| What
is a transgenic animal?
A
trangenic animal carries foreign DNA (genes) stably
integrated within its own genome.
These genes encode heterologous proteins of therapeutic
interest (antibodies, vaccinating antigens...) which
can be secreted in the milk of transgenic animals and
then purified. The transgenic animal becomes an outstanding
way for producing efficiently the protein of interest
in large quantities
within a framework that is fast, safe, and less expensive
than other bioreactors.
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Why
choosing transgenic animals' milk for recombinant protein
or vaccine production?
The mammary gland is an organ naturally
designed to produce complex glycosylated proteins in
high concentration (140 g/l for rabbit milk) in order
to feed newborn rabbits. Mammary gland epithelial cells
possess the machinery needed to properly fold and assemble
complex glycosylated proteins. Mammary gland epithelial
cells perform post-translational modifications
such as glycosylation
and gamma carboxylation.
For many recombinant proteins of human origin these
post-translational modifications are essential to guaranty
proper biological activity and acceptable
pharmacokinetics.
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What
are the advantages of transgenic animal compared with
traditional production methods?
Several
methods are currently used for industrial production
of proteins:
Bacterial systems (E. Coli), are commonly
used and very efficient. They offer low costs of production.
But there utilization remains limited to the production
of simple non-glycosylated proteins that do not require
sophisticated folding processes. Purification of proteins
derived from bacterial fermentation is also often difficult
to set up.
Fungal systems allow efficient production
of some secreted proteins. Glycosylation in these systems
add a number of mannoses which strongly affect the pharmacokinetics
properties of the protein.
Baculovirus systems can produce a wide
range of proteins but have yet to be scaled-up to industrial
levels.
Mammalian cell culture is the standard
method for producing glycosylated proteins, (i.e. monoclonal
antibodies). It offers properly folded glycosylated
proteins. A large number of recombinant proteins are
manufactured this way. However capital investment to
build mammalian cell culture facilities and operational
costs remain the main hindrance of this technology.
Transgenic plant systems are powerful
for very large scale production as transgenic fields
represent an important biomass. However, glycosylation
in these systems add a number plant-specific sugars
(including xylose) that are immunogenic for human patients.
Transgenic animal technology
appears to be a good alternative solution for producing
complex glycosylated proteins. It combines the high
expression levels met with bacterial systems, the high
quality level of post-translational
modifications observed in cell culture
and offers lower product costs.
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Why
rabbit?
Because of its ability to generate a large number of
transgenic founders and because of a low rabbit
embryo cost. This provides our customer with
the opportunity to significantly increase the chance
to obtain one or several transgenic rabbit lines producing
sufficient quantities of biologically active protein.
Rabbit can be bred under controlled
sanitary conditions (SPF - Specific
Pathogen Free) which offer good quality guarantees
for human biopharmaceutical productions.
Rabbit is known for its short duration of pregnancy
and fast maturation, thus allowing the production of
transgenic line quicker than with goat, sheep or cattle
and offering a shorter time to market.
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Rabbit is phylogenetically
closer to humans than many other mammals including
ruminants (Cows, goats and sheeps) and rodents (hamsters).
Rabbit mammary gland offers therefore higher
post translational modifications
capabilities making rabbit a model of choice for the
production of human glycosylated proteins. In particular
rabbit mammary gland is known to hypofucosylate
proteins it secretes, and generates proteins with a
mix of NANA
(N-Acetyl Neuraminic Acid) and NGNA
(N-Glycolyl Neurmainic Acid) sialic acids. While human
proteins are 100% NANA sialilated, most mammalian cells
perform high fucosylation rates and 100% NGNA sialic
acids.
Low cost of production: up to 50% cost saving compared
to the industry standard (CHO).
The majority of marketed therapeutic proteins have indications
that do not require more than 10 Kg worldwide on year
basis. Within this context rabbit system appears as
flexible and relevant
bioproduction method.
Suitable for complex glycosylated proteins.
No known prion
diseases in rabbit.
No serious viral disease transmission to humans.
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How
much milk and recombinant protein / vaccine can a rabbit
produce?
One female rabbit can produce up to 250
ml of milk per day of lactation.
In the standard process, only 100-150 ml of milk
is collected from one typical female rabbit per
day of lactation. The volume collected represents
15 litres of milk
per year per female. |
|
| |
Davies
(1983) The composition of milk. Biochem. Lactation,T.B.Mepham.
Elsevier, 71-117.
Jenness (1982) Inter-species comparison of milk
proteins. Dev Diary Chemistry, 1:87-114. |
| 
|
The level of recombinant protein measured in
the milk of our transgenic rabbits varies from
0.1 to
10 g per litre.
This
technology platform can offer production
of dozens kilograms of
recombinant protein
or vaccines
per year.
|
| How
are rabbits milked?
Rabbits are mechanically milked in
a confined milking room in our husbandry. Rabbit
can supply between 10 and 15 litres of milk per
year per female.
BioProtein
Technologies designed, in collaboration with INRA,
a proprietary milking
apparatus that allows efficient rabbit
milking. Such machines have been in operation
for several years and have proved to be highly
successful. |
|
What
kind of proteins can be manufactured?
- Plasma proteins
- Monoclonal Antibodies
- Hormones
- Peptides
- Recombinant vaccines
BioProtein
Technologies has so far focused on the upstream processes
that lead to the generation of animals that produce
the required proteins in their milk. Good results have
been already obtained with different classes of proteins
including:
|
Hormones
Human Growth Hormone active in vitro.
Gonadotropin hormone active in vitro.
Rotavirus Virus-Like Particles
(Rotavirus VLP's)
Obtaining transgenic animals expressing two proteins
of the Rotavirus capsid in their milk.
Activation of the immune response in naive mice
after the injection of transgenic milk.
Monoclonal Antibodies
Production of monoclonal antibodies in transgenic
rabbit milk. Rabbit mammary gland naturally hypofucosylates
the proteins it secretes in milk. This characteristic
gives rabbit an advantage that can be used to
manufacture antibodies using ADCC (Antibody Dependent
Cell Cytotoxicity) mechanisms. |
|
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How
are the recombinant proteins / vaccines produced in
the milk of transgenic rabbits?
Among
all the proteins that occur in milk, only six are specifically
produced by mammary cells. These specific proteins fall
into two biochemical classes: caseins and milk serum
proteins (whey proteins).
The production of a given protein in milk can be obtained
by transferring to rabbit embryos the cDNA encoding
this protein combined with mammary gland specific gene
promoter. Additional regulatory sequences such as enhancers
or insulators can help control the level of gene expression.
The Company has exclusive licenses from INRA (French
Agronomy Research Institute) on the rabbit WAP
(Whey Acidic Protein) gene promoter
and on a gene insulator. BioProtein Technologies also
has exclusive know-how on the transcribed region components
(introns, enhancer and terminator).
These
key elements allow BioProtein Technologies to target
the very efficient production of recombinant proteins
in the milk of transgenic rabbits.
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How
are transgenic animals generated?
The cDNA encoding the therapeutic
protein is optimized in silico and synthesized
de novo prior to be combined with regulatory
components from genes specifically expressed in milk.
The construct is micro-injected into very early stage
embryos which are then transferred to surrogate mothers.
After one month (gestation time in rabbit) the first-generation
offspring (F0 or genetic founders)
is born. Young rabbits that have correctly integrated
the transgene are identified by ear biopsy
followed by PCR analysis.
Founders are selected for their efficiency to produce
the protein of interest in their milk and used to generate
a second generation (F1) of transgenic rabbits.
Transgenic F1 progeny is identified by
ear biopsy followed by PCR analysis. Sexually
mature F1 females are inseminated with non-transgenic
sperm in order to obtain F2 progeny and milk. F1 females'
milk is mechanically collected
and the recombinant protein of interest is characterized
in order to select the best offspring lines for large
scale production and to develop the purification
process strategy.
In order to guarantee production efficiency and consistency
along the whole product life cycle, cell banks will
be established according to regulatory guidelines. These
banks are called Master
Transgenic Bank (MTB) and Working
Transgenic Bank (WTB).
It can consist in freezing sperm from well characterized
transgenic males (Master/Working
Sperm Bank - MSB/WSB), as well as embryo
banks derived from these males (Master/Working
Embryo Bank - MEB/WEB) as required by
EMEA and FDA guidelines. As an example, sperm from one
well characterized transgenic F1 male can be used to
generate one MSB. This
bank will be used to inseminate non-transgenic females
and generate a larger number of F2 transgenic males.
Sperm from these F2 males will be used to prepare the
MSB.
During
the whole product life cycle, WSB and or WEB
will be used to generate reproductive transgenic males
whose mission will be to generate the industrial herd
of transgenic lactating females that will produce the
recombinant protein of
interest in their milk.
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How
soon can recombinant proteins/vaccines be produced by
transgenic rabbits?
Transgenically
produced proteins are secreted in the milk of females
during lactation. Milking typically begins upon delivery
of the offspring. Often, enough milk may be obtained
from founders to evaluate the concentration of recombinant
protein secretion, to begin characterizing the protein,
to start purification process development.
Larger milk quantities for preclinical batches preparation
can be obtained from F1 transgenic females.
The
total time from transgene introduction to
first natural lactation
of founder female rabbits is about 6
months. In parallel, founders are crossbred
with wild type rabbits in order to give birth to offspring
and begin full-scale milk production. Those founders
that are males must produce daughters and these daughters
must have offspring of their own before full-scale milk
collection may begin. The time to first lactation is
about 12 months for transgenic lines derived from founders.
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