Transgenic Technology: a Validated Approach for

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Bioprocessing
Transgenic Technology: a Validated
Approach for Large-Scale Manufacturing
By Yann Echelard,
William Gavin, Ashley
Lawton, Carol Ziomek
and Harry Meade at GTC
Biotherapeutics Inc
Transgenic technology – as a platform for the production of human
recombinant therapeutics – offers significant advantages with respect to
capital expenditure and cost of goods. For the near future, the transgenic
production of monoclonal antibodies represents an ideal technology for
the basis of a successful biosimilars programme.
The global market for therapeutic recombinant proteins
has grown steadily over the last two decades, reaching
more than $75 billion in 2007 (1). The development
of efficient protein expression systems has been the
key to full exploitation of recombinant technology.
Initially, microbial bioreactors were employed and
worked well for the production of relatively simple
polypeptides such as insulin
Figure 1: Schematic representation
and human growth hormone.
of the transgenic production process
However, for proteins with
(A) A milk expression transgene is generated by attaching milkintricate folding requirements
specific regulatory sequences to the gene encoding the therapeutic
protein of interest. For example, for the production of ATryn , the
and/or complex post-translational
goat beta-casein regulatory sequences are attached to the human
modifications, such as monoclonal
antithrombin cDNA (10). (B) Transgenic production animals have
been generated by pronuclear microinjection, a process during which
antibodies, microbial bioreactors
a solution containing the transgene DNA is microinjected into the
pronucleus of a fertilised embryo. The microinjected embryo is then
were found to be unsuitable. This
transferred to a recipient female. A small percentage (usually <10
led to the development of largeper cent) of resulting offspring carry the transgene integrated in
their genome. These transgenic offspring are then characterised,
scale mammalian cell culture – an
and mated to generate a herd of transgenic animals producing the
protein of interest in their milk. (C) Somatic cell nuclear transfer has
approach that has facilitated the
also been used to generate transgenic founders. The transgene is
development of all the monoclonal
introduced by transfection in the genome of primary cells (usually
foetal fibroblasts). The resulting cells carrying the transgene are then
antibodies that are currently
fused to enucleated oocytes by using a nuclear transfer (cloning)
protocol. The resulting embryos are then transferred to a recipient
commercialised, as well as other
female. The offspring are transgenic since they are derived from the
complex biomolecules.
transfected cells. The advantage of nuclear transfer over pronuclear
microinjection is that it allows pre-selection of the sex of transgenic
However, there are proteins
offspring, as well as the copy-number of the transgene.
that – due to a combination
Mammary glandGene encoding
specific promoter
target protein
of complex structure and large
A
Transgene
therapeutic dosages – have until
now
eluded
recombinant
production
using
bioreactors.
For
Test offspring
Mate transgenic
Collect
for transgene
offspring
example, commercial recombinant
fertilised
embryos
Transfer into
B
production of complex molecules
recipient
Expand
female
herd
such as antithrombin (AT) and
alpha-1 antitrypsin, used in high
Microinject
transgene
doses and currently extracted
from human plasma, has not
yet been achieved commercially
Collect
oocytes
Milk containing
C
in bioreactors. In addition, the
the target
Enucleate
protein
capital investment associated with
Nuclear
Primary
transfer
fibroblasts
Transfer into
recombinant protein production
recipient
female
facilities represents a significant
Transgene
transfection
portion of the development cost of
Transgenic
offspring
new recombinant drugs. The
®
50
construction of a large-scale production platform that allows
for flexible scale-up, while reducing the size of capital
investment, lowers the risk-profile of the manufacturing
infrastructure. Furthermore, the risks associated with the
regulatory approval process, and the very real possibility that
a new drug might fail in the clinic after a significant
investment in manufacturing has already been incurred,
represent another stimulus for the development of flexible
and inexpensive approaches for the manufacture of
therapeutic proteins.
With the careful integration of large animal embryology,
molecular biology, protein chemistry and Good Agricultural
Practices, recombinant production in the milk of transgenic
animals offers a flexible system for the manufacturing of
large amounts of complex therapeutic proteins. The
regulatory approvals of ATryn® (recombinant antithrombin,
rhAT), first by the EMEA (in August 2006) and recently
by the FDA (February 2009), have provided a strong
validation of transgenic technology for the large-scale
production of recombinant biotherapeutics. Beyond
ATryn®, this manufacturing platform is being used for
several biopharmaceuticals currently in development (see
Table 1) and presents an attractive alternative for the costeffective production of biosimilars.
TRANSGENIC PRODUCTION
The targeted expression of heterologous proteins to the
mammary gland of transgenic mice was independently
reported by several groups during the late 1980s (2,3).
This was followed by reports on the generation of
transgenic sheep, goats, cows and pigs carrying milkspecific transgenes for the production of recombinant
proteins for clinical use. The aim was to target the
production of recombinant proteins to the mammary
gland of transgenic farm animals in order to solve the
problems associated with either microbial or animal cell
expression systems. Bacteria often improperly fold
complex proteins, leading to involved and expensive
refolding processes, and both bacteria and yeast lack
adequate mammalian and proteolytic processing. Cell
culture systems require high initial capital expenditures,
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lack scale-up (or down) flexibility, and use large volumes
of culture media. Transgenic livestock can be maintained
and scaled-up in relatively inexpensive facilities, use
animal feed as a raw material, and can achieve impressive
yields of recombinant proteins with mammalian-specific
N- and O-linked glycosylation and γ-carboxylation.
The targeting of a recombinant protein to the milk of
a transgenic animal is achieved by first generating an
expression vector containing the gene encoding the
protein of interest fused to milk-specific regulatory
elements (see Figure 1A). This transgene is then
introduced into the germline of the chosen production
species. Pronuclear microinjection of one-cell embryos
(see Figure 1B) or, alternatively, transfection into a
primary cell population suitable for somatic cell nuclear
transfer (see Figure 1C) have both been used to generate
transgenic founders. Following germline integration,
mammary gland-specific transgenes are predictably
inherited by the offspring of the founder animal. The
expression level of the protein(s) of interest is variable;
concentrations surpassing 1-5g/L are routinely attained
and levels of up to 20g/L have also been achieved. Milk
can easily be obtained using the established large-scale
technologies of the dairy industry.
Several animal species have been used for transgenic
production – notably goats, sheep, pigs, cows and rabbits.
There is usually a trade-off between milk yield and time to
natural lactation. For example, rabbits have a short
gestation interval that allows up to eight lactations per
year. However, only 1-2 litres of milk can be routinely
obtained per lactation. This limits the value of this
expression system to products with a commercial scale in
the low-kilogram range (4). Recombinant protein
production in the milk of transgenic sows has been
reported for coagulation factors, notably Protein C and
human factor IX (5,6). Transgenic ruminants are obvious
candidates for targeting expression of recombinant
proteins to the mammary gland. Transgenic dairy goats,
with an average milk output per doe of 600 to 800 litres
per natural lactation, are well-adapted to the production of
therapeutic proteins; the timeline from initiation of
transgene transfer to natural lactation of resulting
transgenic does is 16 to 18 months. A large number of
production females can easily be generated from a
transgenic male using natural breeding, artificial
insemination or embryo transfer techniques. Relatively
small herds of a few hundred transgenic does can then
easily yield several hundreds of kilograms of purified
product per year (see Figure 2). This level of production
can meet the manufacturing needs of several factors
traditionally derived from plasma fractionation, as well as
for a large number of recombinant antibodies currently
in development (1,7).
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Table 1: Therapeutic proteins produced in the milk of transgenic animals
that are currently in commercial development
Products/Company(ies)
Production animal
Development stage
ATryn®, recombinant human
antithrombin (AT)/GTC Biotherapeutics
Goats
EU: approved for AT HD
US: approved for AT HD
Phase III for heparin resistance
Phase II for DIC in severe sepsis
Rhucin®, C1 Esterase Inhibitor/
Pharming
Rabbits
Completed Phase III trials for HAE
Phase I for organ transplantation
MM-093 (AFP)/Merrimack
Pharmaceuticals and GTC
Biotherapeutics
Goats
Phase II, Autoimmune diseases
Protexia®, pegylated human
butyryl-cholinesterase/PharmAthene
Goats
Phase I, Organophosphates poisoning
Lactoferrin/Pharming
Cows
Phase I nutritional applications
Human growth hormone/BioSidus
Cows
Phase I, Short stature
Recombinant Factor VIIa/LFB
Biotechnologies and GTC Biotherapeutics
Rabbits
Preclinical, Haemophilia with inhibitors
Fibrinogen/Pharming
Cows
Preclinical
Recombinant Factor IX/GTC Biotherapeutics, Pigs
LFB Biotechnologies and ProGenetics
Preclinical, Haemophilia B
Alpha-1 Antitrypsin (AAT)/GTC
Biotherapeutics and LFB Biotechnologies
Goats
Preclinical, AAT hereditary deficiency
Anti-CD20 mAb/GTC Biotherapeutics
and LFB Biotechnologies
Goats
Preclinical, Oncology
CD137 (4-1BB) mAb/GTC Biotherapeutics
Goats
Preclinical, Oncology
Abbreviations
DIC, disseminated intravascular coagulation HAE, hereditary angioedema
AFP, alpha-fetoprotein
mAb, monoclonal antibody
SAFETY ASPECTS OF THE TRANSGENIC
PRODUCTION FARM
Safety for human recombinant therapeutics produced
through transgenic technologies, such as GTC’s ATryn®
recently approved by the FDA, starts at the level of the
farm. It is imperative to have a clear sense of surrounding
properties to understand existing domestic and wild
animal demographics in the surrounding environment.
The facilities should be designed with the species in
mind, and have a high level of control and containment
to include appropriate fencing and barriers to avoid
the release of any animals. A unique and redundant
animal identification system and a comprehensive
documentation system for all procedures involved in
daily animal care and operation of the farm (for example,
Good Agricultural Practices) should be instituted. A
biosecurity programme that encompasses external and
internal biosecurity, animals and personnel, and a pest
management plan (to control birds, rodents and insects
for example) should be developed.
During early stage development and operation of the
farm, consideration should be given to applications for
oversight from animal regulatory agencies, as well as other
state and local authorities (8). Lastly, voluntary
organisations – such as the Association for Assessment and
Accreditation of Laboratory Animal Care, AAALAC
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International – should be
considered for accreditation of the
operation and facilities.
Protein titer in milk
A one-time population of
20g/L
10g/L
5g/L*
100
the facility should be considered
to limit the potential negative
Market
75
supply
effects of subsequent animal
introductions. Animal acquisition
50
should include – at a minimum –
a comprehensive review and
25
understanding of animal history,
Clinical
and the development of a
supply
0
pre-screening disease testing plan.
0
10
20
30
40
50
60
70
80
Consideration should be given to
Goats required (current farm has 1,700 goats)
animal acquisition from countries
* Average transgenic yield of mAb (figures assume 500L
free of diseases of concern for the
milk/goat/year and 50 per cent process yield)
establishment of a disease-free herd,
depending on the species being utilised. Disease entities that
could potentially adversely affect the herd or the final
product need to be addressed. Ultimately, the intent should
be to have and maintain a closed herd. An ongoing disease
testing/surveillance programme and establishment and
documentation of the specific pathogen-free (SPF) status of
the herd should be a priority. Lastly, international expert
veterinary, viral and prion panels are recommended to
review and codify any programme.
All raw materials used in the manufacture of a
recombinant product need to be controlled – and the
same is true for transgenic technologies utilising animals
as the production system. Controls should be included
for water, bedding, feeds and supplements, and any
medicinal agents utilised in or on the animals.
Additionally, all waste material removal must comply
with any existing or specific regional regulations.
For the collection of milk, a significantly heightened
level of control and documentation must be in place. At
a minimum, qualification of an animal to enter a
production collection system should address such
features as: verification of the genetic modification,
verification of the SPF status of the animal; testing of the
milk for the presence of the recombinant protein in the
intended form and concentration; screening for any
adventitious agents of concern (for example, zoonotics)
and other testing as appropriate.
Lastly, consideration should be given to the storage
of milk prior to downstream purification. This review
should include optimal storage temperature and ranges,
types of containers, temperature controls and
recording, back-up power, and finally transportation
(validated) of the collected milk to the storage and/or
downstream purification site. All of this must be
documented and conducted under appropriate QA
oversight for cGMP manufacture.
Product quantity (kg)
Figure 2: Scalability of transgenic herds
producing recombinant proteins in their milk
52
MILK TESTING
Once sufficient milk has been collected and stored for
production of a batch of product, a representative sample
is tested for composition and recombinant protein
content, and screened for adventitious agents, including
viruses (known and unknown). Conventional in vitro
cell line screening for virus, validated for milk, is
performed (in the case of ATryn®) with human, monkey,
hamster and goat cells, assessing cytopathic effects,
haemadsorption and haemagglutination with various
species’ red cells (9). Additionally, specific validated
immunofluorescence assays screen for emerging zoonotic
viruses. To date, no viruses have been detected in the
milk from any goats in the closed, highly controlled
GTC farm.
DOWNSTREAM PROCESSING
Purification of the recombinant protein usually begins
with clarification of the milk by filtration to eliminate
particulate materials, followed by chromatography steps
such as affinity, anion exchange and hydrophobic
interaction columns (HIC) that remove residual milk
proteins, including endogenous versions of the
recombinant protein (10). The drug substance may then
be formulated, filled into vials, lyophilised and capped to
produce the final drug product. The manufacturing
process is also validated for robust removal of a variety of
potential contaminants, including model viruses and
prions (11). Additionally, a variety of impurity assays for
potential milk protein contaminants have been
developed and are utilised for assessing the purity of the
bulk drug substance.
Biochemical and non-clinical characterisation of the
purified recombinant product is critically important. For
ATryn®, it was determined that it had an amino acid
sequence identical to human plasma-derived AT with six
cysteine residues forming three disulphide bridges and 34 N-linked carbohydrate moieties. Recombinant AT,
however, had a more heterogeneous glycosylation profile
resulting in an increased heparin affinity (10), but was
not different in potency assays using excess heparin.
SAFETY AND EFFICACY IN HUMANS
In human clinical trials, as for any recombinant
protein, efficacy is the primary endpoint but safety
of the product is key. Safety assessment for a
transgenically-derived protein not only encompasses
clinical adverse events, but also the immunogenicity of
the recombinant protein and any potential milk
protein contaminants. In the case of ATryn®, no
immunogenicity was observed, either clinically or in
laboratory-based tests, to rhAT or any potential
contaminating goat milk proteins (12-14).
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PRODUCTION OF BIOSIMILARS
More complex and costly manufacturing processes required
to produce recombinant protein therapeutics are primary
drivers behind the relatively high prices of biological drugs,
especially monoclonal antibodies. With the large numbers
of such products in companies’ development pipelines, the
cost of biological drugs as a percentage of total
pharmaceutical costs is already over 20 per cent, and is
anticipated to continue to grow in the future (1); it is clear
that the incessant increase in expenditure is unsustainable.
This societal reality has focused political bodies to introduce
legislation for abbreviated regulatory pathways that have
begun to facilitate the introduction of less expensive
‘generic’ versions of recombinant therapeutics into the
marketplace. Due to their earlier patent expiries, the initial
biosimilar products that have been approved have been less
complex proteins such as human growth hormone and
erythropoietin. The high specific activity and relatively low
dose of these proteins mean that there is little advantage to
be gained through decreasing the cost of goods. The fact
that the total world demand for such products is typically
less than 10kg means that conventional cell culture and
fermentation production techniques are adequate for
their manufacture.
The higher doses of the major monoclonal antibodies
and fusion proteins that are marketed mean that much
larger quantities – typically 250-1,000kg – are required to
satisfy world demand. The capital expenditure required to
build cell culture facilities at this scale are not only beyond
the reach of most companies, but also pose a significant
risk based on the uncertainty of how quickly the markets
for biosimilar antibodies will materialise, and how large
they will ultimately become (how much capacity do you
need to build?), especially when that investment will
primarily occur several years ahead of product approval.
History tells us that getting this equation wrong can have
deadly consequences (for example, Synergen, Xoma,
ImmunoGen, AlphaBeta).
Transgenic production of monoclonal antibodies
represents a significant opportunity for companies to enter
the biosimilar marketplace. GTC has expressed over 20
antibodies with relatively consistent productivity –
approximately 3-7g/L milk (although up to 15g/L has
been achieved (7)). At these levels, a typical goat is capable
of producing at least 1kg of purified antibody each year.
Therefore, a milking herd of 100 goats is capable of
producing 100kg of product; as a reference point, the
current GTC operations (see Figure 3) comprise more
than 1,700 goats. The estimated capital expenditure
required to set up a transgenic farming operation at an
equivalent scale to a cell culture facility are approximately
10-fold less. More significantly, the herd can be scaled up
as demand increases, and therefore the capital expenditure
Innovations in Pharmaceutical Technology
can be phased in line with demand – significantly
minimising the risk of under- or over-estimating ultimate
market size. The relatively high productivity of transgenic
founder-lines, combined with the significantly decreased
capital expense, means that transgenic production is a
technology that offers a very competitive cost-of-goods
compared with conventional production technologies.
Although the glycosylation of transgenic goat-derived
antibodies is not the same as either CHO-cell or humanderived antibodies, it is important to note that the
differences do not affect the half-life, affinity or in vivo
activity of the antibodies (15). In addition, transgenic
goat-derived proteins do not appear to be immunogenic
based on the available data. This is not altogether
surprising, as there are no sugar residues unique to goats
that are not found either in humans and/or CHO-cells; it
is merely the relative amounts of the sugars that are
different. The carbohydrate-pattern present on rhAT
produced in transgenic goats did not invoke an antibody
response as previously indicated (12-14). Based on these
observations, it appears unlikely that the differences in
glycosylation patterns of goat-derived antibodies will
impart clinically significant pharmacokinetic and
antigenicity properties when compared with CHO-cell or
human antibodies.
CONCLUSION
Transgenic technology as a platform for human
recombinant therapeutic protein production is now a
validated, scaleable, high-volume production technology
that offers significant advantages with respect to total capital
expenditure and less risky phasing of capital investment, as
well as a competitive cost-of-goods. As an example for the
near future, the transgenic production of monoclonal
antibodies represents an ideal technology to form the basis
of a successful biosimilars programme.
Figure 3: Aerial view
of the GTC transgenic
production farm,
located in western
Massachusetts
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For The Art of Expression, Academic Press, San Diego,
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4.
Yann Echelard, PhD, is Vice President, Research and
Development, at GTC Biotherapeutics, Inc (Framingham, MA,
USA). He has 25 years’ research experience and has been
involved in transgenic research and the development of protein
expression systems since 1994 when he joined GTC (then
Genzyme Transgenics Corporation). Yann received a PhD in
Microbiology and Immunology from the Université de Montréal
(Canada) and completed post-doctoral training at the Ludwig Institute of Cancer
Research, the Roche Institute and Harvard University (Cambridge, MA, US).
Email: yann.echelard@gtc-bio.com
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Ashley Lawton, PhD, is Vice President, Business Development,
at GTC Biotherapeutics, Inc, where his responsibilities include
the development of a Follow-On Biologics business and the
commercialisation of internal early-stage research programmes.
Through roles at Eli Lilly, Celltech, RepliGen, Genzyme and
Phylos, he has broad expertise in the development and
commercialisation of protein therapeutics. Ashley graduated
from Southampton University (UK) with a BSc in Human Physiology and
Biochemistry and a PhD in Biochemistry. More recently, he graduated from
Boston University (US) with an MBA in General Management.
Carol A Ziomek, PhD, is Vice President of Development and
a Founding Member of GTC Biotherapeutics Inc. In this role,
she manages the technical aspects of product development
for recombinant antithrombin (ATryn®), and leads the multidisciplinary team that has developed the ATryn® viral and
pathogen safety strategies. Carol obtained a BSc in Chemistry
from Wilkes College (Pennsylvania, US), a PhD in Biology
from Johns Hopkins University (Baltimore, MD, US), and performed her postdoctorate at the University of Cambridge (UK).
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6.
William Gavin, DVM, is Vice President of Farm Operations and
Chief Veterinarian at GTC Biotherapeutics, Inc. As GTC’s USDA
Attending Veterinarian, he has responsibility for complete
oversight of GTC’s production animal operations both internally
and externally, as well as founder transgenic animal development.
William has over 20 years of experience in the fields of
embryology, transgenics and nuclear transfer/cloning. He
received a BSc from the University of Massachusetts and a Doctorate in Veterinary
Medicine from Tufts University, School of Veterinary Medicine (N Grafton, MA).
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Echelard Y et al, The first biopharmaceutical
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Harry M Meade, PhD, is Senior Vice President, Research
and Development, and a Founding Member of GTC
Biotherapeutics, Inc. He directs all transgenic molecular
biology research and development efforts conducted within
the company. Harry obtained a BSc in Chemistry and Electrical
Engineering from Union College (Schenectady, NY, US), a PhD
in Biology from the Massachusetts Institute of Technology
(Cambridge, MA, US) and completed his post-doctoral studies at Harvard
University (Cambridge, MA, US). Harry has held scientific positions
with Genzyme Corporation, Biogen and Merck.
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of adalimumab (D2E7) a fully human anti TNF
monoclonal antibody expressed in transfected CHO
cells versus transgenic goats, Clin Immunol, 103(S1),
ppS160-S171, 2002
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