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 GTC BIOTHERAPEUTICS (WWW.GTC-BIO.COM)
into ova what would otherwise take
years with traditional breeding companies appear to be close to produced transgenically for use in
programs: mosquitoes incapable of achieving market approval. In fact, modeling human diseases in
carrying malaria, for example, or one company, GTC Biotherapeutics, preclinical studies or generating
production of leaner meat by beef Inc. (Framingham, MA; www.gtc- antibody candidates for further
cattle. In an early experiment, a bio.com), is undergoing review for development. Transgenic companies
“Super Mouse” was created when a market authorization in Europe for with transgenic platform technologies
rat gene for growth hormone was ATryn, its recombinant form of to produce clinical materials may
injected into and expressed by the human antithrombin expressed in partner with other pharmaceutical
genome of a parent mouse (1). the milk of transgenic goats. and biotechnology companies to
Aside from applications designed In the first successful case of produce their products transgenically
to improve characteristics of a transgenic production of a in addition to developing an in-house
particular species or to create therapeutic protein, mouse embryos pipeline of products. Some of these
“specialized” research animals were injected with a DNA construct. companies are also capable of the
(expressing green fluorescent It was made by inserting the downstream processing development
protein in zebrafish embryos, for promoter and upstream regulatory and manufacturing of transgenic
example, as a marker for genetic sequence from the mouse whey products, at least to clinical scale,
studies) (2), transgenic technology is acidic protein gene (murine milk whereas others rely on the
also achieving increasing success as contains distinctively high levels of manufacturing capabilities of partners.
an alternative to producing proteins this whey protein) into the gene Transgenic production offers
in cell culture and microbial coding for human tissue plasminogen certain advantages compared to
systems. The goal of such work is to activator (tPA). The resultant traditional mammalian cell
produce large quantities of transgenic offspring produced production systems. One source
recombinant proteins in the milk or biologically active tPA in their milk: a compares the average generation of
plasma of transgenic mammals or in heterologous protein of tremendous 0.2–1.0 g/L of recombinant protein
the eggs of transgenic hens. Many therapeutic potential (3). in highly optimized cell cultures to
of these efforts are progressing Several companies are already possible expression levels of
through clinical trials, and a few selling research-grade products 2–10 g/L of milk in transgenic
(including “customized” mice)
40 BioProcess International JUNE 2004 SUPPLEMENT
livestock (4). Another mentions that depending on the species and their
one sheep can produce 2–3 L of age to sexual maturity. Therefore, low
milk per day. If a recombinant successful expression of transgenes in
protein is expressed at a level of transgenic offspring is not a problem
1 g/L, a single sheep could produce when people are working with mice,
up to 20 g of product per week (3). but it is costly when developing
Similar estimates are offered by transgenic livestock. Recently, nuclear
many other companies. Proponents transfer technology has been shown
of transgenic technology also note to significantly reduce the time
that scaling up transgenic required for production of
production involves increasing the recombinant proteins and to more A milking parlor
population of a herd rather than reliably establish “founders” to a
building a mammalian cell breeding herd in which all offspring
production facility that costs tens, born are transgenic.
even hundreds of millions of dollars. Each mammalian system will
The capital costs of building and introduce its own posttranslational
maintaining a farm are also small in modifications, especially
comparison with building and glycosylation patterns (3).
maintaining a typical biotech facility. Mammary tissue can carry out a
Other companies are leveraging the broad range of such modifications,
capabilities of transgenic production but whether those modifications are
to develop recombinant forms of immunogenic to humans depends
Purification skid and operator
proteins, such as blood proteins, on the protein of interest and the
that can be difficult to express using species being used.
bioreactor based methods. Another concern is “leakage” of a
Collecting source material from a target protein into the circulation by
“living bioreactor” also uses a well- way of the mammary epithelial cells
established method: either milking — and as measured by increased
the animals or gathering the eggs, plasma levels of the protein
depending on the species involved. designed to be expressed only in the
Dairy farming already incorporates animal’s milk. Therefore, unless the
hygienic practices, and the transgene construct integrates in an
composition of milk, even as it varies appropriate way in the genome,
from species to species, is well known. certain highly active hormones and
“Known composition,” however, cytokines could have detrimental
means that the milk must undergo effects on the host animal and may
some intermediate processing to not be possible transgenically.
Part of the purification process
remove much of its components (GTC BIOTHERAPEUTICS)
before fluid is introduced as starting CREATING A TRANSGENIC ANIMAL
material to downstream purification JE Smith, author of Biotechnology, • Proving that the foreign DNA
by chromatography. provides a useful sequential list of has been stably and heritably
Among the problems still to be steps toward creating a transgenic incorporated into the DNA of at
worked out are efficiency and the animal, which are generally least some of the newborn offspring.
speed with which a commercial applicable regardless of species: • Demonstrating that the gene is
product can be produced in large • Identification and construction regulated well enough to function
animals. The current methods of of the foreign gene and any in its new environment (1).
producing transgenic animals have a promoter sequences (genetic Figure 1 illustrates one
low rate of live births: The typical engineering) company’s procedure.
success rate is 10–20%, and with the • Microinjection of DNA directly Construction of the Foreign Gene:
use of microinjection techniques, the into the pronucleus of a single Genetic engineering for a protein of
successful expression of transgenes in fertilized egg (or introduction interest has already been discussed in
offspring runs at much less than 50%. through nuclear transfer, a viral previous chapters. The focus of
The next challenge is to identify vector, or other means, as touched transgenic production, however, is
and screen founder animals that on below) the construction of a transgene: a
produce high levels of protein. After • Implantation of these gene foreign to the animal species in
those founders are identified, it can engineered cells into surrogate which it will be expressed. A
take months or years to breed and mothers recombinant DNA construct is
establish a production herd, • Bringing the developing formed by combining a cloned target
embryo to term
SUPPLEMENT JUNE 2004 BioProcess International 41
Figure 1: Making a transgenic product (SCHEMATIC COURTESY OF GTC THERAPEUTICS)
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
protein gene with a regulatory pH is 6.5–6.7 and as high as pH 6.8 recovering more than 90% of drug
sequence (promoter) of a milk- in ewe’s milk” (7). A target protein protein from milk, requiring less
specific gene that will direct its expressed in milk is usually found in (costly) downstream processing and
expression to the mammary gland solution with a colloidal mixture of perhaps resulting in higher yields.
during lactation (3, 5). Transgenic fats and proteins in which are The scalable technique separates
production of proteins in blood suspended casein micelles, somatic (dissolves) clusters of milk caseins,
plasma/serum, urine, and semen has cells, and bacteria from the which make up 70–80% of total milk
also been investigated and may prove lymphatic ducts of the udder (7). protein, in initial processing steps;
feasible for some unique products Although purification methods caseins tend to aggregate, trapping
(e.g., see www.hematech.com for differ from company to company and the therapeutic proteins (8, 9).
production of human polyclonal are still being developed and Speaking of costs, whereas milk
antibodies in transgenic bovine optimized, generally the raw milk is contains fewer proteins than
plasma, and www.polyclonals.com for filtered to remove fat, casein, cells, traditional fermentation broths,
production of humanized polyclonal and other particulates, yielding a clear chicken eggs contain only 12 total
antibodies in rabbits). Some amber-colored fluid. That fluid then proteins — one of those being
companies are working on transgenic undergoes a capture chromatography ovalbumin, which may be useful in
hens, but milk appears to be the step specific for the therapeutic processing or formulation down the
primary choice for production of protein, followed by additional line. A number of companies are
recombinant proteins. Companies chromatography steps to achieve predicting successful production of
have developed proprietary mammary clinical grade purity (7). So once the therapeutics in chicken eggs from
promoters, some of which contain capture is performed, downstream chimeric hens. So far they’re
additional regulatory sequences to processing is indistinguishable for the claiming high, if variable, expression
further direct expression for products of transgenic animals and levels and the potential for
specialized applications — such as to cell culture or fermentation. Because simplified purification. We focus on
secrete a protein that would normally transgenics dramatically lowers the transgenic mammals here only
be membrane-bound. cost of bulk production, the because they are further along in
Why milk? Major milk-specific development as an expression
proteins are caseins and whey system. (For the same reason, we do
proteins, most of which have been not discuss investigations into
cloned and are well characterized. transgenic expression in blood,
According to one publication, the urine, and semen.)
mammary gland — with a cell Microinjection: Pronuclear
density of up to 1000 times that of microinjection, although not the
a mammalian cell-culture bioreactor only method under development,
— can produce greater than 10 was the first method used. In this
grams of recombinant protein per method, the fertilized eggs used to
liter of milk per day. (5). create the transgenes are flushed
Although major differences exist from the oviducts of “superovulated
in milk composition from species to Chromosomes from a transgenic donor females”: females that have
animal after fluorescence in situ
species, generally “milk is hybridization (FISH); the red and green
been mated with fertile males and
approximately 85–90% water, the dots in the upper left show integration that, depending on the species, may
of the transgene. (GTC BIOTHERAPEUTICS)
42 BioProcess International JUNE 2004 SUPPLEMENT
have received pregnant mare serum In somatic cell nuclear transfer, these reasons, it is important to
gonadotropin, fluorogestone acetate, also called therapeutic cloning, a characterize multiple founders to
or prostoglandin (hormonally somatic cell is fused with a select lines with desired phenotypes,
stimulating them to produce lots of enucleated oocyte. The nucleus of and if microinjection is used, several
eggs at once instead of the one or the somatic cell provides the genetic generations may be required before a
two common in large animals). The information, and the oocyte stable transgenic herd is established.
critical step is then to develop the provides nutrients and other energy- The transgene will, however, be
transgene and get it into embryo and producing materials necessary for transmitted to all offspring in nuclear
the embryo into the host female. In the embryo’s development (11). transfer techniques.
this process, the transgene is injected Use of Viral Vectors: In another
into the pronucleus of a fertilized method, the helper cell line from a ANIMALS ON THE PHARM
egg. The technician uses a specially gene of interest is “packaged” into Although a small number of
designed micromanipulation pipette an engineered viral vector: a virus still companies are working to develop
and works under extreme encoded to “infect” but with the commercially viable transgenic
magnification. It is tedious work, and disease-causing gene sequence production of protein therapeutics,
not all injections are successful. removed (replication deficient). The many are working with multiple
Nuclear Transfer: Some companies hope is that, if the virus is injected species and with a number of
are no longer using microinjection into the mammary gland during partnering agreements in place at
and have developed methods to hormone-induced mammogenesis, many different stages. Most
transfer nuclei isolated from females could begin producing the transgenic species are studied for
embryo-derived cells into oocytes protein in milk without having to research applications as well as
with their nuclei removed. The wait through gestation; and their potential commercial pharmaceutical
advantage of nuclear transfer is that offspring would also express the production.
its success rate replaces the time- transgene (5, 12, 13). Production Caveats: Transgenics in general is
consuming process of culling levels thus far are lower than desired a rapidly advancing field, and
nontransgenic offspring from the (5), but in an early success, a gibbon keeping up to date on work in
breeding program, and thereby ape leukemia virus was used to progress is far from easy. Therefore,
accelerates formation of the deliver the structural gene encoding the following examples (presented
transgenic herd. for human growth hormone to a alphabetically by species) attempt
The process is explained goat, and the hormone was expressed only to summarize information
succinctly on the Geron web site: in her mammary epithelial cells. about work in progress that is
Implantation: After fertilized eggs readily available; it is not inclusive,
In this process, the nucleus have been washed from the oviduct nor can it present the complete
containing all of the chromosomal of a superovulated female donor and story of this segment of the
DNA is removed from an egg cell have received the transgene, they are biotechnology industry. Efforts are
and replaced with the nucleus transferred to the oviduct or uterus made here to use material no more
containing all of the chromosomal of a “pseudopregnant” recipient than two years old. Any claims of
DNA from a donor somatic or animal and developed to term. Those cost savings and potential
nonreproductive cell. Fusion recipients are prepared for embryo therapeutic yields are offered to
between the resulting egg cell and transfer by mating with vasectomized emphasize the potential promise of
the donor somatic nucleus results males. The offspring are eventually the expression system, but those
in a new cell which gains a tested through a blood or tissue differ from company to company
complete set of chromosomes sample (usually from the ear or tail) and as the technology and
derived entirely from the donor for presence of the transgene. Then expression efficiencies advance (14).
nucleus. Mitochondrial DNA, the company must wait for the Chickens and Eggs: Chickens and
providing some of the genes for maturity of the animals to test for roosters grow faster than most
energy production, resides outside production of the protein of interest. mammals, can be raised in close
the nucleus and is provided by the When microinjection techniques quarters, and can synthesize high
egg. After a brief culture period, are used, not all offspring will express levels of protein in egg whites. A
the resulting embryo is implanted the transgene, and offspring that do big advantage in working with
into the uterus of a female animal, may express it at different levels or chickens is our familiarity with them
where it can develop and produce even in different organ systems. The gained from years of use in vaccine
the live birth of a cloned consensus indicates that the success and antibody production. Eggs
offspring. The offspring is rate of germ-line transmission of the contain simple and well-
essentially a genetic clone of the transgene averages 50% or less for characterized proteins (ovalbumin is
animal from which the donor microinjection. The insertion site a specific protein already present).
nucleus was obtained. (10) may influence the expression levels or They contain only 12 proteins to be
even result in transgenic animals filtered out compared with as many
showing no expression at all. For as 20,000 in traditional
44 BioProcess International JUNE 2004 SUPPLEMENT
fermentation. Chickens appear to could produce enough fibrinogen, approval of a recombinant
add correct sugars to glycosylated and “35 ⫻ 103” cows could make therapeutic protein produced
proteins and can be raised at a cost enough HSA (16). The transgenically. It is also the first
of around $20 a year per transgenic disadvantages, however, include both transgenic recombinant protein to
chicken (15). One rooster can mate their size (and therefore the cost of complete phase III trials (18).
with 10 hens in eight hours and can their “pharming” habitat) and the Mice: Mice can be easily raised in
produce 100,000 offspring a year. seven to eight years required to a laboratory; gestation takes three
Products in development include produce a milking herd (3). weeks, with sexual maturity reached
vaccines; interferons, commercial Products in development include in one month, so initial results are
cytokines; human serum albumin; HSA, rHSA, and human milk possible in six months or less. They
HSA, insulin, and MAbs (from protein. A research farm in Alapitkä, are also inexpensive to maintain.
germline transgenic chickens in Lapinlahti (Finland) is working to Mouse milk has a higher
development). Additionally, produce lactoferrin for medical use concentration of acidic whey protein
development plans are ongoing (12) (http://opp.ysao.fi/~pemo/future/ — a desired characteristic for some
for proinsulin produced at breeding.htm). applications. Another advantage to
$10/gram (in contrast with $1550 using transgenic mice in research is
to $3100 per gram using current that mice lack the cell-surface
production methods). molecule that serves as the receptor
Companies, Milestones: Avigenics for the polio virus in humans;
(Athens, GA, www.avigenics.com) transgenic mice can express the
holds a patent on its “Windowing human gene for polio and develop
Technology” for injecting foreign symptoms of the disease.
genetic material through an aperture Products in Development: Mice
in an egg shell; TranXenogen are mostly used in basic research for
(Shrewsbury, MA; www.tranxenogen. transgenesis feasibility studies and as
GTC BIOTHERAPEUTICS (WWW.GTC-BIO.COM)
com) holds a gene-testes transfection disease models. Knockout mice
technology and was the first to Companies: created with a nonfunctional gene
express MAbs in the whites of • GTC Biotherapeutics, Inc. are tools for studying gene
chimeric chicken eggs (proof of (with about a dozen partners) (17) functions.
principle); TransGenRx (Dallas, TX; • The Dutch company Pharming Mice may yield small amounts of
www.tgrx.com) and Viragen have BV (Leiden, The Netherlands; milk compared with larger species,
proprietary gene transfer vectors. www.pharming.com) was the main but they are still powerful little
Viragen (Plantation, FL; company working with development “bioreactors.” Peptides derived from
www.viragen.com) works with a of transgenic cows with the creation antineoplastic urinary protein
vector obtained from Oxford of Herman, the bull, designed to (ANUP) were shown to reduce
BioMedica plc (San Diego, CA, and breed progeny that produce tumor burden by 70% in nude mice
Oxford, UK; www.oxfordbiomedica. lactoferrin. implanted with human cervical
co.uk) with an exclusive license from • Hematech, LLC (Westport, cancer cells (an avian transgenic
the Roslin Institute (Edinburgh, CT; www.hematech.com) is working platform is in development for
UK; www.ri.bbsrc.ac.uk). Also of on production of human polyclonal related recombinant protein
interest, GenWay Biotech (San antibodies in transgenic bovine production). Other research with
Diego, CA; www.genwaybio.com) is plasma. transgenic mice includes expression
(among other activities), producing Goats: Goats are smaller than of malaria protein for possible
gene-specific IgY (chicken) cattle and also produce a large vaccine; MAbs and Ig fusion
antibodies. amount of milk in a shorter time. proteins; alpha-1 proteinase
Cows: The prospect of obtaining Expression though natural lactation inhibitor; antithrombin III;
the large amounts of milk produced takes 15–18 months, but it can be angiogenin; beta interferon; cystic
by dairy cows made them early induced earlier. fibrosis transmembrane regulator;
candidates for studies into transgenic Products in development include Factor X; glutamic acid
production. Dairy cattle produce 23 alpha-1 proteinase inhibitor ; MAbs, decarboxylase; glucocerebrosidase;
g of protein/kg of body weight Ig fusion proteins, ATryn HGH, HSA, tPA, myelin basic
during peak lactation. A 1997 article (recombinant human antithrombin protein; proinsulin; prolactin; soluble
estimated that one transgenic cow III); and tPA. CD4-HIV receptor; and fibrinogen.
could produce the annual US market Companies, Milestones: GTC’s Companies, Milestones: The first
needs for Factors VIII and IX; two submission of a market transgenic mice were developed in
cows could produce enough protein authorization application to the 1981. TranXenoGen holds a
C, three cows could produce EMEA for Atryn is the first worldwide license for ANUP;
enough antithrombin III, 17 cows application submitted in the United Invitrogen is manufacturing,
States or Europe for review and
46 BioProcess International JUNE 2004 SUPPLEMENT
marketing, and distributing GTC’s purified therapeutic protein can be Products in Development: Sheep
patented transgenic expression obtained annually from 400 were the first transgenic livestock in
system (pBC1 kit) for inserting transgenic female rabbits. 1985. Products produced in sheep
genes into mouse DNA. A number Heterologous proteins produced in milk include fibrinogen (the major
of other companies and rabbit milk and serum have achieved constituent, with thrombin and
governmental, industrial, and an average yield of 20 gm/year Factor XIII, of fibrin sealants used
university laboratories are producing from four to five liters of annual in wound sealing); human Factor
various forms of knockout mice and production of milk (20). VII, Factor IX, and activated
other forms of transgenic mice for The first transgenic rabbits were protein C, which prevents blood
research. produced in 1985. The lipid clots; and alpha-1-antitrypsin
metabolism in rabbits is closer to (AAT).
that of humans than is that of mice, Companies: PPL Therapeutics
so rabbits are good models for was the main company involved,
studies of athrosclerosis. Rabbits using Roslin Institute technology.
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
WWW.SARDI.SA.GOV.AU/PAGES/LIVESTOCK/ good model for skin cancer studies
PIGS/SERVICES/ABOUT_PPPI_4.HTM
Rabbits also grow to be fairly
Pigs: Pigs grow quite large and do large (compared with mice and rats,
require an investment in space and at least) so maintaining a large
food, but their 114-day gestation number of rabbits for commercial
period and one-year generation production still requires a sizable BPI’s senior technical editor meets
interval facilitate propagation and financial commitment — but again, Dolly, the cloned sheep (1999)
expansion of transgenic lines. not a commitment approaching the
Primarily the full-sized domestic cost of building a manufacturing Other Species: Frogs, nematodes,
swine are used. facility. and marine invertebrates (sea urchins
Products in Development: Pigs are Products in development include and mollusks for example) have
mostly used for xenotransplantation recombinant human C1 inhibitor been used to study various promoter
research, but transgenic pigs have for hereditary angioedema, human elements and gene transfer
produced human hemoglobin and erythropoietin, extracellular technology. Although fish have been
human protein C. Pigs are proving superoxide dismutase, human alpha- used at the research scale to produce
to be valuable research models for antitrypsin (produced in blood); growth hormone, transgenic fish are
retinitis pigmentosa and other eye human interleukin 2; tPA; being developed mostly for
diseases; future applications may chymosin; alpha glucosidase, and applications in aquaculture.
benefit from similarities between human growth hormone; Cytoplasmic injection is possible
human and pig digestive and chimerized MAbs for use as with fish (not so in mammals)
cardiovascular systems. radioimmunotherapeutic agents because embryo development
Companies: Most groups working against cancer, MAbs against happens externally; 35–80%
with transgenic pigs are university- Hodgkin’s disease and renal cell microinjection survival; 10–70%
based; one example: Duke University carcinoma; and human calcitonin. transgenic production.
Medical Center, North Carolina State Companies include Therapeutic Chesapeake PERL, Inc. (College
University, and NIH are collaborating Human Polyclonals Inc. (Mountain Park, MD; www.c-perl.com) received
on research into treatment of retinitis View, CA; www.polyclonals.com), a $2 million, three-year National
pigmentosa (19). Pharming BV, and BioProtein Institute of Standards and
Rabbits: Rabbits appear to be Technologies (Massena, France, Technology Advanced Technology
attractive candidates as transgenic and Cambridge, MA; Program (ATP) grant to genetically
animals. They are cost-effective to www.bioprotein.com). transform caterpillars to produce
raise, they reach sexual maturity Sheep: When the COL1A1 gene humanized glycoprotein
after five to six months, average from connective tissue cells modifications. The company uses a
eight offspring per pregnancy, and (fibroblasts) was combined with a baculovirus to express recombinant
can produce up to 40 embryos vector and fused with enucleated proteins in whole insect larvae by
following superovulation. Their milk sheep eggs, two lambs secreted milk incorporating biological pathways
has a high protein content, and they containing 650 µg/mL. Fibroblasts into caterpillars (“Transpillars”) to
can produce up to 250 mL of milk a secrete Type 1 collagen, the absence produce mammalian glycoprotein
day. Kilogram-scale quantities of of which in humans causes structures rather than those naturally
osteogenesis imperfecta.
48 BioProcess International JUNE 2004 SUPPLEMENT
occurring in insects. The company 1991 Points to Consider in preserve the quality of the breeding
hopes that using Transpillars to Human Somatic Cell Therapy and stock; and that milk collection,
manufacture therapeutic proteins Gene Therapy handling, storage, and transport
may increase the number of likely 1992 Nucleic Acid follow SOPs (7).
drug targets available for production Characterization of Cell Lines Used
in the C-PERL system (21). to Produce Biologicals A PROMISING TECHNOLOGY
1993 Points to Consider in The future production of
THE REGULATORY ISSUES Characterization of Cell Lines Used recombinant proteins in transgenic
Regulatory agencies in the United to Produce Biologicals animals looks very promising.
States and Europe require that 1994 Points to Consider in Methods of producing transgenic
transgenically produced therapeutics Manufacture and Testing of animals and their offspring differ
be safe, pure, well-characterized Monoclonal Antibody Products for from company to company and
(identity), and of demonstrated Human Use according to the therapeutic protein
potency — following Good 1995 Points to Consider in the of interest. Because most target
Manufacturing Practices (GMPs). Manufacture and Testing of proteins are expressed under the
Freedom from potential animal Therapeutics Products for Human control of milk-specific gene
pathogens, demonstrated lot-to-lot Use Derived from Transgenic regulatory elements in a variety of
consistency, and elimination of Animals. This is the major species, certain species produce
immunogenicity are also elements regulatory document pertaining to particular types of protein more
necessary for regulatory approval. transgenic animals and covers the effectively than others. Additionally,
Although viral removal steps for following five points: generation and the amount of published literature
transgenics are analogous to those characterization of the transgene regarding transgenics development
for cell-culture-derived products, construct, creation and specific to individual species and
and although the mammary gland characterization of the founder recombinant proteins is large and
itself may filter out systemic animal and its propagation, growing. Some companies have
pathogens and viruses from the milk maintenance of transgenic animals been issued significant patents for
reservoir (7), concern remains about and production herds, purification their proprietary vectors and/or
unknown milk-borne animal and characterization of transgenic expression systems. Still others are
pathogens, just as there may be products, and preclinical safety close to or already entering late-
concerns about unknown pathogens evaluations. stage clinical trials, indicating that
in any recombinant system — 1997 Points to Consider in the the first marketed therapeutic
including CHO systems. Manufacture and Testing of product produced transgenically
Transgenic “pharmers” must be Monoclonal Antibody Products for may not be far in our future (22).
familiar with 21 CFR regulations — Human Use Regulatory and public acceptance
regulations applicable to all Other agencies involved in of therapeutic products produced in
biologicals (parts 58, 210, 211, overseeing maintenance of the milk of transgenic animals may
600, and 680). They must also transgenic herds include the United not prove as sensitive as regulatory
operate under Good Agricultural States Department of Agriculture positions over transgenically
Practices (GAP) ensuring protection (USDA), which has authority over modified plant and insect species
of their animals from exposure to animal-disease testing criteria within that might escape into wild
potential disease vectors. As far as the United States (also overseeing populations. Also these valuable (in
prion diseases are concerned, there animal import/export criteria). The most cases pampered) “living
seems to be little to no risk of USDA also ensures animal health bioreactors” will not be allowed to
transmission through milk. and welfare through oversight of the enter the food chain. Public
The following regulatory Animal Welfare Act (AWA) (6). acceptance of genetically modified
documents are those relevant to Transgenic “pharming” must crops and animals is fraught with
therapeutic proteins produced in comply with Good Agricultural legitimate concerns over issues such
transgenic animals in the United Practices (GAP) and the accreditation as “genetic drift” and the associated
States. The corresponding CPMP requirements of the American need to ensure isolation and control.
document went into effect in 1995 Association of Laboratory Animal Care A transgenic mosquito or tse-tse fly
and is titled Use of Transgenic International (AAALAC-International). or even some species of fish would
Animals in the Manufacture of GAP considerations include certifying indeed be more difficult to contain
Biological Medicinal Products for that animals are scrapie-free; that the than the larger animals used in
Human Use. facility is separated from other therapeutic protein production.
1985 Points to Consider in livestock species; that SOPs are in Even the term, living bioreactor,
Production and Testing of New place for animal care, identification, reflects a position that bothers many
Drugs and Biologics Produced by and tracking; that animal feed contain people: that of turning a living
Recombinant DNA Technology no animal by-products; that sperm creature into a tool for human
and embryo banks are maintained to
50 BioProcess International JUNE 2004 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.

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Series. Cambridge University Press: Cambridge, UK, 1996, p. 174.
2 Amsterdam, A; Lin, S; Hopkins, N. Transient and Transgenic
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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: 2213-
2224.
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.

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