cell culture


growth media. Furthermore, plants can
produce large functional pharmaceutical
proteins in contrast to bacterial cells [1].
Plant biotechnologists in collaboration
with vacuum scientists and genetic engineers are working to develop a scalable
vacuum-enabled agro-infiltration technology transient expression platform that is
applicable for commercial pharmaceutical protein production. The scalability of
vacuum infiltration makes it a superior
candidate for gene delivery for large-scale
production of pharmaceutical proteins.
Researchers have demonstrated a vacuum
infiltration procedure and examined its
scalability in several studies with pharmaceutically important proteins such as VLP
and mAb based models. Such vacuum infiltration is considered to be more robust
as they can infiltrate a large number of
plants in a short period of time. Researchers have also shown the superior advantage of vacuum infiltration that can facilitate the development of a highly scalable
protein production platform for fast, economical and safe manufacturing of human pharmaceuticals in plants. In vacuum
infiltration, a desiccator is connected to a
vacuum pump to provide the vacuum for

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infiltration (Figure 1A). Plants are then


placed upside down on a shelf and plant
leaves are subsequently submerged into
the infiltration media containing Agrobacterium (Figure 1B). The process involves
a vacuum at 100 mbar which is applied
for 1 min and subsequently the release
valve is slowly opened to release the vacuum. Agro-infiltration is then achieved by
the exposure of the plants to the vacuum.
The next step involves the air in the interstitial space of the leaves that is drawn out
by the vacuum, and Agrobacteria in the
infiltration medium subsequently enters
and replaces the air in this space when the
vacuum is being released. Through this
research, the researchers thus showed that
the accumulation level and the temporal
expression pattern of target proteins were
not altered by the switch from syringe to
vacuum infiltration [1]. Thus, the promise of agro-infiltration based on syringe
and vacuum infiltration is a manifold that
provides an efficient, robust and scalable
gene-delivery technology for the transient expression of recombinant proteins
in plants. The development of this technology is believed to greatly facilitate the
realization of plant transient expression
systems as a technologically superior

October 2016 Vacuum Technology & Coating

Figure 1. Vacuum agro-infiltration set up that consists of N. benthamiana leaves with Agrobacterium tumefaciens. A. tumefaciens containing
target gene construct was re-suspended in infiltration buffer and loaded into a desiccator that was connected to a vacuum pump (A) The entire
leaf system of a 6-week old plant was then submerged into the infiltration buffer (B). Agro-infiltration was achieved by applying and releasing a
vacuum through the pump [Source: Adv Tech Biol Med. 2013 Jun; 1(1): 103].

platform for the commercial production


of pharmaceutical proteins [1].
The researchers employed the entire
shoot of a plant that was subject to infiltration with the vacuum method and the
expression of the target protein could be
detected over the entire plant, as illustrated by the green fluorescent protein (GFP)
marker shown in Figure 2 [1, 2]. It was
demonstrated that as many as six plants
could be vacuum infiltrated within a short
time of about 3 min from start to finish
[2]. Further research and developmental
works on vacuum infiltration were carried
out including several scale-up studies using the vacuum infiltration for the production of pharmaceutical proteins. To facilitate these studies, a vacuum tunnel was
designed that was able to accommodate
16 trays of plants per infiltration cycle
with automation [3].
Vacuum infiltration tunnel technology
was explored by biotechnology companies to deliver transgenes on a large
manufacturing scale. Vacuum infiltration
was attempted commercially to process
several metric tons of N. benthamiana
plants per hour by Kentucky Bioprocessing, LLC (Figure 3) [2]. However, in
order to achieve consistent infiltration of
plant material, it is important to keep the
vacuum pressure stable and vacuum duration optimized for each plant species at
a specific infiltration scale. The scale-up
vacuum infiltration allowed the researchers to produce gram level of pharmaceu-

tical grade of NVCP VLPs under current


Good Manufacture Practice (cGMP)
guidelines that was sufficient both in
quality and quantity for a phase I human
clinical trial [3].
Concluding Remarks
Vacuum-enabled infiltration technology has provided a robust route to utilize
plants for manufacturing commercially important pharmaceutical proteins

with superior scalability, safety, speed,


and cost-effective benefits. Such vacuum-based gene delivery system is a superior alternative to mammalian, insect,
and bacterial cell cultures and that can
be leveraged for effective expression
and production of commercial grade recombinant pharmaceutical proteins as
advanced human biological products.
As described in this column, specifically
agro-infiltration based on vacuum infiltration has shown tremendous promise

Figure 2. Expression of the green fluorescent protein (GFP) in vacuum agro-infiltrated leaves.
N. benthamiana leaves were infiltrated with A. tumefaciens harboring the GFP gene in MagnICON vectors. Post agro-infiltration leaves were photographed in a dark room under UV light
for 6 days [Source: Adv Tech Biol Med. 2013 Jun; 1(1): 103].

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Figure 3. Commercial scale production of N. benthamiana plant (A) and agro-infiltration (B). [Source: Adv Tech Biol Med. 2013 Jun; 1(1): 103
and Kentucky Bioprocessing, LLC].

that has provided an efficient, robust


and scalable gene-delivery technology
platform for the transient expression of
recombinant proteins in plants. It is believed that along with deconstructed viral
vectors, vacuum infiltration will greatly
facilitate the implementation of plant
transient expression systems as a prominent commercial production platform for
the rapid, economical and safe production
of pharmaceutical proteins. We anticipate
a rapid development in research and developments in this area involving a mul-

tidisciplinary collaborative approach that


will include biotechnologists, vacuum
and pharmaceutical experts, biologists
and protein scientists to work together on
this exciting technology development for
the next generation production of human
biological products that are important to
treat complex human diseases.
References for Further Reading
1. Qiang Chen, Huafang Lai, Jonathan Hurtado, Jake Stahnke, Kahlin Leuzinger, and
Matthew Dent. Agroinfiltration as an Effec-

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tive and Scalable Strategy of Gene Delivery


for Production of Pharmaceutical Proteins,
Adv Tech Biol Med. 2013 Jun; 1(1): 103.
2. Leuzinger K, Dent M, Hurtado J, Stahnke
J, Lai H, Zhou X, Chen Q. Efficient agroinfiltration of plants for high-level transient
expression of recombinant proteins. J Vis
Exp. 2013 Jul 23;(77).
3. Lai H, Chen Q. Bioprocessing of plant-derived virus-like particles of Norwalk virus
capsid protein under current Good Manufacture Practice regulations. Plant Cell Rep.
2012;31:573584.

October 2016 Vacuum Technology & Coating