FOCUS ON...
Transgenics
Development of a Plant-Made
Pharmaceutical Production Platform
Using Single-Use Materials and an Aquatic Plant
Keith M. Everett, Lynn Dickey, John Parsons, Rachel Loranger, and Vincent Wingate
S
ince the late 1980s, studies
have shown that plants can
manufacture functional
transgenic pharmaceutical
compounds. Advantages attributed to
plant-made pharmaceutical (PMP)
approaches are compelling, and PMP
production continues to attract
interest from investors and the
biopharmaceutical industry (Table 1).
Proposed PMP benefits include proven
scalability, high production capacity,
limited exposure to human or animal
pathogens, lower capital expenditures
(CapEx), and decreased operating
costs. Those putative advantages have
proven to be significant business forces
driving continued investor support for
PMP ventures. PMP production’s
lower cost relative to cell culture
provides an opportunity to subsidize
more research for additional product
development.
Placed in wider context, the
business value, for example, of
potentially large production capacity
coupled to lower CapEx requirements
and manufacturing costs is
underscored by concerns over the
expanding gap between production
volume and patients’ needs for
potentially life-saving drugs. In 2010,
178 drug shortages were reported to
the US FDA, 132 of which involved
sterile, injectable drugs. In 2011, the
agency has continued to see an
increased number of shortages,
especially those involving older sterile
injectable drugs (1). So the disparity
between production and capacity
continues to expand.
16 BioProcess International
10(1)
January 2012
Photo 1: Biolex transforms the aquatic plant, Lemna minor, to produce industrial quantities of PMPs.
Many situations can create a drug
supply shortage, including natural
disasters, limited profitability,
regulatory activities, and populationbased demands. One compelling
solution to the growing difference
between drug production capacity and
patient needs is taking advantage of
the simplicity, lowered cost of goods
(COGs), increased speed, and greater
capacity offered by PMPs.
Other PMP manufacturing
advantages are operational and relate,
for example, to the risk of animal
by-product exposure to cultures, feed
streams, and/or active pharmaceutical
ingredients (APIs). Manufacturers
must demonstrate that their processes
and APIs are free of biologically active
animal by-products if they are to be
approved. For example, prions and
viral particles associated with animalbased products may cause serious
adverse reactions in humans when
injected or ingested. Such exposure
can be minimized in well-designed
processes using plant-based production
cultures and USP Class VI certified
components.
With PMP systems, however, the
serious concern over animal
by-product contamination is reduced
or removed completely. They have less
inherent animal by-product exposure,
thereby eliminating the need for the
additional expense of removing
associated contaminants. The end
result can be simpler, less-expensive
processes with more rapid production
and development timelines.
Historical Perspective
of PMP Industry Growth
The possibility of transgenic plantbased pharmaceutical production
emerged in the 1980s as advances in
Table 1: PMPs in clinical trials as of March 2010*
Company/
Institution
Arizona State
University
Plant
Potato
Potato
Potato
Tobacco
Undisclosed
Product
Vaccine
Vaccine
Vaccine
Vaccine
Oral vaccine
Lemna
Alpha interferon
Lemna
BLX-155
Dow
Agrosciences
Plant nonnicotine
tobacco
Vaccine
Medicago
Tobacco
Protalix
Plant cell
Vaccine
Vaccine
Glucocerebrosidase
Sembiosys
Plant cell
Safflower
Safflower
Biolex
Indication
Escherichia coli
Hepatitis B
Norwark virus
Norwark virus
Undisclosed
Clinical Stage
Phase 1
Phase 1
Phase 1
Phase 1–2
Phase 1 planned
start mid 2007
Hepatitis B and C Phase 2b (×2 trials:
and cancer
480 and formulated
dose), poised for
phase 3 initiation
Fibrinolytic “clot Phase 1 ready
buster”
Newcastle
USDA approved in
disease in
February 2006
poultry
H5N1 influenza
H1N1 influenza
Gaucher’s
disease
Acetylcholinesterase Biodefense
Insulin
Diabetes
Apolipoprotein AI
Cardiovascular
Phase 2a
Phase 1, preliminary
On sale, namedpatient basis
Phase 1
Phase 3 in planning
Preclinical
complete
*
Adapted from Marshall B. PMPs in Clinical Trials and Advanced PMIPs; molecularfarming.com, updated by
the author June 2011
genetic engineering reached a critical
point and plant genetic transformation
became possible. Initial success in
transforming plants with genes
encoding pharmaceutical compounds
engendered a great deal of speculation
about the future of PMP
manufacturing and its potential
benefits to human health, global
nutrition, veterinary medicine, and
so forth (2).
Since the successful production of
the first PMP compound in 1986,
plant transformation strategies,
upstream processes, and PMP-related
regulatory laws have been redefined
and improved continually. Although
conventional pharmaceutical
manufacturing platforms such as yeast
and mammalian cell culture continue
to yield approved drug products, PMP
products have been a rarity. It is not
unusual for new technologies to take
some time to affect pharmaceutical
markets. Despite the acknowledged
gradual evolution of the PMP sector,
a number of plant-based recombinant
drug products are currently poised to
move into the final stages of drug
approval (Table 1).
Experts have suggested several
explanations for the differences
between the PMPs’ outlook in the late
1980s and current projections. During
18 BioProcess International
10(1)
January 2012
the late 1980s and 1990s, PMP
opportunities seemed vast and
immediate because scientists in
industry and academia focused
primarily on yield rather than
regulatory aspects. Early business
models that sprang from this initial
excitement proved incomplete. Early
PMP researchers envisioned an open,
field-based production factory that
could be rapidly implemented with
simplified scalability, resulting in an
equally rapid turnaround in
investment capital. That initial vision
was gradually brought down to Earth
during the following decade because
of mounting concerns over genetic
containment.
Because of documented incidents of
accidental release of or contamination
by genetically modified organisms
(GMOs, plants in this case), the
public has become quite sensitive to
the potential for unchecked genetic
contamination of the environment.
That concern arises from uncertainty
regarding what potential damage or
catastrophic consequences the release
of putative artificial genetic material
may cause. Such concerns are
legitimate, and manufacturers must
verify that proper precautions are in
place to contain their GMOs. The
necessary containment measures for
Photo 2: Typical BioQuate connector; each is
unisex and connects to other BioQuate DAC of
any tubing size (www.bioquate.com/demo/).
a given enclosed process are dictated
by individual system and facility
requirements. Open systems must
consider the requirements for proper
and sufficient buffer areas — where
these requirements have been or can
be established. Some requirements for
open systems are still in development,
making open GMO production
systems particularly challenging from
a regulatory standpoint.
Another reason the PMP sector’s
growth has slowed is the US
Department of Agriculture’s (USDA)
Animal and Plant Health Inspection
Services (APHIS) rigorous
enforcement of genetic material
containment regulations. To protect
public interests, regulatory agencies
carefully monitor current and future
safety concerns associated with
GMOs that can produce PMPs and
implement appropriate regulations.
Complying with those legitimately
prescribed rules has challenged PMP
manufacturing growth.
Regulations designed to protect the
public food supply and ecological
genetic stability have created industry
concerns over the cost and complexity
of effective containment strategies (3).
Facing progressively tighter
environmental regulations, many
PMP research operations are
investigating the use of greenhouses,
enclosed hydroponic-based systems,
and other designs in which
environmental contamination is
minimized to reduce regulatory
complexity (4).
Some companies continue to
develop crop-based production
platforms (e.g., Sembiosys, Medicago,
Photo 3: Biolex master plant bank vessels and
the BioQuate disposable aseptic connector used
for making critical connections and transfers in
the LEX System upstream process
Table 2: Scale has little effect on the LEX
System culture growth rate or yield per unit
fresh weight. As indicated, scaling up the
process gives repeatable biomass-to-yield
ratios. Correlations between production
volume and yield can falter when typical
production systems are scaled. However, in
this case, the Lex System technology shows no
decline in yied proportional to scale.
Scale
(Grams of Biomass)
MPB (1 g)
Inoculation bag (300 g)
4 × 8 ft. bag (6,000 g)
Yield (% Total
Soluble Protein)
6.7
6.8
6.5
and iBio). Other approaches attempt
to adapt platform species to
regulatory requirements through
enclosed, cell-based liquid culture
platforms using modified bioreactors,
lighted tanks, and other aspects of
conventional cell culture processes
(e.g., Protalix). Notably, another class
of processes in development suggests
that systems familiar to the FDA
provide the only feasible route to
pharmaceutical manufacturing and
regulatory success. Biolex
Therapeutics Inc. of North Carolina
is actively exploring such
technological opportunities.
PMPs are typically derived from
methods of transgenic protein
expression in plants. Among the
differing plant platforms are two
relatively distinct categories of
transformation approaches: stable
transgene expression and transient
transgene expression. The Biolex stable
transgene manufacturing process,
termed LEX System (or Lemna
Expression System), has been proven
through good manufacturing practice
(GMP) production runs for now
completed and successful Locteron
phase 1, 2a, and 2b clinical trials.
20 BioProcess International
10(1)
January 2012
Photo 5: Seed-bag support rack (left) as compared with a production-bag support rack (right)
Leveraging Single-Use Products:
Benefits and Compliance
Photo 4: The customized MPB vessel is
designed to integrate aseptically with the
upstream manufacturing process.
A Novel Approach
Biolex is a privately owned, clinicalstage biopharmaceutical company that
uses proprietary technology to develop
pharmaceutical proteins, including
glycan-engineered monoclonal
antibodies, biosimilars, follow-on
biologics, and veterinary medicines (5).
The LEX System process uses
genetically transformed cultures of
Lemna minor (duckweed) in
manufacture of those products. As a
plant-based production system, it
addresses the need for control,
containment, and cost reduction. The
system has produced pharmaceutical
compounds proven effective through
phase 2 clinical studies. The current
drug product made with LEX System
technology (developed in conjunction
with Netherlands-based Octoplus
NV) is Locteron, a controlled-release
interferon formulation for treating
Hepatitis C (6–8).
Having used a relatively common
species of aquatic monocot (Photo 1)
to create a PMP manufacturing
process, Biolex integrated its process
into a single-use systems (SUS)
approach. Adding SUS materials to
the original PMP approach further
reduced compliance concerns by nearly
eliminating any need for cleaning
validation upstream.
Combining PMP production processes
with a new generation of single-use
products presented an opportunity to
the disposables industry. The synergies
of those technologies became all the
more compelling as requirements for
plant genetic containment increased
their hold on the PMP industry. In
2004, Biolex began testing bags for use
as manufacturing culture vessels,
biomass sequestering and filtration
systems, and manifolds.
Each stage of the production
process is designed to be closed and
aseptic to build on the already
animal-product-contact-free aspect of
plant-based cultures. To further
reduce cost and complexity, the
design ensures that no classified areas
are required for the upstream process.
LEX System technology is designed
to prevent contamination from all
microorganisms and biocontaminants
through the application of disposable
aseptic connectors and gammasterilized single-use seed bags,
production bags, harvest bags, and
tubing assemblies. In addition,
harvest has been simplified through
application of a proprietary single-use
design to create a fully disposable,
low-cost material separation and
sequestration step. With a tested and
proven aseptic process, this
technology stands exemplifies
pharmaceutical process containment
and control.
The single-use process addresses
animal-product-free requirements,
including extractables and leachables
control through strict use of USP
Class VI materials in the process
stream. Using Class VI disposables
hands off to SUS vendors a large part
Figure 1: Process flow diagram of the first step in upstream manufacturing; transfer from the
master plant bank to the seed bags
Seed bag inoculation:
bags undergo direct inoculation
through Q-Slant
BioQuate
aseptic connector
Secondary air ballast
Lights
Q-Slant
Solid waste to
decontamination autoclave
Cooling
blowers
Media bag in tote
Slants, tubing, etc.
Seed bag rack: 32 total bags
8 shelves, 4 bags/shelf
Media
*Slant removed from chamber for each seed bag to be inoculated
Figure 2: Contents of the seed bags are transferred to the production bags through aseptic
connectors. The production bags are incubated for the requisite time and then in-process test
samples are removed and sent to quality assurance for analysis.
Media prefilter
Flow sensor
Peristaltic pump
Inline air filter
Research-grade
compressed air
supply for
biological functions
Primary air ballast
Primary air ballast
Production bag inoculation: 4 × 8 ft. bags
aseptically inoculated with one seed bag each
Production bag rack holds eight
4 × 8 ft. bags, 1 bag/shelf
of the compliance, documentation,
and regulatory control burden.
Additional regulatory support is
handled by in-house and external
material and product testing, vendor
auditing, and document management.
All FDA and EMEA regulations
pertaining to SUS are strictly
followed, and compliance control is
built into the upstream process
USP Class VI Cytotoxicity and
Physicochemical Standards; USP Class VI
Material Certificate, signed and dated
(S&D).
Documentation with Certificate of
Origin of Materials provided for all
product contact materials; (S&D)
TSE, BSE free — for example, a
statement should be provided that
bovine-derived materials from bovine
spongiform encephalopathy (BSE)
countries or any products exposed to
transmissible spongiform
encephalopathy (TSE) agents (as
defined by the US Department of
Agriculture, 9 CFR 94.11) are not used or
manipulated in the same facility as any
of the materials of construction for the
bag assembly (S&D)
All materials of construction must be
animal-derived-component free (ADCF),
(S&D)
Media
bag
in tote
Recombinant plant line GMP pilot plant at
Biolex Therapeutics, Inc.
Single-use regulatory guidelines cover
bag materials in general and specific
product-contact layers (11).
All Single-Use, GMP Materials
Primary air ballast
Master plant-bank controlled
environment chamber*
Regulatory Guidelines
through a rigorous documentation
and SOP management system.
One of two distinct advantages of
this production approach is that it
transfers part of the disposables quality
responsibility to vendors. The second
advantage is that consistent application
of single-use products throughout
upstream processes eliminates all
cleaning validation requirements for
process stream hardware. That is a
significant cost savings for Biolex and
goes far toward offsetting the cost of
disposables-based operations.
The LEX System Process
Lemna (commonly known as
duckweed) is an aquatic
monocotyledonous plant that thrives
in the top 1 cm of still, freshwater
systems. Several of its characteristics
make it ideally suited for PMP
production (4), including its
• ability for clonal reproduction
• high protein content
• rapid growth rate
Extractable and leachable screening
results and documentation must be
available as needed for pre- and
poststerilization testing
Materials are gamma stable
Materials are UV stable
Additional Requirements for
Product-Contact Layers
In addition to the above requirements,
the following requirements are also
applicable for bag product-contact
layers:
Hemolysis ISO 1993-4 2002
Physicochemical USP28
Cytotoxicity MEM Elution, USP 28
Bacterial endotoxin; LAL <0.25 EU/mL
extraction media, pass
Moisture permeability, E-96-90 method,
<0.3 g/100 in 24 hours
• classification as a nonindustrialized
plant, not a food crop
• growth in a wide range of
conditions
• ability to produce secreted or
tissue-bound products.
In addition, Lemna’s process
advantages include
• viral inactivation steps are not
required
January 2012
10(1)
BioProcess International
21
• scale has no effect on yield per
unit weight or protein quality
• no pesticides, herbicides, or
antibiotics are used in production
cultures.
Clonal reproduction simplifies line
management, propagation, and the
process feed stream. The absence of
pollen or flowers simplifies genetic
containment. Lemna’s high total
soluble protein (TSP) content
provides higher expression and target
protein concentrations per unit
biomass than other PMP platforms
(9). Another strategically
advantageous characteristic is its
culture growth rate. Lemna doubles
its biomass every 36 hours under
normal growth conditions, a doubling
rate that exceeds other plant systems
biomass build rates.
filtered ambient air. Other important
environmental parameters are media
composition and temperature.
In processes, facilities, and
hardware designs, only those four
critical environmental factors are
Process Control
considered. This simplicity goes far
Photosynthesis converts sunlight to
toward limiting overall process and
energy for cellular processes, in which hardware design complexity and final
carbon dioxide is converted to
facility costs. The LEX System
biomass and cellular products such as
upstream process is in definite
target transgenic proteins.
contrast with complex photobioreactor
Consequently, to exploit Lemna’s
systems that require monitoring of
growth rate and high levels of
many physical indices, precision
expression, cultures must receive
controls, and cleaning validation.
appropriate amounts of light and
Design of experiment (DoE)
software established critical ranges for
environmental parameters. Studies
tested ranges for yield, repeatability,
and robustness. A study of failure
mode and effects analysis (FMEA)
used a scaled-down model system.
That model addressed the range of
possible failures, effects of each failure
on product quality and yield, and
critical points for process failure
responses. Those process ranges and
failure data provided process design
standards and established system
robustness. A scaled-up
manufacturing system confirmed
those results.
In addition to environmental
testing, DoE addressing media
composition, storage life, and
postharvest FMEA added further
critical guidelines for media
MINIMIZE
RISK
MINIMIZECONTAMINATION
CONTAMINATION
RISK
preparation and vessel filling
• On-line
during
batch
processing
On-lineaseptic
asepticQC
QCsampling
sampling
during
batch
processing
processes. Criteria for product storage
•
Pre
assembled
single
use
set
with
7-sample
bags
and
aseptic
connector
•MINIMIZE
Pre assembled
single use set with 7-sample
bags and aseptic connector
CONTAMINATION
RISK
were developed through HPLC
• On-line aseptic QC sampling during batch processing
AUTOMATED
PROCESS
SAMPLER
quality analysis of storage regimes for
AUTOMATED
PROCESS
SAMPLER
• Pre assembled single
use set with 7-sample
bags and aseptic connector
• Day-to-day reliable performance, following customer sampling SOP
• Day-to-day reliable performance, following customer sampling SOP
harvested media and tissue. Those
• Walk away operation, process and sample data monitored and archived
•AUTOMATED
Walk away operation,
process and
sample data monitored and archived
PROCESS
SAMPLER
• Critical process parameters and events alarmed, and archived
data were added to the overall process
•• Day-to-day
Critical process
parameters
and
events
alarmed,
and
archived
reliable performance, following customer sampling SOP
design to set hold-step requirements
• Walk away operation, process and sample data monitored and archived
ADDITIONAL
FEATURES
• Critical process parameters
and events alarmed, and archived
and facilitate raw-product transfer
ADDITIONAL
FEATURES
• Input signals from auxiliary equipment can be archived
• 21CFR
Input signals
from
auxiliary
equipment
can bemulti-level
archived password protection
method controls.
compliant
software
includes
user log-on,
•ADDITIONAL
21CFR
compliantFEATURES
software includes user log-on, multi-level password protection
and
e-signature
Intensive collaboration among
• Input
signals from auxiliary equipment can be archived
and e-signature
• 21CFR compliant softwareMONITORING
includes user log-on, multi-level password protection
upstream process development,
ENVIRONMENTAL
and e-signature
ENVIRONMENTAL
MONITORING
•
Registration and (bar code) labeling
of environmental monitoring samples
quality assurance (QA), and
collected
during
batch
•ENVIRONMENTAL
Registration
and
(barprocessing
code)
labeling of environmental monitoring samples
MONITORING
regulatory and operations functions
• On-board
storage
enclosure
for
EM
samples
can
be
made
available
(optional)
collected during batch processing
• Registration and (bar code) labeling of environmental monitoring samples
throughout development of the
• On-board storage enclosure for EM samples can be made available (optional)
collected during batch processing
production format was necessary to
• On-board storage enclosure for EM samples can be made available (optional)
ensure minimized requirements for
redesigns. This ensured careful
considerations of regulations from the
e-mail: [email protected] www.awst.com
FDA and European agencies at all
e-mail: [email protected] www.awst.com stages of development. For example,
e-mail: [email protected] www.awst.com
the potential for animal-product
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Imagepurposes
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exposure was designed out of the
process, as were any activities difficult
to validate or hard to control and
document. Lot definition
requirements also played an important
role in developing the best and most
compliant lot characterization.
Regulators have responded to this
single-use, plant-based process with
several comments and observations (10):
• simplicity, few critical process
parameters (CPP)
• no animal-derived components in
the process or media
• no viral inactivation or prion
clearance measures required
• closed system: upstream
processing can be in unclassified area
• most special GMP guidances for
transgenic plants do not apply
Primary air ballast
Figure 3: Process flow diagram of the final step in upstream manufacturing is a closed, aseptic,
completely disposable harvest process based entirely on single-use product-contact materials.
Production bags are harvested under aseptic conditions, and media are pumped out of 4-ft × 8-ft .
bags through a peristaltic pump and into a disposable filter bag. From the filter bag, media cleared
of fronds and debris are pumped aspectically to 500-L totes for transfer to downstream processing.
Peristaltic
pump
Solid waste to
decon. autoclave
Bags, biomass, tubing
Harvest
material
in 500-L tote
Production bag rack holds eight
4 × 8 ft. bags, 1 bag/shelf
Harvest material
to downstream
• no open field production
• no herbicides, pesticides,
antibiotics, or environmental
bioburden
• no pollen or seeds due to clonal
propagation.
Single-Use Component
Benefits and Requirements
This plant-based process uses
proprietary culture bags designed to
meet vertical and horizontal stacking
density and scaling requirements. All
filters, aseptic connectors, ports, and
pressurized systems undergo extensive
in-housing testing for baseline
characteristics, flow volume and rate,
burst pressure, barrier effectiveness,
and product consistency.
The “Regulatory Guidelines” box
provides an example of regulatory
materials of construction (MoC)
requirements for the disposable,
production-scale vessels.As indicated
in that list, animal-contact–free MoC
are of primary importance. All
products are certified animalproduct–free before any purchases for
GMP operations. Each approved
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item is shipped from its vendor with a certificate of
authenticity (C of A), Certificate of conformity (C of C),
lot number, and sterilization records.
One important component that helped Biolex design its
enclosed, aseptic upstream process was the latest generation
of aseptic connectors (Photo 2). When properly used, these
connectors (BioQuate ReadyMate) are reported to provide
an aseptic/sterile connection while requiring no classified
areas or external sterile air-flow system.
Based on that design, process connections between
bags, for example, could be made simply. Aseptic integrity
of the components could be ensured despite gray-space
operation. This type of aseptic connector proved a critical
factor in lowering facility costs and simplifying process
and hardware design. Due to their strategic role — and to
prove compliance and control — it was necessary for those
connectors to undergo a comprehensive testing regimen
that included bacterial challenges, consistency testing,
durability analysis, and additional FMEA testing.
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Upstream production begins with a master plant bank
(MPB), the plant analogue of the more commonly known
“master cell bank” (Photo 3). The LEX System process has
demonstrated that small numbers of fronds can be stored in
agar slants at 8–10 °C for up to a year with full viability
and no genetic drift. To further control and preserve the
plant bank, LEX System technology relies on a proprietary
cryopreservation-based living line archival system.
Although conventional archive systems have proven plant
viability through nine years, cryopreservation systems have
the potential to store recoverable plant material for much
longer, possibly 20 years or more.
This upstream process begins when the contents of
slants are transferred to “seed bags” (Photo 4, Figure 1).
Once inoculated and supplied with media, the seed bags
are placed on a lighted rack and attached to a compressed
air supply. Other than temperature control through rack
design and production-suite air handling, the only
environmental support these cultures require are
compressed air for biological processes and light for
photosynthesis. The support rack is a proprietary,
customized, self-contained lighting system with bag
restraint, temperature control, and air distribution built
in (Photo 5). Each rack can provide temperature feedback
to the HVAC system for complete homeostatic control.
When the seed bag incubation cycle is nearly complete,
in-process testing (IPT) samples are used to detail the
integrity of the cultures. All bags with bioburden are
removed from the process stream. Once seed-bag
incubation is complete, those bags are connected to
production bags through aseptic connectors (Figure 2). The
contents of the seed bag are decanted into the production
bag, and the connection between the two is sealed and cut
(Figure 2). Used seed bags are deactivated through
sterilization and processed for waste or materials recycling.
As production cultures reach maturity, samples are
removed through the sample port and sent to QC for
analysis. Bioburden-free bags continue in the process
Figure 4: Comparing cost and speed of LEX System technology and a common pharmaceutical
manufacturing platform based on a detailed design and cost model for a monoclonal antibody at
200 kg/year output and industry benchmarks for a similarly sized CHO facility
Faster Scale-up
(Design/Build/
Validate)
LEX System
LEX
Mammalian
(CHO) system
CHO
0
6
3 Years
LEX
LEX
Mammalian (CHO) system
Lower CapEx
$0
$200 million
$400
$100/g
$200
LEX System
Lower COGS
Mammalian (CHO) system
$0
stream to harvest. Because the process
is robust, contamination is very rare.
During harvest of the production
bags, pumps move media and tissue
through a proprietary, disposable
“harvest bag” (Figure 3). While the
harvest bag captures and encloses
solid materials, media f low through
the bag’s filtration panel and are
pumped to a large collection tote for
transfer to downstream processing.
In a sealed single-use tubing
network, the content of each bag is
protected from contamination
throughout the harvest process (as
with the rest of the upstream
process). If target proteins are
secreted into the media, fronds
sequestered in the harvest bag are
decontaminated and discarded per
USDA regulations. This production
process requires none of the stirring
or active control of media
constituents, pH, or CO2 tension
that many conventional batch
processes require.
Once recovered, media are
removed and sent downstream for
clarification and additional
purification into API. If a target
protein is tissue bound, then harvest
bags containing biomass and
product are transferred to
downstream processing. That
involves simple disruption to extract
protein from the biomass. Often a
low-pH precipitation can
significantly reduce host proteins
and remove phenolic compounds.
Depending on scale, either
centrifugation or depth filtration
clarifies the extract. Standard,
relatively low-cost equipment is
used for those steps, and no viral
clearance is required. The rest of
the downstream process is typical
and depends on the target protein.
For example, protein A capture is
used for antibodies followed by one
Syringe line under barrier isolation
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or two additional chromatography
steps. Either biomass or media can
be frozen and stored for later
processing, if desired.
Effective PMP System
LEX System technology offers several
advantages over other systems (Figure
4). It minimizes the risk of accidental
genetic release of product or
contamination of food sources.
Although nontoxic and edible, Lemna
is not commonly used as a food
source. A further advantage is that no
pesticides, herbicides, or antibiotics
are used in Lemna cultures. In
addition, the single-use aspect of this
production system has also nearly
eliminated cleaning validation
activities from upstream processes.
The current single-use process has
handed off materials and product
validation responsibility to vendors and
manufacturers of disposable products.
This has provided additional benefits
of reduced work-force requirements and
documentation burden.
Using disposable aseptic connectors
eliminates the need for classified areas
in upstream processes. This reduces
facility and operating costs and process
complexity. Aseptic connectors
provided for a process that can specify
and achieve low contamination levels,
well within process requirements. In
receiving a contaminant-free feed
stream, downstream processing
benefits as well.
LEX System technology has
merged plant-based pharmaceutical
protein production with single-use
materials. The benefits and advantages
to this approach have been
demonstrated through investor
support and FDA acknowledgement
of successful clinical trials. A crucial
synergy has manifested itself in the
careful adaptation of single-use
products to the Lemna-based PMP
technology.
References
1 Office of the Press Secretary, The White
House. Fact Sheet: Obama Administration Takes
Action to Reduce Prescription Drug Shortages in the
US; 31 October 2011; www.whitehouse.gov/thepress-office/2011/10/31/fact-sheet-obamaadministration-takes-action-reduce-prescriptiondrug-sh.
2 Molecular Farming: Plant Bioreactors. Bio
Pro, Baden-Württemberg; www.bio-pro.de/
magazin/thema/00178/index.html?lang=en.
3 Thomson JA. Seeds for the Future: The
Impact of Genetically Modified Crops on the
Environment. Cornell University Press, Ithaca,
NY; 2006.
4 Gasdaska JR, Spencer D, Dickey L.
Advantages of Therapeutic Protein Production
in the Aquatic Plant Lemna. BioProcessing J
March–April 2003: 49–56.
5 Cox KM, et al. Glycan Optimization of
a Human Monoclonal Antibody in the Aquatic
Plant Lemna minor. Nature Biotech. 24 2006:
1591–1597.
6 Dzublyk I, et al. Phase 2a Study to
Evaluate the Safety and Tolerability and
Antiviral Effect of Four Doses of a Novel,
Controlled-Release Interferon alfa-2b
(LocteronTM) Given Every Two Weeks for 12
Weeks in Treatment-Naïve Patients with
Chronic Hepatitis C (Genotype 1). J.
Hepatology 46, 2007: 4S:232A.
7 Lawitz E, et al. Early Viral Response of
Controlled-Release Interferon alpha2b and
Ribavirin vs. Pegylated Interferon alpha 2b and
Ribavirin in Treatment-Naïve Genotype1
Hepatitis C: 12 Week Results (SELECT-2
Trial). J. Hepatology 52, 2010: S114.
8 Long WA, et al. Timing and Frequency
of Depression During HCV-Treatment with
Controlled Release INFa2b (CR2b) vs.
Pegylated IFNa2b (PEG2b): Results from
SELECT-2, a Randomized Open-Label
72-Week Comparison in 116 Treatment-Naïve
Patients with Genotype 1 HCV. J. Hepatology
54, 2011: S181–182.
9 Cox KM. Production of Antibodies in
Plants. An Z and Strohl W, Eds. Therapeutic
Antibodies: From Theory to Practice. Wiley, Inc:
New York, NY; September 2009.
10 Private communication between Biolex
and regulatory authorities
11 Biolex internal documents. •
Corresponding author Keith Everett is
director of process design and research at
Biolex Therapeutics, Inc, 158 Credle St.,
Pittsboro, NC 27312; 1-919-542-9901, ext.
2032; [email protected]. Lynn Dickey,
PhD is vice president of research and
technology development, John Parsons is
research scientist of plant biology, Rachel
Loranger is upstream research coordinator,
and Vincent Wingate is associate director
of upstream process development and plant
biology, all at Biolex Therapeutics.
To order reprints of this article, contact
Rhonda Brown ([email protected])
1-800-382-0808. Download a low-resolution
PDF online at www.bioprocessintl.com.
January 2012
10(1)
BioProcess International
26