| 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.
Many recombinant proteins of human origin require these
post-translational modifications for proper function
or 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 they are limited to the production of simple non-glycosylated
proteins not requiring sophisticated folding process.
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 may offer properly
folded and modified proteins, but the low yields per
cost of production facility are a hindrance.
Transgenic plant systems, are powerful
for very large scale production as transgenic fields
represent a important biomass. However, glycosylation
in these systems add a number plant-specific sugars
(including xylose) that are imunogenic for human patients.
Transgenic
animal technology appears to be a good alternative
for producing complex glycosylated proteins. It combines
both the expression levels met with bacterial systems
and the post-translational modifications that can be
achieved with tissue culture. Compared to cellular expression
it offers lower product costs.
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Why
rabbit?
Ability to generate a large number of transgenic founders
due to a low cost of rabbits embryos. 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 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.
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Attractive in terms of time to market.
Low cost of production: up to 50% cost saving compared
to the industry standard (CHO).
80% of the therapeutic proteins marketed have indications
that do not require more than few Kg of worldwide yearly
production. Rabbit is the most suitable animal to produce
proteins quantities of up to 10 kg per year.
Rabbits are genetically closer to humans than any other
dairy animals and as a result are the model of choice
for the production of human therapeutic proteins.
Suitable for complex glycosylated proteins.
No known prion disease 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
1 to 10
g per litre.
Thanks
to our husbandry, we can guarantee a production
of kilograms of
recombinant protein
or vaccines
per year (using hundreds of lactating females).
|
| How
are rabbits milked?
Rabbits are mechanically milked in a confined
milking room in our husbandry. Milk is directly
filtered on a 0.22 µm membrane, clarified
(fat & casein removal) and stored in steril
plastic bag at -20°C under GMP
requirements before being processed
by Eurogentec, our DSP partner ---
> More...
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
- MAbs
- Hormones
- Peptdies
- VLP-based 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
Chimerization of two murine antibodies.
Transgenic mouse producing a chimerized monoclonal
antibody in milk.
Transgenic rabbits for monoclonal antibody production. |
|
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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 whey proteins.
The production of a given protein in milk can be obtained
by transferring to rabbits the gene 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
can recombinant vaccines be produced in the milk of
transgenic rabbits?
Rotavirus
Virus-Like Particles (Rotavirus-VLP's)
are high molecular weight protein complexes consisting
of hundreds of Rotavirus proteins of two types: VP2
and VP6. These proteins are derived from the Rotavirus
capsid, and assemble naturally to form virus-like particles,
mimicking the rotavirus structure, with the same ability
to trigger the immune system but without the risk of
infection. Rotavirus-VLP's
represent a very powerful
antigen carrier due to their high molecular
weight and the repetition of the antigen motif in each
particle.
Recombinant
chimeric proteins can be produced with any antigen of
interest (whether small or large peptide or even large
functional molecules) inserted within VP2 and VP6 protein
without disrupting the Rotavirus-VLP structure, in order
to produce safe and effective
vaccine solutions.
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How
are transgenic animals generated?
The
gene encoding the therapeutic protein is combined by
our molecular biology team with a milk-specific promoter.
The construct is introduced into very early stage embryos
which are then transferred to surrogate mothers.
After the birth of the first-generation
offspring (F0), young rabbits that have
correctly integrated the transgene are identified by
ear biopsy and PCR analysis.
Founders will be 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 via tissue biopsy
followed by a PCR analysis. Sexually mature transgenic
F1 females are then inseminated with non-transgenic
sperm.
Their milk is mechanically collected
and the recombinant protein is characterized in order
to select the best offspring lines for large scale production
and to develop the purification process strategy
(GLP, pre-GMP, GMP).
In parallel, sperm from F1 transgenic males - Master
Sperm Bank, MSB - is collected and cryo-preserved
in liquid nitrogen, as recommended by the FDA
Guidelines and the European Regulation.
This sperm will be used to artificially inseminate non-transgenic
females to generate the second filial offspring (F2).
Sperm from F2 transgenic males - Working
Sperm Bank, WSB - is recovered and will
serve, for 15 to 20 years, at generating transgenic
F3 females which will produce industrial amounts of
recombinant proteins or vaccines
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 the
birth of 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 and even to
begin preclinical studies.
The
total time from transgene introduction to
first natural lactation
of founder female rabbits is about six
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 11 months for transgenic lines derived from founders.
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FREQUENTLY ASKED QUESTIONS ::::: 
