CrisBi
Breaking the
productivity
barriers of
baculovirus
technologies
CrisBioTM technology
For decades, the vaccines have been produced in cell
cultures or fertile eggs. The huge demand of vaccines and
the urgent need to reduce the vaccine production costs, to
make them accessible to the whole human population and
farm animals, made crucial the development of new
production processes.
Four main production platforms are used for the production
of the next generation vaccines (bacteria, yeast, mammalian
or insect cells). ALGENEX has developed the “fifth
production element” to obtain the subunit vaccines, the
CrisBioTM technology platform.
ALGENEX has developed and patented this innovative
technology platform, which take advantage of a multicellular
living organism, the Lepidoptera Trichoplusia ni (T. ni ). This
highly efficient technology combines T. ni chrysalises with
the proprietary improved baculovirus vectors TopBac® for
the production of recombinant subunit vaccines. The
scientific team at ALGENEX has optimized and
industrialized CrisBioTM technology, reaching production
yields that exceed 20 times the productivity of insect cells
cultured in bioreactors. CrisBioTM technology is currently
used by ALGENEX to produce several virus-like particle
(VLP) vaccines in collaboration with international veterinary
companies.
CrisBi
CrisBioTM rationale
Trichoplusia ni chrysalises (pupae) can be produced in a costefficient and unlimited manner. These chrysalises function as
biocapsules containing insect cells in perfect physiological
conditions ready to be infected by a recombinant baculovirus
(AcMNPV-TopBac®) expressing the protein of interest. After 4-7
days of incubating the infected pupae at the appropriate
temperature, the recombinant protein is produced up to
milligram scale per pupa. The recombinant protein is then
extracted and purified by conventional means. The proteins
expressed in pupae are fully functional, presenting the same
postranslational modifications and folding obtained in insect
cell cultures. The production is not influenced by the many
factors affecting cell cultures. Billions of encapsulated
cells in perfect
physiological conditions
ready for protein
production
Significant
reduction in CAPEX
and OPEX in
comparison to
bioreactor-based
production
No mammalian
compounds are used
in the production
process (biosafety)
Cell diversity increases
productivity for even the
most difficult-to-produce
proteins
CrisBio Biocapsule
Linear scaling-up
Production yields not
subjected to pH,
media nutrients or
oxygen levels
2 DNA sequence
Baculovirus generation
3 weeks
2 weeks
Baculovirus stock
Scaling-up
production in less
than 2 months
1 week
Protein purification
1 week
Protein production
in pupae
CrisBioTM
3.- Semi-automated processes: Most
companies using insects as living
biofactories employ insect larvae instead
o f p u p a e f o r p ro t e i n p ro d u c t i o n .
Movement and soft bodies difficult its
robotic manipulation such as baculovirus
vector inoculation. CrisBioTM overcomes
this difficulty because the pupae are inert
and can be stacked and stored at 4ºC for
at least a week before injected with a
baculovirus by a specifically designed
robot. The storage room needed to
incubate the infected pupae is much more
smaller than that required for infected
larvae. Healthy or infected pupae can be
easily transported to vaccine production
factories by land or air without any
deleterious effect in productivity.
14"
12"
10"
4.- TopBac® and
synergism: ALGENEX is proprietary of the patented improved
8"
baculovirus expression cassette TopBac®. Baculoviruses modified by this genetic construct increase
their productivity up to 6 times in insect pupae. CrisBioTM is the only technology which combines 6"the
4"
best baculovirus expression vectors with a living insect. It allows to reach unprecedented
2"
productivities, up to gram quantities of recombinant protein per liter of insect extract. CrisBi
0"
Unicellular
Multicellular organism
CrisBioTM Biocapsule
Bioreactor
Inocula(on)robot)(CBIR-3000))
Robot CBIR-3000
Inocula(on)speed:)3,000)pupae)/hour)
Inoculation speed:
3,000 pupae/hour
mg/g"of"pupae"biomass"
1.- Fast development times: less than two months
are required to scale-up the production of any
recombinant protein (extremely important in
pandemic situations). This scaling-up is linear and
depends only on the virus vector inoculum quantities
and the number of chrysalises infected and incubated
at the same conditions (high production consistence
among infected pupae). The virus inoculum can be
produced in cells or pupae, being the last one more
productive (up to 109 pfu/pupa).
mg/g"of"pupae"biomass"
CrisBioTM advantages
2.- Productivity and enablement: cell
diversity increases productivity. The pupa
tissue heterogeneity, not possible in cell
cultures, provides alternate cellular
environments for high level expression of
even the most difficult-to-produce
proteins.
14"
12"
10"
8"
6"
Conven-onal"Bac$Conven-o
TopBac"
TopBac"
4"
2"
0"
3 Upstream and downstream processes are extremely simple and can be performed by
conventional means by trained personnel. There are no relevant differences in
downstream to obtain a purified protein in comparison to cell culture procedures.
CrisBioTM processes
Upstream standard
process
Robotized inoculation
Baculovirus stock
T. ni Pupae
Protein extraction
Downstream standard
process
Frozen pupae
CrisBi
Frozen pupae
Incubation
Vaccine
formulation
Centrifugation
First filtration
process
Protein
purification
Tangential flow
filtration
4 CrisBioTM supply
In some aspects, the production platform
CrisBioTM is similar to the egg-based production,
but differs in its versatility. Eggs can be used
only for viruses that replicate efficiently in them.
In addition, egg supply for vaccine production is
limited and also expensive considering the
productivity per unit. Egg-derived vaccines
frequently contain elements that may cause
allergic reactions and eggs may support the
replication of unapparent pathogens that could
infect the vaccine receivers. In contrast, there are
not reports either of allergic reactions produced
by T. ni-derived products or about pathogens
replicating in this Lepidoptera
that may affect mammals. Frequently, a single
vaccine dose is obtained from a single egg (e.g.
influenza vaccine). In contrast, the T. ni pupae
may produce any recombinant subunit vaccine
and dozens of vaccine doses can be produced
by a single baculovirus infected pupa. The
operating procedure of healthy egg and pupa
transfer to the vaccine production laboratory is
almost the same, but with the difference of
space required in such transport and the lability
of the material. Insect pupae can be delivered
infected and frozen to the vaccine production
factory, ready to extract the vaccine antigen.
Modular GMP laboratory
Bacmid'
A"
Top%
PI#
(0.7ml)#
Entomology unit
producing clean pupae
Without%top%
B"
Pupa%inocula+on%
PII#
(2.6ml)#Top%
PIII#
Produc?on#batch#
(24ml#108#)# in#insect#cells#(Sf9)#
Alveole%
Alveoles%
CrisBi
Company producing
vaccines
With%top%
Inocula(on)robot)(CBIR-3000))
Inocula(on)speed:)3,000)pupae)/hour)
Robot%CBIR)3000%
Frozen
pupae
Frozen infected pupae
Clean
pupae
Pupae
Frozen
PIII#
PII#
7#in#larva#
(220ml)#
infection (210ml#3x10
pupae
±#109#in#cells)#
Pupae&produc+on&
Produc?on#batch#
in#insect#pupae#
Vaccine antigen
Pupae&inocula+on& Vaccine
Vaccine&an+gen&
antigenVaccine&
formula+on&
5 Our business model
[email protected] CrisBi
- Feasibility
CrisBioTM) studies including
optimization of your recombinant
protein production
-Co-development agreements of
protein based products, specially in
the fields of diagnostic and vaccines
(glycoproteins, VLPs, etc..) and
-Supply Agreements bringing the
insect biomass (pupae) or clarified
extracts to your manufacturing site as
raw materials for your Vaccines.
6