BIOTECHNOLOGY
Transgenic technology
in the production of
therapeutic proteins
Transgenic technology represents a new generation of biopharmaceutical
production system to meet the medical needs of the new millennium.
Dr Patricia F Dimond, Genzyme Transgenics Corporation
T
ransgenic technology enables the high level
expression of recombinant human
therapeutic proteins in the milk of
transgenic animals. The technology has the
potential to transform the way in which
biopharmaceuticals are produced by providing a
robust, highly efficient process that substantially
lowers capital expenditure and production costs.
In particular, the technology enables the economical,
high-volume production of proteins such as
monoclonal antibodies - therapeutics that may be
required in relatively large and/or repeated doses
for chronic illnesses such as cancer and
autoimmune diseases. Specifically, the recent FDA
approvals of antibodies as human therapeutics is
driving demand for production systems that can
cost-effectively deliver these molecules at the 100
kilogram per year scale or greater.
Benefits of transgenic production
The production of recombinant human proteins in
the milk of transgenic animals presents several
advantages over mammalian cell culture for
proteins required at high levels. Expression levels
in the milk of transgenic animals are typically 10100 fold higher than those in Chinese Hamster
Ovary (CHO) cell culture systems; for example,
Genzyme Transgenics Corporation (GTC) has
demonstrated expression of recombinant
antibodies at levels between 1g/L and 10g/L in
transgenic mice and goats, compared with less than
0.1g/L to 1g/L for CHO cell cultures.
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Transgenically produced recombinant proteins
have the same amino acid sequence as native
human proteins because they are synthesised by the
cells of the mammary gland from a recombinant
version of the native gene. Recombinant proteins
expressed in milk have mammalian glycosylation
patterns. While these patterns may differ from human
patterns, the various different, complex proteins that
have been expressed in the milk of transgenic animals
exhibit the appropriate folding, assembly and biological activity of the native molecule.
Transgenic production enables the development
of certain scarce and valuable human proteins. For
example, therapeutic proteins currently purified
from pooled human plasma - a source subject to
periodic shortages - are now being produced
transgenically (antithrombin III, human recombinant
alpha-1-proteinase inhibitor, human serum
albumin). Pooled human plasma also carries a
theoretical risk of human viral disease transmission,
such as hepatitis C. Transgenic production of such
proteins may avoid the potential risks associated
with transmission of viral diseases through the
human blood supply, as well as guarantee an
uninterrupted supply for patients suffering from
hereditary deficiencies.
Additionally, certain proteins cannot easily be
produced in cell culture because the cells making
them do not secrete them into tissue culture
supernatant, thereby complicating the purification
process. To overcome this impediment for one
potential therapeutic, GTC has transgenically
produced glutamic acid decarboxylase, a possible
drug for the treatment of diabetes.
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Transgenic technology also offers a highly
flexible production system that can be applied to a
wide range of recombinant protein types, ranging
from human plasma proteins to soluble cell surface
receptors. To date, GTC has produced 50 diverse
proteins in the milk of transgenic animals such as
mice, rabbits and goats including, for example,
monoclonal antibodies for the treatment of cancer and
autoimmune diseases, as well as other proteins
including beta interferon, human growth hormone,
tissue plasminogen activator, insulin and prolactin.
Transgenic production also provides significant
capital flexibility in the development process.
Given the time and risks associated with the
development of new biopharmaceuticals, costly
investment in cell culture facilities can restrict
product development choices. The capital costs
for the transgenic production of 100kg of a
monoclonal antibody are $6 million for herd
scale-up, farm and dairy costs, but excluding
purification facility costs. Typical costs associated
with cell-culture based bioreactor plants approach
at least $50 million, and more often reach the $200
million level. Furthermore, expanded transgenic
production output to supply clinical trial and
marketing needs can be met by breeding and
milking more animals, rather than by a significant
investment in new production facilities.
The transgenic production process
A transgenic animal is an animal whose cells
incorporate sequences of DNA that are not normally
part of its genome. To produce a transgenic animal,
a segment of DNA, or expression vector, is
To date,
GTC has
produced 50
diverse proteins
in the milk of
transgenic
animals such
as mice, rabbits
and goats …
Figure 1. Typical transgenic development plan.
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BIOTECHNOLOGY
Transgenic
production also
provides
significant
capital flexibility
in the
development
process
constructed consisting of a promoter DNA
sequence and a DNA sequence that codes for the
desired recombinant protein. The presence of the
promoter segment directs production of the
protein to the mammary gland. The recombinant
proteins controlled by milk promoters are then
generally expressed only in the mammary gland
during lactation.
Transgenic animals are produced by
introducing - usually through microinjection - the
expression vector into an early-stage embryo. The
embryo is then transferred to a surrogate mother.
Following the birth of animals resulting from these
embryo transfers, animals carrying the transgene for
the protein are identified. The female offspring of
these “founder” animals form the production herd.
Production lines established from a single transgenic
Figure 2a. Transgenic purification process.
founder ensure that all animals in the herd exhibit
consistency in recombinant protein expression levels
and characteristics that are stable from lactation to
lactation and generation to generation.
While GTC has chosen goats as its production
species, transgenic mice, rabbits, sheep, pigs and
cows have been developed. GTC routinely
achieves about 5-10% transgenic animals among
live births subsequent to the microinjection
procedure. These first generation transgene
carriers, or founder animals, may be male or
female. If the founder is female, then the time
from transgene introduction to the first natural
lactation is 18 months for goats. Lactation may be
induced in females at three months of age,
however, thereby supplying material for testing and
early clinical trial supply. If the founder is male,
then he must produce transgenic daughters, which
in turn must produce transgenic daughters before
full-scale milk production can begin. The time to
first lactation is about 2.5 years for transgenic lines
derived from male founders.
More recently, transgenic female animals have
been produced by somatic cell nuclear transfer
(cloning).
By combining nuclear transfer
technology with lactation-induction in prepubertal
goats, it may be possible to considerably shorten
the time line associated with founder production
and subsequent herd development (Figure 1).
GTC reported the development of three cloned
female goats carrying a transgene targeting the
expression of antithrombin III (rh AT III). These
goats were produced from a female cell line
originating from goats that were transgenic for rh
AT III produced by microinjection. Expression of
rh AT III in the milk of the cloned animals was
consistent with the expression found in the milk of
other transgenic females from the same line
obtained by natural breeding.
Therapeutic protein recovery from milk
Figure 2b. Key purification technologies.
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Milk as a source material of recombinant proteins
offers a much higher protein concentration - typically
10-fold to 1,000 fold greater than tissue culture
supernatant. Furthermore, milk does not contain
active proteases that can break down the protein of
interest. Because this high concentration decreases
the volume of material input into the purification
process, the need for materials and equipment such
as buffer salts, chromatography columns and
custom materials for chromatographic capture
steps is significantly reduced. The concentration
of protein in milk also remains consistent over the
production cycle, whereas in cell culture
bioreactors, the protein concentration varies
considerably with the stage of growth and state of
the cells that are producing it.
Therapeutic proteins are generally more readily
purified from milk than from tissue culture
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Milk as a source
material of
recombinant
proteins offers
a much higher
protein
concentration
…
supernatants. Recombinant protein typically
represents a minor component of supernatant that
must be separated from cells, cellular debris, lipids,
DNA, host proteins, enzymes, pyrogens and other
potential contaminants or impurities. Serumsupplements to culture supernatants further
complicate the purification problem by
adding exogenous bovine proteins, including
immunoglobulins, and dyes.
By contrast, milk provides a relatively “clean”
feedstream, consisting of about 87% water, 4% fat
and 9% non-fat solids. The soluble phase contains
the normal milk proteins (casein and whey
protein), mineral salts and vitamins; the non-soluble
phase contains fat globules, protein aggregates and
cells. Recombinant proteins usually accumulate in
the soluble whey fraction of milk, thereby
facilitating their recovery. Standard dairy procedures
remove as much as 95% of fat by centrifugation,
and casein can be precipitated at low pH or with
enzyme treatment. Ultrafiltration can be used to
remove fat, casein and any cellular components of
the milk, as well as provide a barrier to microbial or
viral contaminants. The protein of interest can
then be purified by chromatography. Figure 2
depicts the generic process for protein recovery
from milk. GTC has developed proprietary
procedures for protein purification from milk that
typically attain 65% yields at 99.99% final
product purity.
Transgenic production economics
Therapeutic
proteins are
generally more
readily purified
from milk than
from tissue
culture
supernatants
The most significant driver for transgenic production
of biopharmaceuticals is the need for recombinant
human proteins in quantities that cannot be
produced economically from either scarce or
expensive alternative sources. Currently, these
proteins are produced from pooled human blood
plasma. Given the relatively low levels of protein
production typical of cell culture bioreactors,
transgenic production offers a safe, viable and
economic alternative for the recombinant production
of blood proteins such as rh AT III, human serum
albumin and other proteins important in the
clotting cascade. This case may be the most
compelling for rh AT III and human serum
albumin, where yearly market demand exceeds 50
kilograms and 440 metric tons, respectively.
Furthermore, the recent regulatory approvals for
monoclonal antibodies as therapeutics for chronic
diseases - including cancers, rheumatoid arthritis
and heart disease - are driving the need for
production systems that can produce proteins
economically at the hundreds of kilograms scale.
Monoclonal antibody production provides a
good model for an examination of the relative costs
of large-scale recombinant protein production
performed in either cell culture or transgenic
animals. To date, 14 monoclonal antibodies have
been approved for human or diagnostic use in the
US, with an additional two dozen therapeutic
antibodies in clinical trials and another 200 in
preclinical development.
Annual production requirements for a given
antibody therapeutic approach the 100 kg scale.
Table 1 depicts a comparison between producing
100 kg of recombinant monoclonal antibody in
Chinese hamster ovary (CHO) cells or in the milk
of transgenic goats. For production at this scale,
170,000 litres of CHO cell culture supernatant
containing 1 gram of antibody per litre are
required, compared with 21,000 litres of milk
containing about 9 grams of protein per litre from
a transgenic goat. Ultimately, one gram of
CHO-produced antibody costs from $300 to
$3,000, compared with $105 for transgenicallyproduced protein.
Safety considerations
Genzyme Transgenics Corporation has taken a
leading role in working with regulatory agencies to
establish criteria to ensure appropriate control over
both the manufacturing process for transgenic
biopharmaceuticals and the products themselves.
GTC operates its farm and milking procedures
CHO cells
Transgenic
Production days/year
200 (20 runs)
300 (2 litres/goat/day)
Reactor capacity required
8,500-litre vessels
35 goats
Unit product cost
$300-$3,000
$105
Capital expenditures
$20-50 million
$2-5 million
(plus seed culture)
Table 1. The economics of transgenic MAb production (100kg scale).
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under “Good Agricultural Practices” (GAP), which
provide the highest animal care standards for
reduction of disease transmission.
GTC has established methods to prevent,
monitor and control viral infections in its herds,
and maintains a US Department of Agriculturecertified “scrapie-free” goat herd for the
production of rh AT III, as well as for other
products in development. With respect to
minimisation of viral contamination risk, rigorous
control is exercised at the level of the animal, the
milk and the final product. Animal control
measures, for example, include use of defined breeding
stock, identification and tracking of individual
animals, frequent health screening and provision of a
defined feed source that does not contain any animal
products. Milk controls include monitoring of milk
sources, collection and handling. Product safety is
assured by specific steps in the protein purification
process that remove and/or inactivate bacterial, viral
and prion agents.
Conclusion
The production of recombinant proteins in the
milk of transgenic animals offers an enabling
technology that supports the production of large
quantities of biopharmaceuticals at highly
competitive costs. Transgenic technology further
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enables the production of unique molecules that
cannot be produced by other means, and protein
recovery at high yield and purity. Transgenicallyproduced proteins show appropriate protein
structure, stability, bioactivity and safety. In
summary, transgenic technology represents the
next generation of biopharmaceutical production
systems, providing a unique capability for the
supply of biotherapeutics to address unmet
medical needs as we enter the new millennium.
Patricia F Dimond, PhD, joined Genzyme
Transgenics Corporation in 1996 as Director of
Corporate Development and Communications; she is
also responsible for development and funding of the
company’s idiotypic cancer vaccine programmes for
B-cell lymphoma and myeloma. Dr Dimond
received a Masters degree in membrane biophysics
from Georgetown University and a PhD in cellular
biochemistry from Catholic University in
Washington, DC. She completed a National
Institutes of Health (NIH) post-doctoral research
fellowship in the Department of Biochemistry at
Boston University School of Medicine. Dr Dimond
has over 15 years’ experience in the biotechnology
industry, including positions in communications and
corporate development at Becton-Dickinson, TSI
Corporation and PerSeptive Biosystems.
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