C
H A P T E R
FOUR
Transgenic Animals
Walking Bioreactors
by S. Anne Montgomery
W
ork on transgenic
expression systems
using animals began
in the early 1980s,
primarily as a way of
improving the genetic characteristics
of livestock. Transgenic animals
acquire genetic material (sometimes
from another species) through
human intervention rather than
through normal sexual reproduction.
The hope was to accomplish with
microinjection of functional genes
into ova what would otherwise take
years with traditional breeding
programs: mosquitoes incapable of
carrying malaria, for example, or
production of leaner meat by beef
cattle. In an early experiment, a
“Super Mouse” was created when a
rat gene for growth hormone was
injected into and expressed by the
genome of a parent mouse (1).
Aside from applications designed
to improve characteristics of a
particular species or to create
“specialized” research animals
(expressing green fluorescent
protein in zebrafish embryos, for
example, as a marker for genetic
studies) (2), transgenic technology is
also achieving increasing success as
an alternative to producing proteins
in cell culture and microbial
systems. The goal of such work is to
produce large quantities of
recombinant proteins in the milk or
plasma of transgenic mammals or in
the eggs of transgenic hens. Many
of these efforts are progressing
through clinical trials, and a few
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GTC BIOTHERAPEUTICS (WWW.GTC-BIO.COM)
companies appear to be close to
achieving market approval. In fact,
one company, GTC Biotherapeutics,
Inc. (Framingham, MA; www.gtcbio.com), is undergoing review for
market authorization in Europe for
ATryn, its recombinant form of
human antithrombin expressed in
the milk of transgenic goats.
In the first successful case of
transgenic production of a
therapeutic protein, mouse embryos
were injected with a DNA construct.
It was made by inserting the
promoter and upstream regulatory
sequence from the mouse whey
acidic protein gene (murine milk
contains distinctively high levels of
this whey protein) into the gene
coding for human tissue plasminogen
activator (tPA). The resultant
transgenic offspring produced
biologically active tPA in their milk: a
heterologous protein of tremendous
therapeutic potential (3).
Several companies are already
selling research-grade products
(including “customized” mice)
produced transgenically for use in
modeling human diseases in
preclinical studies or generating
antibody candidates for further
development. Transgenic companies
with transgenic platform technologies
to produce clinical materials may
partner with other pharmaceutical
and biotechnology companies to
produce their products transgenically
in addition to developing an in-house
pipeline of products. Some of these
companies are also capable of the
downstream processing development
and manufacturing of transgenic
products, at least to clinical scale,
whereas others rely on the
manufacturing capabilities of partners.
Transgenic production offers
certain advantages compared to
traditional mammalian cell
production systems. One source
compares the average generation of
0.2–1.0 g/L of recombinant protein
in highly optimized cell cultures to
possible expression levels of
2–10 g/L of milk in transgenic
SUPPLEMENT
livestock (4). Another mentions that
one sheep can produce 2–3 L of
milk per day. If a recombinant
protein is expressed at a level of
1 g/L, a single sheep could produce
up to 20 g of product per week (3).
Similar estimates are offered by
many other companies. Proponents
of transgenic technology also note
that scaling up transgenic
production involves increasing the
population of a herd rather than
building a mammalian cell
production facility that costs tens,
even hundreds of millions of dollars.
The capital costs of building and
maintaining a farm are also small in
comparison with building and
maintaining a typical biotech facility.
Other companies are leveraging the
capabilities of transgenic production
to develop recombinant forms of
proteins, such as blood proteins,
that can be difficult to express using
bioreactor based methods.
Collecting source material from a
“living bioreactor” also uses a wellestablished method: either milking
the animals or gathering the eggs,
depending on the species involved.
Dairy farming already incorporates
hygienic practices, and the
composition of milk, even as it varies
from species to species, is well known.
“Known composition,” however,
means that the milk must undergo
some intermediate processing to
remove much of its components
before fluid is introduced as starting
material to downstream purification
by chromatography.
Among the problems still to be
worked out are efficiency and the
speed with which a commercial
product can be produced in large
animals. The current methods of
producing transgenic animals have a
low rate of live births: The typical
success rate is 10–20%, and with the
use of microinjection techniques, the
successful expression of transgenes in
offspring runs at much less than 50%.
The next challenge is to identify
and screen founder animals that
produce high levels of protein. After
those founders are identified, it can
take months or years to breed and
establish a production herd,
SUPPLEMENT
depending on the species and their
age to sexual maturity. Therefore, low
successful expression of transgenes in
transgenic offspring is not a problem
when people are working with mice,
but it is costly when developing
transgenic livestock. Recently, nuclear
transfer technology has been shown
to significantly reduce the time
required for production of
recombinant proteins and to more
reliably establish “founders” to a
breeding herd in which all offspring
born are transgenic.
Each mammalian system will
introduce its own posttranslational
modifications, especially
glycosylation patterns (3).
Mammary tissue can carry out a
broad range of such modifications,
but whether those modifications are
immunogenic to humans depends
on the protein of interest and the
species being used.
Another concern is “leakage” of a
target protein into the circulation by
way of the mammary epithelial cells
— and as measured by increased
plasma levels of the protein
designed to be expressed only in the
animal’s milk. Therefore, unless the
transgene construct integrates in an
appropriate way in the genome,
certain highly active hormones and
cytokines could have detrimental
effects on the host animal and may
not be possible transgenically.
CREATING A TRANSGENIC ANIMAL
JE Smith, author of Biotechnology,
provides a useful sequential list of
steps toward creating a transgenic
animal, which are generally
applicable regardless of species:
• Identification and construction
of the foreign gene and any
promoter sequences (genetic
engineering)
• Microinjection of DNA directly
into the pronucleus of a single
fertilized egg (or introduction
through nuclear transfer, a viral
vector, or other means, as touched
on below)
• Implantation of these
engineered cells into surrogate
mothers
• Bringing the developing
embryo to term
A milking parlor
Purification skid and operator
Part of the purification process
(GTC BIOTHERAPEUTICS)
• Proving that the foreign DNA
has been stably and heritably
incorporated into the DNA of at
least some of the newborn offspring.
• Demonstrating that the gene is
regulated well enough to function
in its new environment (1).
Figure 1 illustrates one
company’s procedure.
Construction of the Foreign Gene:
Genetic engineering for a protein of
interest has already been discussed in
previous chapters. The focus of
transgenic production, however, is
the construction of a transgene: a
gene foreign to the animal species in
which it will be expressed. A
recombinant DNA construct is
formed by combining a cloned target
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Figure 1: Making a transgenic product (SCHEMATIC
protein gene with a regulatory
sequence (promoter) of a milkspecific gene that will direct its
expression to the mammary gland
during lactation (3, 5). Transgenic
production of proteins in blood
plasma/serum, urine, and semen has
also been investigated and may prove
feasible for some unique products
(e.g., see www.hematech.com for
production of human polyclonal
antibodies in transgenic bovine
plasma, and www.polyclonals.com for
production of humanized polyclonal
antibodies in rabbits). Some
companies are working on transgenic
hens, but milk appears to be the
primary choice for production of
recombinant proteins. Companies
have developed proprietary mammary
promoters, some of which contain
additional regulatory sequences to
further direct expression for
specialized applications — such as to
secrete a protein that would normally
be membrane-bound.
Why milk? Major milk-specific
proteins are caseins and whey
proteins, most of which have been
cloned and are well characterized.
According to one publication, the
mammary gland — with a cell
density of up to 1000 times that of
a mammalian cell-culture bioreactor
— can produce greater than 10
grams of recombinant protein per
liter of milk per day. (5).
Although major differences exist
in milk composition from species to
species, generally “milk is
approximately 85–90% water, the
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COURTESY OF
GTC THERAPEUTICS)
pH is 6.5–6.7 and as high as pH 6.8
in ewe’s milk” (7). A target protein
expressed in milk is usually found in
solution with a colloidal mixture of
fats and proteins in which are
suspended casein micelles, somatic
cells, and bacteria from the
lymphatic ducts of the udder (7).
Although purification methods
differ from company to company and
are still being developed and
optimized, generally the raw milk is
filtered to remove fat, casein, cells,
and other particulates, yielding a clear
amber-colored fluid. That fluid then
undergoes a capture chromatography
step specific for the therapeutic
protein, followed by additional
chromatography steps to achieve
clinical grade purity (7). So once the
capture is performed, downstream
processing is indistinguishable for the
products of transgenic animals and
cell culture or fermentation. Because
transgenics dramatically lowers the
cost of bulk production, the
Chromosomes from a transgenic
animal after fluorescence in situ
hybridization (FISH); the red and green
dots in the upper left show integration
of the transgene. (GTC BIOTHERAPEUTICS)
processing and purification stages
tend to be the most expensive part of
the manufacturing process in both
labor and materials. The overall cost
of purification of transgenically
produced material is about the same
as that for bioreactor-produced
material.
Lowering the Cost of Processing: As
an example of work being done to
further improve the downstream
processing of transgenic proteins
contained in milk, BioSante
Pharmaceuticals, Inc. (Lincolnshire,
Il; www.biosantepharma.com) has
patented calcium phosphate
nanoparticle (CAP) technology for
recovering more than 90% of drug
protein from milk, requiring less
(costly) downstream processing and
perhaps resulting in higher yields.
The scalable technique separates
(dissolves) clusters of milk caseins,
which make up 70–80% of total milk
protein, in initial processing steps;
caseins tend to aggregate, trapping
the therapeutic proteins (8, 9).
Speaking of costs, whereas milk
contains fewer proteins than
traditional fermentation broths,
chicken eggs contain only 12 total
proteins — one of those being
ovalbumin, which may be useful in
processing or formulation down the
line. A number of companies are
predicting successful production of
therapeutics in chicken eggs from
chimeric hens. So far they’re
claiming high, if variable, expression
levels and the potential for
simplified purification. We focus on
transgenic mammals here only
because they are further along in
development as an expression
system. (For the same reason, we do
not discuss investigations into
transgenic expression in blood,
urine, and semen.)
Microinjection: Pronuclear
microinjection, although not the
only method under development,
was the first method used. In this
method, the fertilized eggs used to
create the transgenes are flushed
from the oviducts of “superovulated
donor females”: females that have
been mated with fertile males and
that, depending on the species, may
SUPPLEMENT
have received pregnant mare serum
gonadotropin, fluorogestone acetate,
or prostoglandin (hormonally
stimulating them to produce lots of
eggs at once instead of the one or
two common in large animals). The
critical step is then to develop the
transgene and get it into embryo and
the embryo into the host female. In
this process, the transgene is injected
into the pronucleus of a fertilized
egg. The technician uses a specially
designed micromanipulation pipette
and works under extreme
magnification. It is tedious work, and
not all injections are successful.
Nuclear Transfer: Some companies
are no longer using microinjection
and have developed methods to
transfer nuclei isolated from
embryo-derived cells into oocytes
with their nuclei removed. The
advantage of nuclear transfer is that
its success rate replaces the timeconsuming process of culling
nontransgenic offspring from the
breeding program, and thereby
accelerates formation of the
transgenic herd.
The process is explained
succinctly on the Geron web site:
In this process, the nucleus
containing all of the chromosomal
DNA is removed from an egg cell
and replaced with the nucleus
containing all of the chromosomal
DNA from a donor somatic or
nonreproductive cell. Fusion
between the resulting egg cell and
the donor somatic nucleus results
in a new cell which gains a
complete set of chromosomes
derived entirely from the donor
nucleus. Mitochondrial DNA,
providing some of the genes for
energy production, resides outside
the nucleus and is provided by the
egg. After a brief culture period,
the resulting embryo is implanted
into the uterus of a female animal,
where it can develop and produce
the live birth of a cloned
offspring. The offspring is
essentially a genetic clone of the
animal from which the donor
nucleus was obtained. (10)
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In somatic cell nuclear transfer,
also called therapeutic cloning, a
somatic cell is fused with a
enucleated oocyte. The nucleus of
the somatic cell provides the genetic
information, and the oocyte
provides nutrients and other energyproducing materials necessary for
the embryo’s development (11).
Use of Viral Vectors: In another
method, the helper cell line from a
gene of interest is “packaged” into
an engineered viral vector: a virus still
encoded to “infect” but with the
disease-causing gene sequence
removed (replication deficient). The
hope is that, if the virus is injected
into the mammary gland during
hormone-induced mammogenesis,
females could begin producing the
protein in milk without having to
wait through gestation; and their
offspring would also express the
transgene (5, 12, 13). Production
levels thus far are lower than desired
(5), but in an early success, a gibbon
ape leukemia virus was used to
deliver the structural gene encoding
for human growth hormone to a
goat, and the hormone was expressed
in her mammary epithelial cells.
Implantation: After fertilized eggs
have been washed from the oviduct
of a superovulated female donor and
have received the transgene, they are
transferred to the oviduct or uterus
of a “pseudopregnant” recipient
animal and developed to term. Those
recipients are prepared for embryo
transfer by mating with vasectomized
males. The offspring are eventually
tested through a blood or tissue
sample (usually from the ear or tail)
for presence of the transgene. Then
the company must wait for the
maturity of the animals to test for
production of the protein of interest.
When microinjection techniques
are used, not all offspring will express
the transgene, and offspring that do
may express it at different levels or
even in different organ systems. The
consensus indicates that the success
rate of germ-line transmission of the
transgene averages 50% or less for
microinjection. The insertion site
may influence the expression levels or
even result in transgenic animals
showing no expression at all. For
these reasons, it is important to
characterize multiple founders to
select lines with desired phenotypes,
and if microinjection is used, several
generations may be required before a
stable transgenic herd is established.
The transgene will, however, be
transmitted to all offspring in nuclear
transfer techniques.
ANIMALS ON THE PHARM
Although a small number of
companies are working to develop
commercially viable transgenic
production of protein therapeutics,
many are working with multiple
species and with a number of
partnering agreements in place at
many different stages. Most
transgenic species are studied for
research applications as well as
potential commercial pharmaceutical
production.
Caveats: Transgenics in general is
a rapidly advancing field, and
keeping up to date on work in
progress is far from easy. Therefore,
the following examples (presented
alphabetically by species) attempt
only to summarize information
about work in progress that is
readily available; it is not inclusive,
nor can it present the complete
story of this segment of the
biotechnology industry. Efforts are
made here to use material no more
than two years old. Any claims of
cost savings and potential
therapeutic yields are offered to
emphasize the potential promise of
the expression system, but those
differ from company to company
and as the technology and
expression efficiencies advance (14).
Chickens and Eggs: Chickens and
roosters grow faster than most
mammals, can be raised in close
quarters, and can synthesize high
levels of protein in egg whites. A
big advantage in working with
chickens is our familiarity with them
gained from years of use in vaccine
and antibody production. Eggs
contain simple and wellcharacterized proteins (ovalbumin is
a specific protein already present).
They contain only 12 proteins to be
filtered out compared with as many
as 20,000 in traditional
SUPPLEMENT
fermentation. Chickens appear to
add correct sugars to glycosylated
proteins and can be raised at a cost
of around $20 a year per transgenic
chicken (15). One rooster can mate
with 10 hens in eight hours and can
produce 100,000 offspring a year.
Products in development include
vaccines; interferons, commercial
cytokines; human serum albumin;
HSA, insulin, and MAbs (from
germline transgenic chickens in
development). Additionally,
development plans are ongoing (12)
for proinsulin produced at
$10/gram (in contrast with $1550
to $3100 per gram using current
production methods).
Companies, Milestones: Avigenics
(Athens, GA, www.avigenics.com)
holds a patent on its “Windowing
Technology” for injecting foreign
genetic material through an aperture
in an egg shell; TranXenogen
(Shrewsbury, MA; www.tranxenogen.
com) holds a gene-testes transfection
technology and was the first to
express MAbs in the whites of
chimeric chicken eggs (proof of
principle); TransGenRx (Dallas, TX;
www.tgrx.com) and Viragen have
proprietary gene transfer vectors.
Viragen (Plantation, FL;
www.viragen.com) works with a
vector obtained from Oxford
BioMedica plc (San Diego, CA, and
Oxford, UK; www.oxfordbiomedica.
co.uk) with an exclusive license from
the Roslin Institute (Edinburgh,
UK; www.ri.bbsrc.ac.uk). Also of
interest, GenWay Biotech (San
Diego, CA; www.genwaybio.com) is
(among other activities), producing
gene-specific IgY (chicken)
antibodies.
Cows: The prospect of obtaining
the large amounts of milk produced
by dairy cows made them early
candidates for studies into transgenic
production. Dairy cattle produce 23
g of protein/kg of body weight
during peak lactation. A 1997 article
estimated that one transgenic cow
could produce the annual US market
needs for Factors VIII and IX; two
cows could produce enough protein
C, three cows could produce
enough antithrombin III, 17 cows
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could produce enough fibrinogen,
and “35 ⫻ 103” cows could make
enough HSA (16). The
disadvantages, however, include both
their size (and therefore the cost of
their “pharming” habitat) and the
seven to eight years required to
produce a milking herd (3).
Products in development include
HSA, rHSA, and human milk
protein. A research farm in Alapitkä,
Lapinlahti (Finland) is working to
produce lactoferrin for medical use
(http://opp.ysao.fi/~pemo/future/
breeding.htm).
GTC BIOTHERAPEUTICS (WWW.GTC-BIO.COM)
Companies:
• GTC Biotherapeutics, Inc.
(with about a dozen partners) (17)
• The Dutch company Pharming
BV (Leiden, The Netherlands;
www.pharming.com) was the main
company working with development
of transgenic cows with the creation
of Herman, the bull, designed to
breed progeny that produce
lactoferrin.
• Hematech, LLC (Westport,
CT; www.hematech.com) is working
on production of human polyclonal
antibodies in transgenic bovine
plasma.
Goats: Goats are smaller than
cattle and also produce a large
amount of milk in a shorter time.
Expression though natural lactation
takes 15–18 months, but it can be
induced earlier.
Products in development include
alpha-1 proteinase inhibitor ; MAbs,
Ig fusion proteins, ATryn
(recombinant human antithrombin
III); and tPA.
Companies, Milestones: GTC’s
submission of a market
authorization application to the
EMEA for Atryn is the first
application submitted in the United
States or Europe for review and
approval of a recombinant
therapeutic protein produced
transgenically. It is also the first
transgenic recombinant protein to
complete phase III trials (18).
Mice: Mice can be easily raised in
a laboratory; gestation takes three
weeks, with sexual maturity reached
in one month, so initial results are
possible in six months or less. They
are also inexpensive to maintain.
Mouse milk has a higher
concentration of acidic whey protein
— a desired characteristic for some
applications. Another advantage to
using transgenic mice in research is
that mice lack the cell-surface
molecule that serves as the receptor
for the polio virus in humans;
transgenic mice can express the
human gene for polio and develop
symptoms of the disease.
Products in Development: Mice
are mostly used in basic research for
transgenesis feasibility studies and as
disease models. Knockout mice
created with a nonfunctional gene
are tools for studying gene
functions.
Mice may yield small amounts of
milk compared with larger species,
but they are still powerful little
“bioreactors.” Peptides derived from
antineoplastic urinary protein
(ANUP) were shown to reduce
tumor burden by 70% in nude mice
implanted with human cervical
cancer cells (an avian transgenic
platform is in development for
related recombinant protein
production). Other research with
transgenic mice includes expression
of malaria protein for possible
vaccine; MAbs and Ig fusion
proteins; alpha-1 proteinase
inhibitor; antithrombin III;
angiogenin; beta interferon; cystic
fibrosis transmembrane regulator;
Factor X; glutamic acid
decarboxylase; glucocerebrosidase;
HGH, HSA, tPA, myelin basic
protein; proinsulin; prolactin; soluble
CD4-HIV receptor; and fibrinogen.
Companies, Milestones: The first
transgenic mice were developed in
1981. TranXenoGen holds a
worldwide license for ANUP;
Invitrogen is manufacturing,
SUPPLEMENT
marketing, and distributing GTC’s
patented transgenic expression
system (pBC1 kit) for inserting
genes into mouse DNA. A number
of other companies and
governmental, industrial, and
university laboratories are producing
various forms of knockout mice and
other forms of transgenic mice for
research.
WWW.SARDI.SA.GOV.AU/PAGES/LIVESTOCK/
PIGS/SERVICES/ABOUT_PPPI_4.HTM
Pigs: Pigs grow quite large and do
require an investment in space and
food, but their 114-day gestation
period and one-year generation
interval facilitate propagation and
expansion of transgenic lines.
Primarily the full-sized domestic
swine are used.
Products in Development: Pigs are
mostly used for xenotransplantation
research, but transgenic pigs have
produced human hemoglobin and
human protein C. Pigs are proving
to be valuable research models for
retinitis pigmentosa and other eye
diseases; future applications may
benefit from similarities between
human and pig digestive and
cardiovascular systems.
Companies: Most groups working
with transgenic pigs are universitybased; one example: Duke University
Medical Center, North Carolina State
University, and NIH are collaborating
on research into treatment of retinitis
pigmentosa (19).
Rabbits: Rabbits appear to be
attractive candidates as transgenic
animals. They are cost-effective to
raise, they reach sexual maturity
after five to six months, average
eight offspring per pregnancy, and
can produce up to 40 embryos
following superovulation. Their milk
has a high protein content, and they
can produce up to 250 mL of milk a
day. Kilogram-scale quantities of
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purified therapeutic protein can be
obtained annually from 400
transgenic female rabbits.
Heterologous proteins produced in
rabbit milk and serum have achieved
an average yield of 20 gm/year
from four to five liters of annual
production of milk (20).
The first transgenic rabbits were
produced in 1985. The lipid
metabolism in rabbits is closer to
that of humans than is that of mice,
so rabbits are good models for
studies of athrosclerosis. Rabbits
also replicate HIV very well through
expression of the human CD4 gene
in their T lymphocytes. They
express rabbit papilloma and EJ-ras
genes, which may make them a
good model for skin cancer studies
Rabbits also grow to be fairly
large (compared with mice and rats,
at least) so maintaining a large
number of rabbits for commercial
production still requires a sizable
financial commitment — but again,
not a commitment approaching the
cost of building a manufacturing
facility.
Products in development include
recombinant human C1 inhibitor
for hereditary angioedema, human
erythropoietin, extracellular
superoxide dismutase, human alphaantitrypsin (produced in blood);
human interleukin 2; tPA;
chymosin; alpha glucosidase, and
human growth hormone;
chimerized MAbs for use as
radioimmunotherapeutic agents
against cancer, MAbs against
Hodgkin’s disease and renal cell
carcinoma; and human calcitonin.
Companies include Therapeutic
Human Polyclonals Inc. (Mountain
View, CA; www.polyclonals.com),
Pharming BV, and BioProtein
Technologies (Massena, France,
and Cambridge, MA;
www.bioprotein.com).
Sheep: When the COL1A1 gene
from connective tissue cells
(fibroblasts) was combined with a
vector and fused with enucleated
sheep eggs, two lambs secreted milk
containing 650 µg/mL. Fibroblasts
secrete Type 1 collagen, the absence
of which in humans causes
osteogenesis imperfecta.
Products in Development: Sheep
were the first transgenic livestock in
1985. Products produced in sheep
milk include fibrinogen (the major
constituent, with thrombin and
Factor XIII, of fibrin sealants used
in wound sealing); human Factor
VII, Factor IX, and activated
protein C, which prevents blood
clots; and alpha-1-antitrypsin
(AAT).
Companies: PPL Therapeutics
was the main company involved,
using Roslin Institute technology.
BPI’s senior technical editor meets
Dolly, the cloned sheep (1999)
Other Species: Frogs, nematodes,
and marine invertebrates (sea urchins
and mollusks for example) have
been used to study various promoter
elements and gene transfer
technology. Although fish have been
used at the research scale to produce
growth hormone, transgenic fish are
being developed mostly for
applications in aquaculture.
Cytoplasmic injection is possible
with fish (not so in mammals)
because embryo development
happens externally; 35–80%
microinjection survival; 10–70%
transgenic production.
Chesapeake PERL, Inc. (College
Park, MD; www.c-perl.com) received
a $2 million, three-year National
Institute of Standards and
Technology Advanced Technology
Program (ATP) grant to genetically
transform caterpillars to produce
humanized glycoprotein
modifications. The company uses a
baculovirus to express recombinant
proteins in whole insect larvae by
incorporating biological pathways
into caterpillars (“Transpillars”) to
produce mammalian glycoprotein
structures rather than those naturally
SUPPLEMENT
occurring in insects. The company
hopes that using Transpillars to
manufacture therapeutic proteins
may increase the number of likely
drug targets available for production
in the C-PERL system (21).
THE REGULATORY ISSUES
Regulatory agencies in the United
States and Europe require that
transgenically produced therapeutics
be safe, pure, well-characterized
(identity), and of demonstrated
potency — following Good
Manufacturing Practices (GMPs).
Freedom from potential animal
pathogens, demonstrated lot-to-lot
consistency, and elimination of
immunogenicity are also elements
necessary for regulatory approval.
Although viral removal steps for
transgenics are analogous to those
for cell-culture-derived products,
and although the mammary gland
itself may filter out systemic
pathogens and viruses from the milk
reservoir (7), concern remains about
unknown milk-borne animal
pathogens, just as there may be
concerns about unknown pathogens
in any recombinant system —
including CHO systems.
Transgenic “pharmers” must be
familiar with 21 CFR regulations —
regulations applicable to all
biologicals (parts 58, 210, 211,
600, and 680). They must also
operate under Good Agricultural
Practices (GAP) ensuring protection
of their animals from exposure to
potential disease vectors. As far as
prion diseases are concerned, there
seems to be little to no risk of
transmission through milk.
The following regulatory
documents are those relevant to
therapeutic proteins produced in
transgenic animals in the United
States. The corresponding CPMP
document went into effect in 1995
and is titled Use of Transgenic
Animals in the Manufacture of
Biological Medicinal Products for
Human Use.
1985 Points to Consider in
Production and Testing of New
Drugs and Biologics Produced by
Recombinant DNA Technology
50
BioProcess International
JUNE 2004
1991 Points to Consider in
Human Somatic Cell Therapy and
Gene Therapy
1992 Nucleic Acid
Characterization of Cell Lines Used
to Produce Biologicals
1993 Points to Consider in
Characterization of Cell Lines Used
to Produce Biologicals
1994 Points to Consider in
Manufacture and Testing of
Monoclonal Antibody Products for
Human Use
1995 Points to Consider in the
Manufacture and Testing of
Therapeutics Products for Human
Use Derived from Transgenic
Animals. This is the major
regulatory document pertaining to
transgenic animals and covers the
following five points: generation and
characterization of the transgene
construct, creation and
characterization of the founder
animal and its propagation,
maintenance of transgenic animals
and production herds, purification
and characterization of transgenic
products, and preclinical safety
evaluations.
1997 Points to Consider in the
Manufacture and Testing of
Monoclonal Antibody Products for
Human Use
Other agencies involved in
overseeing maintenance of
transgenic herds include the United
States Department of Agriculture
(USDA), which has authority over
animal-disease testing criteria within
the United States (also overseeing
animal import/export criteria). The
USDA also ensures animal health
and welfare through oversight of the
Animal Welfare Act (AWA) (6).
Transgenic “pharming” must
comply with Good Agricultural
Practices (GAP) and the accreditation
requirements of the American
Association of Laboratory Animal Care
International (AAALAC-International).
GAP considerations include certifying
that animals are scrapie-free; that the
facility is separated from other
livestock species; that SOPs are in
place for animal care, identification,
and tracking; that animal feed contain
no animal by-products; that sperm
and embryo banks are maintained to
preserve the quality of the breeding
stock; and that milk collection,
handling, storage, and transport
follow SOPs (7).
A PROMISING TECHNOLOGY
The future production of
recombinant proteins in transgenic
animals looks very promising.
Methods of producing transgenic
animals and their offspring differ
from company to company and
according to the therapeutic protein
of interest. Because most target
proteins are expressed under the
control of milk-specific gene
regulatory elements in a variety of
species, certain species produce
particular types of protein more
effectively than others. Additionally,
the amount of published literature
regarding transgenics development
specific to individual species and
recombinant proteins is large and
growing. Some companies have
been issued significant patents for
their proprietary vectors and/or
expression systems. Still others are
close to or already entering latestage clinical trials, indicating that
the first marketed therapeutic
product produced transgenically
may not be far in our future (22).
Regulatory and public acceptance
of therapeutic products produced in
the milk of transgenic animals may
not prove as sensitive as regulatory
positions over transgenically
modified plant and insect species
that might escape into wild
populations. Also these valuable (in
most cases pampered) “living
bioreactors” will not be allowed to
enter the food chain. Public
acceptance of genetically modified
crops and animals is fraught with
legitimate concerns over issues such
as “genetic drift” and the associated
need to ensure isolation and control.
A transgenic mosquito or tse-tse fly
or even some species of fish would
indeed be more difficult to contain
than the larger animals used in
therapeutic protein production.
Even the term, living bioreactor,
reflects a position that bothers many
people: that of turning a living
creature into a tool for human
SUPPLEMENT
benefit (as has been done with agricultural uses of
livestock for food and clothing — though even those
traditional practices have their detractors). Responsible
discussion of moral and ethical issues (see Chapter 6)
must continue inside and around the biotechnology
industry, especially until environmental safety issues have
been addressed.
REFERENCES
1 Smith, JE. Biotechnology (Third Edition). Studies in Biology
Series. Cambridge University Press: Cambridge, UK, 1996, p. 174.
2 Amsterdam, A; Lin, S; Hopkins, N. Transient and Transgenic
Expression of Green Fluorescent Protein (GFP) in Living Zebrafish
Embryos. CLONTECHniques 1995 (July), [email protected].
3 Walsh, G. Proteins: Biochemistry and Biotechnology. John Wiley
and Sons, Inc.: New York, NY, 2002, pp. 73–77.
4 www.csun.edu/~hcbio027/biotechnology/lec14/lec14.html.
5 Genzyme Transgenics Corporation. Transgenically Produced
Biopharmaceuticals: Production of Recombinant Proteins in the Milk of
Transgenic Animals. www.genzyme.com/transgenics.
6 US Gov CFR site, 9 CFR, parts 1–3; also Gavin, WG. The
Future of Transgenics. Regulatory Affairs Focus, May 2001, pp.
13–19.
7 Meade, HM; et al. Expression of Recombinant Proteins in the
Milk of Transgenic Animals. Gene Expression Systems: Using Nature for
the Art of Expression. Academic Press: 1999, p. 415.
8 BioSante Pharmaceuticals, Inc. Receives Patent for New
Method of Processing Drug Proteins.
www.biospace.com/news_story.cfm?StoryID=5228804&full=1.
9 CAP Milk Proteins Isolation.
www.biosantepharma.com/products/milkpatent.html.
10 www.geron.com/02.03_nt.html.
11 www.molbio.princeton.edu/courses/
mb427/2001/projects/09/transfer.htm.
12 Metzenberg, S. Transgenic Animals – for Basic Research and
Biotechnology. Online Lecture Notes from California Sate University
Northridge, Biology 470 — Biotechnology.
www.csun.edu/~hcbio027/biotechnology/lec14/lec14.html.
13 Kimball, JW. Transgenic Animals (2004).
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/W/
Welcome.html.
14 Most of the information in the “Animals on the Pharm”
section, unless otherwise noted, comes from Transgenic Animals:
Generation and Use. Houdebine, LM, Ed. Harwood Academic
Publishers: France, 1997.
15 See www.louisianaip.org/
story.pl?NewsID=30 and www.latechnologyguide.com/news01.php.
16 Wall, RJ; Kerr, DE; Bondioli, KR. Transgenic Dairy Cattle:
Genetic Engineering on a Large Scale. J. Dairy Sci. 1997, 80: 22132224.
17 www.genzymetransgenics.com/products/strategic.html.
18 www.genzymetransgenics.com/products/atryn.html; and
www.genzymetransgenics.com/pressreleases/pr022704.html.
19 http://rp.mc.duke.edu/how.asp?TextOnly=No.
20 de Martynoff, G; Fouassier, A. Using Transgenic Rabbits for
Industrial Scale-up: From Gene to Industrial-Scale GMP-Standard
Therapeutic Proteins. Genetic Eng. News 2003, 23(13): 39–42.
21 Protein Therapeutics Made By Insects. BioProcess International
2003, 1(12), p. 10.
22 Animal Pharming: The Industrialization of Transgenic
Animals, December1999, www.aphis.usda.gov/
ceah/cei/animal_pharming.htm. SUPPLEMENT