Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Broad/Platform Technologies
21
Process Scale-up
Homeostatic system
Scalable from 15?l to standard bioreactors.
Rapid process development
In contrast, cell-based technologies involve;
proteins that are often toxic to cell-based systems;
improper protein folding; require specialized post-
translational modification; are often limited by
recombinant DNA safety (RAC) guidelinesl have
potential for viral and prion contaminants; may
contain other antigenic contaminants; and often
provide low yield.
Patents: FABs proprietary cell free protein syn-
thesis technology was primarily developed by Dr.
James R. Swartz, School of Engineering, Stanford
University, and exclusively licensed to FAB.
Exemplary patents include U.S. 7,312,049,
Total amino acid stabilization during cell-free
protein synthesis, Dec. 25, 2007, assigned to
Stanford University, concering S30 cell-free ex-
tracts of a E. coli bacteria containing inactivated
genes for tryptophanase, arginine decarboxylase,
L-serine deaminase
and gamma-glutamylcysteine synthase;
7,041,479, Enhanced synthesis of active proteins
containing disulfide bonds, concerning enhanced
in vitro synthesis of polypeptides containing
disulfide bond and; 6,994,986, In vitro synthesis
of polypeptides by optimizing amino acid metabo-
lism.
Licensing information: Contact Fundamental
Applied Biology, Inc. (FAB).
Products made using this tech.: Cell-Free has
been successfully used by FAB and its partners
for the production of:
Novel vaccine constructs
Peptides prone to proteolysis
Therapeutic proteins that are difficult to ex-
press in soluble form
Therapeutic proteins that are prone to variants
and difficult to purify.
Further info.:
1. Kanter G, Yang J, Voloshin A, Levy S, Swartz
JR, Levy R. Cell-free production of scFv fusion
proteins: an efficient approach for personalized
Broad/Platform Technologies
59
USA, 86:3699-3703. (1989).
Hatfield G. W. and Gutman G. A. Codon Pair
Utilization Bias in Bacteria, Yeast and Mammals.
In Transfer RNA in Protein Synthesis. (Edited by
D. L. Hatfield, B. J. Lee, and R. M. Pirtle). Pub-
lished by CRC Press, Boca Raton, FL, 1993. A
comprehensive review of codon pair bias and its
relationship to codon context effects on transla-
tion.
Kittle, J.D. Radical Changes in the Engineering
of Synthetic Genes for Protein Expression. Bio-
Pharm International. 2006:12-18 (2006).
141
White collar complex (WCC)
promoter; UV light induction -
universal
Organizations involved:
Dartmouth College - Licensor, primary
Description: Vectors including the white collar
complex (WCC) promoter from the filamentous
fungus Neurospora can activate gene expression
in vivo using only light as an inducer. This pro-
vides a means to induce gene expression using a
reliable, readily available, inexpensive, non-toxic,
and non-pleiotropic inducer - ultraviolet light.
The WCC is comprised of two proteins, WC-1
and WC-2 that readily heterodimerize and strong-
ly bind the cofactor FAD. The WCC is able to ab-
sorb light and subsequently activate transcription
of a gene operatively-linked to a light-responsive
regulatory sequence which is bound by the WCC.
In a host cell, transcription of a gene, operatively-
linked to one or more light-responsive regulatory
sequences, is stimulated by WC-1/WC-2 by alter-
ing the fluence or wavelength of light exposed to
the host cell.
Use with: universal (generic); E. coli; plants;
mammalian cells; insect cells
Use to make: proteins; glycoproteins
Background: Current methods of controlling
gene expression such as inducible promoters, heat
shock, hormones, or drugs/chemicals such as tet-
racycline or IPTG, have generally suffered from
exogenous inducer molecules evoking pleiotropic
effects which complicate analyses. Dartmouth
researchers have identified ths two-protein com-
plex that reliably activates transcription of genes
linked to one or more short DNA light-responsive
regulatory sequences.
Patents: Exemplary U.S. patents include U.S.
6,733,996, Methods for regulating gene expres-
sion using light, Froehlich, et al., May 11, 2004,
assigned to Dartmouth College. The full claims
are:
1. A method of regulating expression of a gene in
a host cell comprising: a) contacting a host cell
containing FAD and a gene operatively-linked
to a light-responsive regulatory sequence with a
recombinant WC-1/WC-2 transactivator which
binds FAD and the light-responsive regulatory
sequence, and b) exposing the host cell to light to
stimulate activity of the recombinant WC-1/WC-2
transactivator so that the gene operatively-linked
to the light-responsive regulatory sequence is
expressed.
2. The method of claim 1 wherein the light-re-
sponsive regulatory sequence comprises SEQ ID
NO:7.
3. An isolated light-responsive regulatory se-
quence consisting of SEQ ID NO:2, SEQ ID
NO:3, or SEQ ID NO:4.
4. A kit comprising a first container means which
contains a recombinant WC-1/WC-2 transactiva-
tor and a second container means which contains
an isolated light-responsive regulatory sequence.
Licensing information: Dartmouth is seeking an
industrial partner to further refine and market this
technology.
Further info.: Froehlich, et al. ((2002) Science
297(5582):815-819) and He, et al. ((2002) Sci-
ence 297(5582):840-843).
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
60
142
Minos transposon cell
transformation - insect larvae;
also eukaryotes
Organizations involved:
Minos Biosystems - Licensor, primary
Institute for Molecular Biology and Biotech-
nology/FORTH - R&D
Description: An isolated DNA sequence which
encodes a transposase protein (or a portion of a
transposase protein) is useful for a transposon-
based method for eukaryotic cell transformation,
including for producing transgenic animals and
for stably transfecting cells in vitro. Applications
include the large scale, low cost production of
high value proteins in mass reared insect larvae.
Isolated transposable elements or the isolated
DNA sequences hybridize to the DNA sequence
of Minos 1 under stringent hybridization condi-
tions.
Transposable elements or transposons are
natural genetic elements capable of jumping or
transposing from one position to another within
the genome of all species. Some transposons will
only function within a limited number of host
organisms while others are more promiscuous
and will function in different organisms including
insects, worms, fish and mammals.
The Minos-1 transposable element is a 1775
bp dispersed repetitive sequence inserted within
the transcribed spacer in one of the repeats of
Drosophila hydei. The element is characterized by
255-bp long perfect inverted repeats and the pres-
ence of two long, non-overlapping open reading
frames (ORFs) on the same strand. The longest
of the ORFs shows approximately 30% sequence
identity with TcA, but does not begin with an
ATG codon. The cloned element represents a de-
fective member of the Minos family, as is the case
with all previously sequenced Tc1-like elements,
with the possible exceptions of Tc1 and TCb1.
Use with: insect larvae; dipteran insects includ-
ing Ceratitis capitata, Anastrepha, Dacus oleae,
Cochliomyla hominivorax, Lucila cuprina, Si-
mulium, Anopheles, Aedes, and Musca domes-
tica; eukaryotes
Use to make: proteins; glycoproteins
Benefits: Minos BioSystems uses proven trans-
poson technology to create recombinant insect,
e.g., Medfly, larvae producing functional human
protein therapeutic. Production of proteins in
insect larvae, has many advantages over compa-
rable plant, animal or cell culture based systems.
The process is contained, rapidly scalable, has
low capital and product cost, high volume output
and an excellent biosafety profile. Annual yields
are potentially over 10 tons of product for each
manufacturing facility capable of producing 1 bil-
lion flies per week.
Patents: Exemplary patents include Eukaryotic
transposable element, 5,348,874 and 6,225,121,
Savakis, et al., Sept. 20, 1994, assigned to Insti-
tute for Molecular Biology and Biotechnology/
FORTH and exclusively licensed to Minos.
Licensing information: Contact Minos Biosys-
tems.
Further information: Stable Expression of Hu-
man Growth Hormone Over 50 Generations in
Transgenic Insect Larvae, Transgenic Research,
16:99-107, 2007.
143
Coconut Express cell-free
translation - plants
Organizations involved:
Plant BioScience Ltd - Licensor, primary
Description: Coconut Express is a method for
low cost, cell-free, high level, protein expres-
sion using the liquid syncytium from coconut
endosperm (a.k.a., coconut water). This method
removes the need for time-consuming cell trans-
formation steps, and has the further advantage of
performing the glycosylation modifications appro-
priate for eukaryotic proteins.
Use to make: Protein, glycoproteins, likely includ-
ing Mabs
Patents: Reportedly filed, but no relevant U.S.
or international patents or published application
Broad/Platform Technologies
61
assigned to the company retrieved.
Licensing information: Contact Plant BioSci-
ence Ltd .
Prokayotes
144
Bacterial cell expression
technology (BCE); araB
promoter - antibody fragments;
E. coli; prokaryotes
Organizations involved:
Xoma Corp. - Licensor, primary
Description: The Bacterial cell expression tech-
nology (BCE), including the araB promoter, en-
ables bacterial, particularly E. coli, expression and
secretion of antibody domains, including antibody
fragments and single-chain antibodies, directly
from bacterial cells as fully functional, properly
folded molecules.
Use with: E. coli; prokaryotes
Use to make: antibodies
Background: XOMA scientists were the first to
demonstrate the secretion of antibody domains
directly from bacterial cells as fully functional,
properly folded molecules.
Besides large-scale protein expression, Bac-
terial cell expression technology (BCE) is an
enabling technology used to discover and screen
recombinant proteins and antibodies for commer-
cial purposes. BCE is also an essential technol-
ogy used in multiple systems for high-throughput
screening of antibody domains. Expression of
antibodies by phage display technology, for
example, depends on the expression and secretion
of antibody domains from bacteria as properly
folded, functional proteins.
Patents: XOMA has reported that it has received
ten U.S. patents to date relating to aspects of its
BCE system, including six patents that broadly
cover the secretion of immunoglobulins from
bacteria, including antibody fragments such as
Fab and single-chain antibodies. Corresponding
foreign patents have also been granted. XOMAs
patent estate is applicable to the practice of anti-
body phage display and other antibody screening
applications.
Exemplary patents include 7,094,579, Eu-
karyotic signal sequences for prokaryotic expres-
sion, Gray, et al., Aug. 22, 2006; and 7,396,661,
Eukaryotic signal sequences for polypeptide
expression and polypeptide display libraries,
Gray, et al., July 8, 2008. These claim methods
and compositions for expressing proteins or
polypeptides in prokaryotic hosts using eukaryotic
signal sequences.
Availability: BCE reagents are available form
Takara Mirus Bio (and perhaps others) for non-
commercial use.
Licensing information: Contact Xoma regarding
commercial use and licensing.
More than 45 companies have licensed BCE.
This seemingly includes most major biopharma-
ceutical developers, and particularly most every
company involved with developing recombinant
antibody fragments, single-chain antibodies, etc.
In some or many cases, BCE may be used for an-
tibody fragment discovery and early development,
switching to eukaryotic system for commercial
manufacture.
XOMA has also provided its BCE technology
in cross-license arrangements that have allowed
XOMA to gain access to other technologies criti-
cal to its antibody discovery and development
programs. XOMA generally does not disclose roy-
alty percentages, up-front or milestone payments,
or other financial terms of specific licenses.
Products made using this tech.: Marketed
biopharmaceuticals manufactured using BCE
include:
a) Cimzia [Certolizumab pegol - CDP 870; tumor
necrosis factor monoclonal antibody Fab, re-
combinant-polyethylene glycol (PEG) polymer
conjugate] marketed for rheumatoid arthritis and
Crohns disease by the UCB Group S.A. with
contract manufacture by Lonza Biologics plc and
BioReliance Corp. Xoma reportedly receives a
royalty of <1% (with Lucentis sales approaching
$1 billon/year).
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
62
b) Lucentis [ranibizumab - vascular endothelial
growth factor (VEGF) monoclonal antibody frag-
ment, recombinant] manufactured and marketed
(in the U.S.) for wet age-related macular degen-
eration (AMD), with international marketing by
Novartis.
145
Subtilisin (psub) fusion protein
tag expression; Profnity eXact
expression - E. coli; bacteria;
yeasts; CHO cells
Organizations involved:
Potomac Affinity Proteins - Assignee, patent
Description: Subtilisin prodomain (psub) fu-
sion protein tags are useful for improved expres-
sion and one-step purification using S189, an
unusually stable form ot the protease enzyme
subtilisin from Bacillus species bacteria. The 75
aa psub is fused to the N-terminal of the desired
protein. The subtilisin prodomain (psub) portion
of the fusion proteins binds to chromatography
media-immobilized subtilisin with high affinity,
and once an activating agent, fluoride, is added,
the subtilisin is activated and cleaves the fusion
protein, releasing the desired protein into solution.
The ligand, S189 subtilisin, both specifically rec-
ognizes and cleaves protein from the bound tag.
Cleavage is claimed to be much more complete
and reliable than other systems.
The systems involve two basic components:
1) a target protein fused to the C-terminus of an
engineered prodomain (protagged protein); 2) a
subtilisin mutant (psub) which is virtually inactive
in the absence of fluoride as a triggering agent.
The ability to isolate the binding and process-
ing steps with a triggering mechanism creates a
processing system with a virtual on-off switch and
allows psub to be used as both the affinity ligand
and processing enzyme for affinity purification
and processing of proteins fused to the tag.
The method consists of passing a crude cell
lysate containing the prodomain-target fusion
protein onto a column of immobilized subtilisin
resulting in strong binding of the target to the
resin. Once contaminating proteins are washed
away, the subtilisin can cleave the target protein
from the bound prodomain by the addition of a
fluoride that converts the enzyme from an inactive
to active form, generating the native protein in a
pure form.
The company has engineered forms of sub-
tilisin and prodomain regions of the enzyme
that possess strong and specific affinity for one
another, and have constructed plasmid vectors
that enable the high-level expression of virtually
any target protein as a fusion with the subtilisin
prodomain.
Background: This method uses a highly modified
form of subtilisin, S189, designed to selectively
break one specific bond in a strategic location---
-and only when it is triggered to do so. Modified
forms of subtilisin, a protease enzyme from Bacil-
lus subtilis, are widely used in laundry detergents.
A number of N-terminal extensions (prodo-
mains) are large enough to fold independently and
have been shown to bind tightly to the active site
of the mature subtilisin protease. Subtilisin BPN
is an extracellular serine proteinase from Bacillus
amyloliquefaciens having a primary translation
product which is a pre-pro-protein. A 30 amino
acid pre-sequence serves as a signal peptide for
protein secretion across the membrane and is hy-
drolyzed by a signal peptidase. The extracellular
part of the maturation process involves folding of
prosubtilisin, self-processing of a 77 amino acid
sequence to produce a processed complex and
finally degradation of the prodomain to create the
275 amino acid mature subtilisin sequence. The
77 amino acid prodomain (in this expression sys-
tem, fused to the protein of interest) is removed
autocatalytically.
Use with: E. coli and other bacteria; also yeasts
and CHO cells
Use to make: proteins; glycoproteins
Benefits: In addition to substantial cost savings,
the method makes it possible to rapidly purify a
variety of different protein targets with a single
type of affinity tag.
Broad/Platform Technologies
63
Patents: Exemplary U.S. applications include
20060134740, Engineered proteases for affinity
purification and processing of fusion proteins,
Bryan, Philip N., June 22, 2006
Availability: An E. coli-based expression system
is marketed as Profinity eXact by Bio-Rad for
non-commercial uses.
Licensing information: Contact Potomac Affin-
ity Proteins regarding commercial use and licens-
ing.
146
Tetracycline-induced
expression repression -
prokaryotes
Organizations involved:
IBA Gene Technologies - Licensor, primary
Max-Planck-Gesellschaft Zur Forderung Der
Wissenschaften E.V. - Assignee, patent
Description: Much like the Tetracycline (Tc; Tet)
Expression Systems widely used in eukaryotes
(see related entry), this system enables tetracy-
cline-induced repression of prokaryotic expres-
sion.
Use with: prokayotes
Use to make: proteins
Patents: This tetracycline promoter based pro-
karyotic expression system is covered by U.S.
5,849,576, Tetracycline promoter for the strin-
gently regulated production of recombinant
proteins in prokaryotic cells, assigneed to Max-
Planck-Gesellschaft Zur Forderung Der Wissen-
schaften E.V.; and EP759997 (BE, DE, FR, UK).
These and other Max-Planck patents have been
exclusively licensed by IBA.
The exemplary claim (1) of 5,849,576 states,
A prokaryotic vector comprising (a) a regulatable
expression control sequence which can be re-
pressed by a tetracycline repressor protein and (b)
a DNA encoding a tetracycline repressor protein
in operative linkage with an expression control
sequence which cannot be repressed by a tetracy-
cline repressor protein.
Availability: Reagents are available for non-com-
mercial use from IBA.
Licensing information: Contact IBA regarding
commercial use and licensing.
Bacteria
147
Bacillus megaterium
expression - E. coli alternative
Organizations involved:
Technical University Carolo-Wilhelmina at
Brunswick - R&D
Description: Bacillus megaterium expression
has been developed, and is an alternative to E.
coli. This includes a polylinker downstream of
the promoter that facilitates versatile cloning in
pHIS1522/pHIS1525 using the BsrGI site directly
before the ATG start codon, allowing cloning
without changing the N-terminus of the protein of
interest. pHIS1525 contains a sequence encoding
the signal peptide of the B. subtilis extracellular
esterase (LipA) enabling efficient secretion of
the target protein into the culture medium, and
target genes can be inserted directly after the
signal peptidase restriction site. Due to use of
the tightly regulated xylose operon, genes can be
induced 130- to 350-fold without proteolysis. A
parallel cloning strategy of the gene of interest in
pHIS1522 and pHIS1525 enables intracellular and
extracellular protein production and a 6xHis tag
(see related entry) upstream the polylinker allows
convenient purification and detection of target
proteins (see related entry). Vectors without tag
sequences (pMM1522 & pMM1525) and vectors
providing a Step-tag (see related entry) are also
available.
Use with: Bacillus megaterium
Use to make: proteins
Benefits: Among bacilli strains (e.g., E. coli), B.
megaterium has the advantage that no alkaline
proteases are present, resulting in expression of
foreign proteins without degradation. In addi-
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
64
tion, there are no endotoxins found in the cell
wall. Protein yields are exceptionally good, even
if inexpensive substrates are used. All B. subtilis
vectors are compatible with B. megaterium as
well. Advantages include:
stable protein expression with high yield
suited for industrial large-scale protein produc-
tion
xylose operon: tightly regulated and efficiently
inducible by xylose (up to 350-fold)
extended polylinker downstream of promoter
allows versatile cloning - additional BsrGI site en-
ables cloning without modifying the N-terminus
no indication of proteolytic instability even up
to 5 hours after induction, since alkaline proteases
(such as e.g., in B. subtilis) are not produced
endotoxins are not found in the cell wall
pHIS1525, pMM1525, pSTREP1525 &
pSTREPHIS1525 vectors provide a signal se-
quence for efficient protein secretion
easy purification and detection using 6xHis-
and/or Streptagged target proteins
Patents: No published patents or applications
were retrieved from simple searching.
Availability: Reagents and kits are available from
MoBiTec for non-commercial uses.
Licensing information: Contact the Technical
University Carolo-Wilhelmina at Brunswick.
Products made using this tech.: HIV gp120 (or
160) for diagnostics is commercially produced by
B. megaterium.
Further information: Hollmann, R., Malten, M.,
Biedendieck, R., Yang, Y., Wang, W., Jahn, D.,
Deckwer, W.-D., (2006). Bacillus megaterium as
a production system for recombinant proteins,
Chemie-Ingenieur-Technik 78, 289-294.
Production and secretion of recombinant pro-
teins using Bacillus megaterium, Ph.D. thesis,
Dr. Yang Yang, Universitt Carolo-Wilhelmina
zu Braunschweig (online at http://bib1lp1.rz.tu-
bs.de/docportal/servlets/MCRFileNodeServlet/
DocPortal_derivate_00004176/Yang_Dissertation.
pdf;jsessionid=0000prPHOl4DoqSiCQeksai5Fso?
hosts=local).
Yang, Y., Biedendieck, R., Wang, W., Gamer, M.,
Broad/Platform Technologies
67
Patents: Patents have issued in the United States,
Australia and Singapore; patent applications are
pending in Europe, Japan, Australia, Israel, Mex-
ico and New Zealand. Exemplary U.S. patents
assigned to the Univ. of British Columbia include
6,861,245; 6,210,948; 5,976,864; and 5,500,353.
Availability: CAULOBACTER S-LAYER
PROTEIN SECRETION KITs are available from
Invitrogen for non-commercial use.
Licensing information: Contact the University
of British Columbia regarding commercia use and
licensing.
Further info.:
Nomellini et al 2007. S-layer-mediated dis-
play of the immunoglobulin G-binding domain
of streptococcal protein G on the surface of
caulobacter crescentus: development of an im-
munoactive reagent. Appl Environ Microbiol.
May;73(10):3245-53
Bingle et al. 2000. Secretion of the Caulobacter
crescentus S-layer Protein: Further localization
of the C-terminal secretion signal and its use for
secretion of recombinant proteins. J. Bacteriol.
182: 3298-3301.
Duncan et al, 2005. Evaluating a new system
for the development of particulate enzymes based
on the surface (S)-layer protein (RsaA) of Caulo-
bacter crescentus; fusion with the -1,4-glycanase
(Cex) from the cellulolytic bacterium Cellulomo-
nas fimi yields a robust, catalytically active prod-
uct. Appl. Biochem. Biotechnol. 127: 95-110.
151
Clostridium Expression
System; NTNH promoter from
Clostridium botulinum
Organizations involved:
University of Wisconsin - R&D
Wisconsin Alumni Research Foundation
(WARF) - Licensor, primary
Description: Clostridium genus bacterial gene
constructions may be expressed in a Clostridium
host. A mobilizable transfer plasmid is provided
which permits the direct transfer of the plasmid
and genes carried on it from E. coli into Clos-
tridium species. A promoter is provided for use
in Clostridium species. Also, a useful host strain
is provided which is nontoxigenic and which per-
mits high levels of expression of clostridial genes
(e.g., botulinum toxins) or other genes using the
clostridial promoter.
Use with: Clostridium bacteria
Use to make: proteins
Patents: Exemplary patents include 5,955,368,
Expression system for clostridium species,
Johnson, et al., Sept/ 21, 1999, assigned to Wis-
consin Alumni Research Foundation (WARF).
The first 5 of 15 claims state:
1. A conjugative transfer plasmid comprising: an
origin of replication effective in E. coli; an origin
of replication effective in Clostridium species; a
gene for an antibiotic resistance marker; and an
origin of conjugative transfer which is capable of
modulating the conjugative transfer of the plasmid
from E. coli into a Clostridium species.
2. The conjugative transfer plasmid of claim 1
wherein the origin of conjugative transfer is RP4
oriT.
3. The conjugative transfer plasmid of claim 1
further comprising a genetic construction effective
to express an antibiotic resistance in Clostridium
botulinum.
4. The plasmid pJIR1457.
5. The plasmid pJIR1456.
Other claims include the NTNH promoter from
Clostridium botulinum and use of C. botulinum
strain LNT01. One useful component for the sys-
tem is a wide host range shuttle vector, pJIR1457,
capable of transferring DNA constructs from E.
coli to clostridial species and to C. botulinum in
particular. Examples include expression of botuli-
num toxin (comparable to non-recombinant toxin,
e.g., used in BOTOX from Allergan).
Licensing information: Contact WARF, the
licensing agent for the University of Wisconsin.
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
68
152
Flavobacterium heparinum
expression - glycoproteins
Organizations involved:
BioMarin Pharmaceutical Inc. - Licensor,
primary
Description: The bacterium Flavobacterium
heparinum, naturally capable of expressing gly-
coproteins, is useful as host cells for recombinant
(glyco)protein expression. Vectors for transfor-
mation of F. heparinium, pIBFX1 and pIBFX2,
are provided. A method enables expression of
homologous and heterologous genes in F. hepari-
num and the coupling of expression to secretion
to produce a biologically active polypeptide or
protein. Vectors contain a functional origin of
replication (OriC) from F. heparinum or another
functional origin of replication, replication (rep)
genes which direct the replication of the vector, a
promoter derived from a gene endogenous to the
F. heparinum host; and also usually a chromosom-
al integration sequence. Vectors may also contain
selective markers and/or a regulated promoter for
the expression of desired gene sequences.
Use with: Flavobacterium heparinum
Use to make: proteins; glycoproteins
Background: The DNA sequences and methods
for the expression of homologous and heterolo-
gous proteins in Flavobacterium heparinum, a
Gram negative, non-pathogenic soil bacterium,
had previously not been investigated, despite the
microorganism naturally producing glycosyl-
ated proteins. In addition, F. heparinum naturally
produces low levels of the glycosaminoglycan de-
grading enzymes that act upon heparin and other
glycosaminoglycans.
Patents: Exemplary patents include U.S
.6,841,375, Flavobacterium heparinum expres-
sion system, Su, H., et al., Jan. 11, 2005, as-
signed to BioMarin Pharmaceuticals Inc. Claim
1 states, . A Flavobacterium heparinum host cell
transformed with a recombinant DNA expres-
sion vector selected from the group consisting of
pIBFX1 and pIBFX2.
Licensing information: Contact BioMarin.
Products made using this tech.: BioMarin
reports, The overexpression of homologous and
heterologous cloned genes in the F. heparinum
host cell, and the suitability of F. heparinum for
the expression of recombinant proteins is demon-
strated by the expression of the enzymes, hepari-
nase I, heparinase II, heparinase III, chondroitin-
ase AC, and chondroitinase B from F. heparinum
; dhfrII and beta-galactosidase encoding genes
from E. coli.
153
Lactococcus lactis htrA-
expression - protease depletion
Organizations involved:
GTP Technology S.A. - Licensor, primary
Institut National de la Recherche Agronomique
(INRA) - Assignee, patent
Sabatier University - Assignee, patent
Description: Lactococcus lactis provides a tightly
controlled expression system, both highly induc-
ible and well repressed, combining inducible
expression and protein stability for heterologous
protein production; providing an alternative to Ba-
cillus subtilis and E. coli. In the absence of HtrA,
the extra-cellular proteolysis of all tested proteins,
in particular heterologous ones, has been com-
pletely abolished, and the yield of intact protein
significantly increased.
The technology minimally consists of 2 ele-
ments: a tightly controlled expression system,
both highly inducible and well repressed and; a L.
Lactis htrA mutant strain, devoid of any related
surface protease (serine protease of the trypsin
type) to increase heterologous proteins yield. A
L. Lactis htrA mutant strain devoid of any surface
proteases has increase heterologous proteins yield,
and allows protein purification with a very safe,
GRAS (Generally Recognized as Safe) bacteria
commonly used in foods. This lactococcal expres-
sion/secretion system uses also both PZnzitR, an
expression cassette tightly controlled by envi-
ronmental zinc, and a consensus signal peptide,
Broad/Platform Technologies
69
SPExp4, for efficient protein production. HtrA
deletion or inactivation may also be used in other
bacteria, e.g., E. coli.
Inactivation of HtrA (or complementation of
the Sec machinery with B. subtilis SecDF ac-
cessory protein) results in the increase in heter-
ologous protein yield, as evidenced by protein
yields (protein amount/biomass) comparable to
those obtained using NICE (see related entry) or
P170 expression systems under similar laboratory
conditions.
The use of bacteria as cell factories to produce
heterologous exported proteins is often limited
by extra-cellular proteolysis. A Lactococcus lactis
mutant strain was developed in which recom-
binant or heterologous exported proteins are
stable. A previously unknown membrane prote-
ase belonging to the HtrA/DegP family (HtrA)
was first identified in L. lactis subsp. lactis strain
IL1403. Inactivation of the chromosomal gene
revealed that HtrA acts as a surface housekeeping
protease by elimination of abnormal and/or mis-
folded proteins, and that HtrA is also responsible
for the maturation of natural exported proteins,
such as the L. lactis bacteriolysin, AcmA. IThese
results suggest that HtrA is the sole extra-cellular
housekeeping protease in L. lactis, in agreement
with the analysis of the complete IL1403 genome
sequence. The mutant htrA strain is an efficient
tool to improve yields of heterologous exported
proteins in L. lactis.
Use with: Lactococcus lactis; bacteria
Use to make: proteins
Background: Lactococcus lactis, the model
lactic acid bacterium (LAB), is a food grade and
well-characterized Gram positive bacterium. L.
lactis can also be used as a protein producer in
fermentors. Many heterologous proteins have
already been produced in L. lactis but only few
reports allow comparing production yields for a
given protein either produced intracellularly or
secreted in the medium. Available data show that
i) secretion is preferable to cytoplasmic produc-
tion; ii) secretion enhancement (by signal peptide
and propeptide optimization) results in increased
production yield; iii) protein conformation rather
than protein size can impair secretion and thus
alter production yields; and iv) fusion of a stable
protein can stabilize labile proteins.
Patents: INRA reports patent file nFR 9816462
entitled: Gram positive bacteria deprived of HtrA
proteasic activity and their uses, extended in
Europe, In the US and in Canada; and patent file
nFR0210805 entitled: Zinc regulated prokary-
otic expression cassettes, extended in Europe, In
the US and in Canada.
Exemplary U.S. applications include
20060228377, Poquet, I., et al., Oct. 12, 2006,
with its 11th claim (the first remaining after can-
cellation of the first 10) stating, A method of pro-
ducing a fermented product, comprising: culturing
a bacterial strain with a fermentation substrate
under conditions suitable to produce a fermented
product, wherein the bacterial strain does not
express a functional HtrA.
Benefits include:
A Safe system. - GRAS (Generaly Recog-
nized As Safe) and endotoxin free system.
Easy to use. Microbial host with rapid growth and
very simple metabolism.
Easy to scale-up. Growth in stirred fermentors
without aeration : scale up to thousands of liters in
one step only.
Easy dowstream process. Efficient secretion of
proteins and limited endogen proteins : the protein
of interested is often more than 50% pure in the
supernatant.
Licensing information: Contact GTP Technology
S.A. or INRA (mercier@paris.inra.fr).
Further info.:
Morello E, Bermdez-Humarn LG, Llull D,
Sol V, Miraglio N, Langella P and Poquet I.
2008 Lactococcus lactis, an efficient cell factory
for recombinant protein production and secretion.
J. Mol. Microbiol. Biotechnol.14:48-58.
Cortez-Perez N.G., Poquet I., Oliveira M., Grat-
adoux J.-J., Madsen S.M., Miyoshi A., Corthier
G., Azevedo V. & Langella P. 2006. Construction
and characterization of a Lactococcus strain defi-
cient in intracellular ClpP and extracellular HtrA
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
70
proteases. Microbiology, 152: 2611-2618.
Protein secretion in Lactococcus lactis : an ef-
ficient way to increase the overall heterologous
protein production, Microb Cell Fact., 2005; 4:2,
2005 January 4.
HtrA is the unique surface housekeeping protease
in Lactococcus lactis and is required for natural
protein processing. Mol Microbiol 35, 1042-51.
Controlled production of stable heterologous pro-
teins in Lactococcus lactis. Appl Environ Micro-
biol 68, 3141-6.
Heterologous protein production and delivery
systems for Lactococcus lactis. Genet Mol Res 2,
102-11.
154
Lactose (lac) promoters;
Isopropyl-beta-galactosidase
(IPTG) induction - bacteria
Organizations involved:
Description: Lactose (lac) promoters enables
regulation of expression by the lac repressor, with
induction of expression by isopropyl-beta-galacto-
sidase (IPTG). This has been and continues to be
widely used.
Use with: for E. coli and other bacteria
Use to make: proteins
Patents: No relevant U.S. patents assigned to
the inventor, Yanisch-Perron, Rutgers University,
were retrieved. First reported in 1983, any related
patents have expired.
Availability: Related reagent available from
many vendors.
Further info.:
Yanisch-Perron et al. Gene 33: 103-109 {1985}.
155
NIsin-Controlled gene
Expression (NICE); Lactococcus
lactis expression; nisA
promoter - antibiotic selection
Organizations involved:
NIZO food research B.V. - Licensor, primary
Description: Thie nisin-controlled gene expres-
sion (NICE) system with antibiotic selection is
used in the gram-positive host, Lactococcus lactis,
and provides a well-characterized and established
inducible expression system. It is considered one
of the most successful and widely used Gram-pos-
itive gene expression systems. NICE has more
than 10-years track record in academic research
and is supported by some examples of large scale
production (e.g., the production of lysostaphin)
This system exploits the autoregulatory char-
acteristics of the production of the lantibiotic nisin
by this bacterium for controlled expression of
homologous and heterologous genes that can be
triggered by addition of nisin as an inducer. The
NICE system includes several nisRK-express-
ing production hosts and a variety of expression
vectors that allow translational or transcriptional
fusion of the gene of interest to the tightly con-
trolled nisA promoter.
Use of a dual plasmid-based cassette system
or derivatives of this system has shown that the
NICE system could also be functionally imple-
mented in a wide range of alternative gram-posi-
tive hosts, including B. subtilis
The NICE system has been used successfully
for the construction of nisin-controlled autolytic
lactococcal strains and several conditional mu-
tants, as well as in industrial-scale protein and
peptide production.
Use with: Lactococcus lactis
Use to make: proteins
Background: Nisin is a 34-amino acid anti-mi-
crobial peptide (lantibiotic) with various unusual
amino acids and five ring structures.
Patents: Related patents include U.S. 5,928,946,
Lactic acid bacteria producing lantibiotics similar
Broad/Platform Technologies
71
to nisin A; and 5,914,248, Method for controlling
the gene expression in lactic acid bacteria, both
assigned to Stichting Nederlands Instituut Voor
Zuivelonderzoek (NIZO).
Licensing information: Contact NIZO regarding
commercial use and licensing.
Further info.: Autoregulation of nisin biosynthe-
sis in Lactococcus lactis by signal transduction, J
Biol Chem. 1995 Nov 10;270(45):27299-304.
Trends Biotechnol., Controlled overproduc-
tion of proteins by lactic acid bacteria, 1997
Apr;15(4):135-40.
Optimization of the Lactococcus lactis nisin-con-
trolled gene
expression system NICE for industrial appli-
cations, Microbial Cell Factories 2005, 4:16
doi:10.1186/1475-2859-4-16.
156
P170 Expression
System;Lactococcus lactis
expression
Organizations involved:
Bioneer Corp. - Licensor, primary
Description: The P170 Expression System using
the P170 promoter for inducible expression in
Gram positive lactic acid bacteria, particularly
Lactococcus lactis, with induction of expression
when a certain threshold of lactate is reached in
the culture, with the protein of interest fused to a
secretion signal facilitating secretion of the gene
product into the culture supernatant. This type of
auto-induction eliminates the addition of exog-
enous components to induce gene expression. The
extracellular export of the protein product reduces
the number of steps needed for the subsequent
downstream processing, helping to reduce the
overall cost of the process. A fermentation me-
dium has been developed that is devoid of animal-
derived components.
Despite all of these advantages, the production
of lactic acid - the primary end product of glucose
metabolism - remains a limiting effect on biomass
production in L. lactis. Lactic acid inhibits growth
even when the acid is neutralized by addition
of base to keep pH constant. As a consequence,
the yield in biomass production is below that of
other expression systems, with cell densities of
approximately 20 OD600 units. With this limited
cell density, Bioneer has produced up to 200-300
mg/L of secreted protein, a level acceptable for
some high value proteins. Methods for higher pro-
duction levels are being developed. Lactococcus
lactis P170 expression system may be combined
with Reverse electro enhanced dialysis (REED)
for lactate control.
Use with: Lactococcus lactis
Use to make: proteins
Benefits: The P170 Expression System is par-
ticularly well suited for industrial scale production
of pharmaceutical proteins. Lactic acid bacteria
do not produce endotoxins, which makes them at-
tractive protein production host organisms. In ad-
dition, several lactic acid bacterial strains includ-
ing L. lactis strains do not produce extracellular
proteases and are capable of secreting peptides,
polypeptides or proteins ensuring high gene prod-
uct stability facilitating subsequent purification.
Patents: Exemplary patents include U.S.
7,358,067, Fermentation method for production
of heterologous gene products in lactic acid bac-
teria, Vrang, A., et al., April 15, 2008, assinged
to Bioneer A/S. Claim 1 states, 1. A method of
producing a heterologous peptide, polypeptide
or protein in a Lactococcus lactis bacterium, the
method comprising the steps of: (i) construct-
ing a recombinant Lactococcus lactis bacterium
comprising a nucleotide sequence coding for the
heterologous peptide, polypeptide or protein and
operably linked thereto, appropriate regulatory
nucleotide sequences to control the expression of
the coding sequence, (ii) cultivating said recom-
binant bacterium under fed-batch or continuous
cultivation conditions in a chemically defined
medium, to express the nucleotide sequence, and
(iii) harvesting the recombinant bacterium or
the peptide, polypeptide or protein, wherein the
concentration of glucose is kept at a pre-selected
concentration of at least about 0.5 g/L by con-
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
72
trolled feeding of glucose.
Licensing information: Contact Bioneer regard-
ing commercial use and licensing.
157
Pfenex Expression;
Pseudomonas fuorescens
biovar I (MB101) - E. coli
alternative
Organizations involved:
Dowpharma, Dow Chemical Co. - Licensor,
primary
Description: Pfenex is an expression system us-
ing a natural isolate of Pseudomonas fluorescens
bacterium (strain MB101) as host cells for recom-
binant protein manufacture. Pfenex has proven
to be a robust and cost effective platform for the
production of numerous classes of therapeutic
proteins, including various types of antibody
derivatives/fragments. High cell densities in the
fermentor along with high specific protein yields
result in high target protein titers, e.g., yields over
25 grams/L have been reported. Very high cell
densities are also achievable. An extensive tool
box of unique host strains has also been devel-
oped. These strains have phenotypes selected to
impact the amount of target protein produced and
its solubility and activity. High throughput meth-
ods allow dozens of different expression strategies
and host cell combinations to be rapidly tested
in parallel. Also, efficient periplasmic secretion
of proteins has been developed. This capability
allows the formation of disulfide bonds, critical to
the production of active antibody derivatives, and
also the development of simplified downstream
processes. The technology is easily employed in
traditional E. coli and other fermentation, recov-
ery and purification settings with no need for
additional, unique equipment. Pfenex may join or
replace E. coli as a first-line expression technol-
ogy, particularly as the complexity of new thera-
peutics continues to increase.
Pfenex can achieve soluble, correctly folded
proteins with a yield improvement that can be
over ten times greater than E. coli. With Pfenex,
the vast majority (75-80%) of proteins expressed
so far have folded completely the first time - there
are very few proteins that have not and even then
they have still come out partially folded.
Pfenex is able to express single chain antibod-
ies (scFVs) at high yield and in some cases as
soluble active protein . Active scFvs have been
expressed in both the cytoplasm and periplasm. P.
fluorescens expresses cytoplasmic, soluble, active
scFv at
3g/L.
Many high performance production strains
have been developed. In most cases, Dowpharma
can identify 10-20 different forms of Pfenex for
a given protein and deliver a result within eight
to twelve weeks, while with an insoluble protein
using E. coli this process can take up to a year,
Proteins are expressed in high yields with correct
disulfide bond formation, reduced proteolytic deg-
radation and enhanced solubility/activity. Dozens
of modified host strains can be tested in parallel
to identify expression strategy/host cell combina-
tions that result in improved target protein accu-
mulation and/or stability.
Pfenex Expression Technology has been
developed with regulatory requirements in mind,
specifically eliminating the need for antibiotics
for plasmid stability, while also avoiding ani-
mal-derived products using a defined medium.
In developing Pfenex, Dowpharma claims that
it continues to invest a significant amount of
research and development in identifying high-
performing strains of Pseudomonas fluorescens,
gene mapping these strains and improving them
by application of sophisticated genomic tools.
Dowpharma can quickly and easily modify the
strains to increase the expression of a particular
customers biotherapeutic.
Protein production in P. fluorescens can be
manipulated at the genetic level so that the organ-
ism can secrete proteins into the perisplasm (the
space between the inner and outer membranes of
the cell). Secretion can be of value since simpler
methods can be used to recover the protein (me-
Broad/Platform Technologies
73
chanical cell breakage is not required). Secretion
is typically mediated by a short amino acid leader
sequence on the amino terminus of the secreted
proteins allowing transport across the inner mem-
brane. This sequence is removed at the periplas-
mic face by a signal peptidase.
Use with: Pseudomonas fluorescens
Use to make: proteins; antibody fragments
Background: Pseudomonas fluorescens is an
abundant and non-infectious component of the
microbial flora of soil, water, and plants. This
obligate aerobe is nonpathogenic (BL-1). Deriva-
tives of P. fluorescens strain MB101 have been
used for a number of years to produce a range of
protein types, including industrial enzymes, one
of which has received GRAS notification from the
FDA for a food processing application. Pseu-
domonas fluorescens biovar I MB101 has been
safely used to commercially produce a variety
of proteins at scales up to thousands of liters, for
over 15 years.
Benefits: Money is saved using Pfenex primar-
ily because of the much greater yields it is able
to produce per fermentation batch compared to
E. coli. Because protein comes out in a soluble
form, it is easier to handle and allows a clearer
path through the purification process. Pfenex
technology also fits directly into customers
existing E. coli equipment, allowing for a direct
swap out, requiring no extra investment and it is
operationally very similar to the E. coli process.
A downside of Pfenex is that like all prokaryotes,
including E. coli, Pseudomonas cant glycosylate
proteins (although various glycosylation modifi-
cations may enable this; see related entries).
Claimed benefits include:
Increased protein expression
Secretes proteins to the periplasmic space
Maintains solubility
Increased downstream yield
Scale up is easily
Resolving of protease issues
Improve activity characteristics
Lower cost of goods
Genome fully sequenced by Dow
Broad/Platform Technologies
91
interest at a higher level than the corresponding
non-mutant strain under the same conditions.
Licensing information: Contact Dyadic, which
offers CMO services.
178
CoMed system; Universal Yeast
vectors; pCoMed vectors;
Arxula adeninivorans-derived
TEF1 promoter
Organizations involved:
PharmedArtis GmbH - Licensor, primary
Description: The CoMed system, including
Universal Yeast vectors, is a modular yeast strain/
vector system. Particular combinations of vector
elements result in a Universal Yeast vector that
can be addressed to a range of yeast platforms,
i.e., different yeast expression systems, includ-
ing in parallel for testing. A derivative CoMe-
dImmune system is under current development,
designed for the production of antibodies and
derivatives. Co-transformation and co-assessment
can be performed with several yeasts at the same
time, including in Hansenula polymorpha, Arxula
adeninivorans and other yeasts.
Since vector systems of different yeast spe-
cies are based on different basic vectors it has
been very difficult to exchange single cassettes
between the yeast systems. To reduce this dis-
advantage, the CoMed vector system was estab-
lished containing the pCoMed basic vector for
integration of ARS, selection markers, rDNA
sequences and expression cassettes. The single
modules are flanked by identical restriction sites
and are integrated in the same location of the ba-
sic vector. In this system, various modules can be
integrated. Due to high conservation in yeasts of
the included coding regions targeting of all yeast
species is feasible. If for instance the combination
of rDNA and the ALEU2 gene is chosen, a range
of yeasts with this auxotrophy can be targeted.
The same holds for the insertion of a dominant
selection marker like the E.coli-derived hph gene
conferring resistance to hygromycin B in all yeast
species tested so far. The expression cassette is
inserted in a final step as fragments derived from
pre-constructed plasmids. A range of such cassette
elements exists harbouring a promoter of choice,
among others the A. adeninivorans-derived TEF1
promoter mentioned before, and a S. cerevisiae
PHO5 terminator separated by a multiple cloning
site. This promoter was found to be functional in
all yeast species tested so far. A selection of ARS
sequences is available that will result in either epi-
somal (S. cerevisiae) or chromosomally integrated
plasmids (Hansenula polymorpha). However in-
clusion of such a sequence may reduce the range
of addressible hosts.
By easy exchange of modules, such a vector
can be converted into a plasmid that is optimal for
an individual platform, for instance by inserting
an expression cassette with a promoter element
that elicits efficient gene expression in methy-
lotrophic yeasts only. Variants of the CoMed
basic vector for the production of antibodies and
derivatives are under development. In yet another
design, it is possible to linearize the plasmids
in a way that leaves behind all bacterial DNA
sequence
Use with: yeasts
Use to make: proteins; glycoproteins; antibod-
ies
Patents: IP exclusively licensed by PharmedArtis
GmbH includes EP05/004550.9, Recombinant
protein expression system, claiming a modu-
lar universal vector system for yeasts; and
WO01/385400, Vectors and methods for produc-
ing recombinant proteins in fungi, claiming a
method of rDNA integration and rDNA target-
ing element components of the universal vector
system
Licensing information: Contact PharmedArtis
regarding commercial use and licensing.
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
92
179
Fungal expression systems
Description: Fungi normally secrete high concen-
trations of proteins, making them candidates for
heterologous protein expression. One can gener-
ally culture fungus under conditions that induce
expression of target gene(s), extract mRMA and
construct cDNA libraries in E. coli; use libraries
in conjunction with a selectable marker/reporter to
transform yeast or other fungal host, and culture
in medium that allows selection of desired mutant
phenotypes.
As can be seen from the related entries, a vari-
ety of yeast and other commercially-viable fungal
expression systems are available.
Fungi, unlike bacteria, generally use promoters
from same species.
180
GlycoFi technology; Next
Generation Biotherapeutics
- Pichia pastoris; yeasts;
glycosylation; antibodies
Organizations involved:
Dartmouth College - R&D
GlycoFi, Inc. - Licensor, primary
Merck & Co., Inc. - Parent co.
Description: Various mannosidase-family genes
inserted into yeast provide proteins with mamma-
lian- or human-like glycosylation patterns, includ-
ing antibodies, with unprecedented control and
uniformity. Yeasts, particularly Pichia pastoris,
or other fungi are modified to produce N-glycans
such as Man5GlcNAc2 or other structures along
human glycosylation pathways. This technol-
ogy is useful to perform the final, most complex
step of human glycosylation and can produce a
broad variety of recombinant proteins, includ-
ing antibodies, with fully human (or human-like)
glycosylation structures. Expressing a protein in
multiple GlycoFi strains results in a library of gly-
coforms (protein-glycan combinations) that can
be analyzed through in vitro and in vivo meth-
ods to identify which glycans confer the desired
properties. Strains generating mixtures of specific
glycan structures are also possible, increasing the
power of this approach. For example, Pichia pas-
toris yeast was transformed with 14 heterologous
genes so that it would secrete human erythropoie-
tin (EPO) with fully complex, terminally sialyated
N-glycans.
GlycoFi has engineered a broad selection of
yeast strains, each capable of attaching a specific
and pre-determined glycan structure to a pro-
tein of interest. Each of these strains is ready to
receive a recombinant gene construct and is able
to produce a uniformly glycosylated recombinant
protein, including fully-assembled antibodies
and Fc-fusions. This process can be scaled from
96-well microtiter plates to large-scale fermentors
while maintaining the same product homogene-
ity. Typically expression levels of secreted protein
are in the 100-2600mg/l range (up to 2.6 g/L) in a
3-day process prior to optimization.
This technology is commonly portrayed (or
hyped) as potentially eliminating the need for pro-
ducing many therapeutic glycoproteins in mam-
malian systems. As validation of this technology,
Merck & Co. acquired GlycoFi, the systems
commercial developers, for over $400 million,
with this technology probably the companies
primary asset (vs. some early-stage therapeutics).
Next Generation Biotherapeutics was GlycoFis
pre-Merck acquisition trademark for this technol-
ogy.
Use with: Pichia pastoris; yeasts; fungi
Use to make: proteins; glycoproteins; antibod-
ies
Benefits: Obviously, being able to manufacture
complex glycoproteins, including antibodies,
in yeast rather than mammalian systems saves
considerable money and time. Other advantages
of GlycoFis strains include demonstrated genetic
stability, robust scale-up within standard cGMP
infrastructures, and thoroughly documented strain
history, including a sequenced genome. GlycoFi
strains ensure rapid transition from lab bench
Broad/Platform Technologies
93
to clinical phase development with typical turn
around time of 3-6 months from gene to gram
quantities of secreted protein.
Patents: GlycoFi, now part of Merck & Co.,
owns or controls over 60 issued and pending
patents in the U.S. and abroad relating to glyco-
sylation engineering and the production of human
glycoproteins with proper glycosylation in yeast
and fungi. GlycoFi has also in-licensed comple-
mentary protein expression technology.
Exemplary patents include U.S. 7,029,872,
Methods for producing modified glycoproteins,
Gerngross, T.U., et al., April 18, 2006, with its
abstract stating [truncated], cell lines having
genetically modified glycosylation pathways that
allow them to carry out a sequence of enzymatic
reactions, which mimic the processing of glyco-
proteins in humans, have been developed. Re-
combinant proteins expressed in these engineered
hosts yield glycoproteins more similar, if not sub-
stantially identical, to their human counterparts.
The lower eukaryotes, which ordinarily produce
high-mannose containing N-glycans, including
unicellular and multicellular fungi are modified
to produce N-glycans such as Man5GlcNAc2
or other structures along human glycosylation
pathways. This is achieved using a combination of
engineering and/or selection of strains which: do
not express certain enzymes which create the un-
desirable complex structures characteristic of the
fungal glycoproteins, which express exogenous
enzymes selected either to have optimal activity
under the conditions present in the fungi where
activity is desired, or...
Availability:
Licensing information: GlycoFi strains are
available for out-licensing on a non-exclusive
basis. Terms of a strain license include an upfront
development and licensing fee, milestone pay-
ments, annual maintenance fees and royalties on
product sales. Specific economic terms are protein
and indication dependent.
GlycoFi offers CRO/CMO services, and works
collaboratively with its customers to identify the
most appropriate glycan structure(s) for a given
application.
181
Hansenula expression (yeast)
Organizations involved:
Rhein Biotech Ltd. - Licensor, primary
Dynavax Technologies Corp. - Parent co.
Description: The Hansenula Expression Platform,
including vectors and control sequences, has been
developed Rhein Biotech Ltd. and is being used
for manufacture of a marketed hepatitis B virus
surface antigen (HbsAg) vaccine. Hansenula (not
neccessarily this specific technology) holds the
record in yeast derived-protein production, with
a productivity of more than 13 g/L in phytase
production.
Use with: Hansenula yeasts
Use to make: proteins; glycoproteins
Patents: Exemplary patents include Recombi-
nant production of proteins in yeast, 5,741,674,
Schweden, et al., April 21, 1998, assigned to
Rhein Biotech. The claims of this patent state:
1. A process for the recombinant production of
proteins which are heterologous in the yeast Han-
senula, which comprises transforming Hansenula
with an expression cassette which comprises the
following structural elements encoded: where
L is a leader sequence, A is an adaptor with the
sequence SEQ ID NO:1, P is a processing signal
and GEN is a structural gene for the required
protein; growing Hansenula in a suitable growth
medium; and recovering said protein; wherein
said protein is correctly processed.
2. A process as defined in claim 1, wherein
the leader sequence of the glucoamylase from
Schwanniomyces occidentalis is used as leader
sequence L.
3. A process as defined in claim 1, wherein the
peptide sequence Lys-Arg is used one or more
times as processing signal P.
Another key patent is 5,389,525, DNA-mole-
cules coding for FMDH control regions and struc-
tured gene for a protein having FMDH- activity
and their uses. This has been cited as forming
the basis for HEPLISAV, a recombinant hepatitis
B vaccine that was developed at Rhein Biotech
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
94
GmbH in the early 1990s. This patents abstracts
states, The invention relates to DNA-molecules
comprising DNA-sequences encoding control
regions and the structural gene for a protein
having formate dehydrogenase (FMDH) activ-
ity. Said DNA-molecules may be combined with
DNA-sequences encoding foreign genes so as to
bring these genes under the stringent control of
the regulation of the FMDH regulatory sequences
and/or may be combined to DNA-sequences cod-
ing for secretory signals. The invention further
relates to recombinant vectors containing said
DNA-molecules and micro-organisms contain-
ing said vectors or DNA-molecules. Furthermore,
the invention relates to a process for producing a
useful substance by producing this substance by
culturing said micro-organisms and recovering the
substance.
U.S. 5672487, Recombinant production of
proteins in yeast, assigned to Rhein Biotech and
BASF, describes the use of a pr-pro-sequence
derived from a Carcinus maenas (v) protein that
is compatible to the commonly used MFa1 leader.
Fusions with this pre-pro fragment facilitate yeast,
including Hensensula, secretion of heterologous
proteins.
Licensing information: Contact Rhein Biotech.
Products made using this tech.: Rhein/Dy-
navax manufactures and markets (in countries not
subject to basic hepatitis B virus surface antigen
patents held by Chiron/Novartis) HEPLISAV
hepatitis B virus vaccine.
182
Hansenula polymorpha (yeast)
expression - E. coli alternative
Organizations involved:
Artes Biotechnology GmbH - Licensor, pri-
mary
Bio-Technical Resources (BTR) - Licensor,
primary
Description: Hansenula polymorpha offers a
cost-effective and commercially-viable expres-
sion system. Hansenula polymorpha is often an
alternative to E. coli for producing therapeutic
proteins or technical enzymes. Recombinant Han-
senula strains harbor many copies of the heterolo-
gous DNA, up to 150 copies per cell., resulting in
highly efficient productivity. Up to four different
genes/proteins may be expressed. Yields of up to
13.5 g/L have been obtained, with low levels of
impurities, high product stability and low protease
activity. Typical yields range from 0.5 to several
g/L. H. polymorpha is a very efficient secretor.
H. polymorpha can use alcohol as its sole carbon
source. The system offers high batch-to-batch
reproducibility.
The combined effect of promoter efficiency
and gene dosage results in Hansenula expression
yields superior to other microbial systems. Han-
senula polymorpha is considered an ideal system
for stable high gene dosage dependent expression
and for co-expression of multiple genes, with
high stability of strains (800 generations tested).
Simple, inexpensive media without methanol can
be used, with short fermentation times, e.g., 70-
120 hours.
Use with: Hansenula polymorpha
Use to make: proteins; glycoproteins
Background: Artes Biotechnology GmbH has
20+ year experience with contract R&D and
manufacture using Hensensula.
Patents: The Hansenula technology platform is
protected by a number of patents covering pro-
moters, signal sequences, chaperones as well as
auxotrophic markers and integration sites. As re-
ported by Artes, related patents licensed, some or
many presumably with exclusivity, or developed
by the company include:
a) MOX promoter: EP 173 378, US 5,240,838
(licensed from Rhein Biotech)
b) FMD promoter: EP 299 108, US 5,389,525
(licensed from Rhein Biotech)
c) TPS promoter: WO 00/65071 (licensed from
Rhein Biotech)
d) rDNA integration: WO 01/38510 (licensed
from Rhein Biotech)
e) ARO7 marker: WO 00/65071 (licensed from
Rhein Biotech)
Broad/Platform Technologies
95
f) CMK secretion: WO 00/68400 (licensed from
Rhein Biotech)
g) GAM-KEX2 leader: EP 716 705, US 5,741,674
(licensed from Rhein Biotech/BASF)
h) CHH-KEX2 leader: EP 725 822, US 5,672,487
(licensed from Rhein Biotech/BASF)
i) Glucose Fermentation Process: EP 0911416
(licensed from Hoffmann-La Roche)
j) Xylose reductase / Xylitol dehydrogenase (pat-
ented by ARTES)
k) Cell permeabilization: WO 01/38544 [licensed
from the Institute of Plant Genetics and Crop
Plant Research (IPK)].
Licensing information: Bio-Technical Resources
(BTR) is the exclusive representative for Artes
Biotechnology in North America. Otherwise, con-
tact the developer, Artes Biotechnology GmbH
Licensees include Proteo Biotech AG (2004).
183
Kluyveromyces lactis
expression; pKLAC1;
Acetamidase/Acetamide
selection; K. lactis GG799 -
yeast
Organizations involved:
DSM Biologics - Licensor, primary
New England Biolabs - Licensor, primary
Description: The yeast Kluyveromyces lactis is
useful as host cells for high yield recombinant
protein expression. K. lactis technology offers
high yield protein expression; rapid high cell den-
sity growth; includes glycosylation; can clone and
express genes toxic to E. coli; competent K. lactis
cells are available; no expensive antibiotics or
methanol required; and easy-to-use protocols for
those inexperienced with yeast systems. Acetami-
dase (amdS) expressed from the pKLAC1 vector
permits transformed cells to utilize acetamide as
a sole nitrogen source in defined medium, al-
lowing for simple selection of transformed cells.
Acetamide selection also promotes formation of
cells containing multiple integrations of pKLAC1
which results in higher yields of protein. The
technology includes the K. lactis GG799 strain, an
industrial isolate that has no auxotrophies, rapidly
grows to high cell density, and efficiently secretes
heterologous proteins; pKLAC1 Vector; and
pKLAC1-malE Control Plasmid.
Proteins may be produced intracellularly or be
secreted using the integrative expression vector
pKLAC1. To achieve protein secretion, a gene
of interest is cloned downstream of the K. lactis
alpha-mating factor secretion domain, which is
eventually processed in the Golgi resulting in
secretion of the desired protein.
Use with: Kluyveromyces lactis
Use to make: proteins; glycoproteins
Benefits: The K. lactis expression system offers
several advantages over other yeast and bacte-
rial protein expression systems. K. lactis has
been used to produce proteins at industrial scale
in the food industry for over a decade due to its
ability to rapidly achieve high culture densities
and abundantly produce recombinant proteins.
Yeast expression is driven by a variant of the
strong LAC4 promoter that has been modified
to lack background expression in E. coli. There-
fore, genes toxic to E. coli can be cloned into
pKLAC1 in bacteria prior to their expression in
yeast. Highly competent K. lactis cells make the
technology easy-to-use for those not accustomed
to working with yeast. Their high transformation
efficiency makes the system suitable for methods
that require large numbers of transformants, for
example, expression cloning using cDNA librar-
ies. Proteins expressed in K. lactis have access to
eukaryotic protein folding and glycosylation ma-
chinery that E. coli cells do not possess, making
it an important alternative to bacterial expression
systems.
Patents: Related patents include U.S. 7,390,636,
Method for construction and use of Kluyvero-
myces lactis promoter variants in K. lactis that
substantially lack E. coli transcriptional capabil-
ity, Taron, C., et al., June 24, 2008, assigned to
New England Biolabs, Inc. The abstract states,
Methods and compositions are provided relating
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
96
to production of recombinant protein in yeast. A
modified PLAC4 is described where one or more
mutations may be introduced into the Pribnow
box-like sequences in the promoter. The modi-
fied promoter when placed upstream of a target
gene in a vector causes a significant reduction of
target gene expression in transformed bacteria but
produces efficient expression of the target gene in
yeast.
Availability: Related reagents are marketed or
non-commercial uses by New England Biolabs,
Inc.
Licensing information: A license to use this
system for manufacture of clinical grade material
or commercial purposes is available from New
England Biolabs, Inc. or DSM Biologics.
Further info.:
Kluyveromyces lactis LAC4 Promoter Variants
That Lack Function in Bacteria but Retain Full
Function in K. lactis, Paul A. Colussi and Chris-
topher H. Taron, Appl Environ Microbiol. 2005
November; 71(11): 7092-7098 (doi: 10.1128/
AEM.71.11.7092-7098.2005).
184
NeuBIOS expression;
Neurospora crassa expression;
NeuKARYON - flamentous
fungi; glycosylation; Cabilly-
Boss workaround
Organizations involved:
Neugenesis Corp - Licensor, primary
University of Hawaii - Assignee, patent
Description: NeuBIOS is a Neurospora crassa
(filamentous fungus)-based expression system
with the ability to fold and trim complex mam-
malian proteins correctly, to glycosylate proteins,
and to secrete them into inexpensive liquid media,
making downstream processing and purification
simple and inexpensive. The fungus-based system
also doesnt express toxins and the system has
no known viruses. Wild strains of Neurospora
typically produce more than a gram per liter of
secreted proteins when grown on a liquid minimal
medium for several days.
Neugenesis is developing a host cell line
capable of producing and secreting recombinant
products at levels measured in grams per liter and
at initial purities of more than 90%. Neugenesis
aims to use NeuBIOS to produce high value re-
combinant mammalian proteins, including human
biopharmaceuticals and proteins for research and
development use. To date, Neugenesis has been
able to produce cytokines, monoclonal antibodies,
a thrombolytic protein, and insulin-like proteins.
The NeuKARYON system for antibody manu-
facture, a version of Neugenesis CombiKARY-
ON technology, uses two individually transformed
strains of N. crassa, one producing high levels
of the light subunit and the other producing high
levels of the heavy subunit, which are fused to
form a multicellular, multinucleate strain (hetero-
karyon), which produces both subunits and pro-
cesses them into the intact monoclonal antibodies.
Variants of each subunit gene are generated by
standard techniques and then are inserted into host
cells to produce libraries of strains. NeuKARYON
has the ability to select for equal production of
each subunit before the cells are fused.
For recombinant monoclonal antibodies
manufacture, Neugenesis claims that using MAb
expression technology embodied in US Patent
5,643,745, we can offer the only known alterna-
tive technology to that owned by Genentech under
US Patent 6,331,415. Thus, this is proposed as
an alternative to the need to license the Cabilly-
Boss patent from Genentech (see related entry).
The expression strains are exceptionally stable
and are designed to secrete these proteins into in-
expensive liquid media, which contains no animal
serum or other potential product contaminants.
This makes downstream processing and purifica-
tion extraordinarily simple and inexpensive, re-
sulting in a low overall cost structure. The expres-
sion system has no known viruses, and produces
no toxins or harmful secondary metabolites.
In July 2007, Neugenesis received a multi-
million dollar Phase I contract from the Defense
Broad/Platform Technologies
97
Advanced Research Projects Agency (DARPA)
to develop expression technologies to acceler-
ate recombinant protein production for the rapid
manufacturing of vaccines and biopharmaceuti-
cals. The goal is to develop a commercially viable
system that can produce up to 3 million doses of a
vaccine or a monoclonal antibody therapeutic in a
12 week period.
Use with: Neurospora crassa (filamentous
fungus)
Use to make: proteins; glycoproteins; antibod-
ies
Background: The conventional recombinant ap-
proach to the production and assembly of hetero-
meric proteins, such as monoclonal antibodies,
has been to transform the genes for both subunits
into a single host cell (e.g., see Cabilly-Boss en-
try). Transformed host cells are then selected for
production of the protein. Neugenesis expression
technology enables a new alternative. Two indi-
vidually transformed strains, one producing high
levels of the first subunit and one producing high
levels of the second subunit, are fused to form a
multicellular, multinucleate strain (heterokaryon),
which produces both subunits, and processes
them into the intact molecule. Important features
of Neugenesis protein expression technology
include the ability to fold and trim complex mam-
malian proteins correctly, resulting in bioactivity.
The systems can add sugar residues to secreted
proteins, thus producing N- and O-linked glyco-
proteins.
Neugenesis technology can produce small and
large proteins, including heteromeric proteins
such as human monoclonal antibodies, certain
hormones, and cell surface receptors. Relevant
compounds produced in Neugenesis systems
include: a human cytokine (rhM-CSF), a hu-
man blood protein (HSA), a mammalian food
processing enzyme (bovine chymosin), a mam-
malian hormone (porcine relaxin), a plant protein
(zeamatin), and mammalian plasminogen activa-
tor (DSPA).
Benefits: NeuBIOS is readily scalable and manu-
facturing requires significantly less infrastruc-
ture than traditional methods. Large amounts of
proteins, including glycosylated proteins, can be
rapidly manufactured. Compared to established
mammalian cell culture production systems,
fungal expression systems hold the potential for
significant cost savings. This cost reduction will
come from reduced capital investments, yield
benefits and lower operating costs in production.
Fungal fermentation holds the potential for yields
exceeding several grams/liter during a 10 day
batch cycle. Development times are significantly
reduced for fungal production hosts (typically
6-12 months compared with 18-24 months for
mammalian hosts).
For antibody producers, in particular, and bio-
pharmaceutical companies, in general, the attrac-
tiveness this technology provides
Proprietary intellectual property (IP) position
is free from dominating/competing IP
Reduced capital investments and production
cost through use of proven and efficient fungal
production systems
Scalability and flexibility - A stable and
robust production process that offers flexibility
in optimization and development of production
processes.
Patents: Exemplary patents assigned to the
University of Hawaii and exclusively licensed
to Neugenesis include 5,834,191 (glucoamylase
promoters and vectors); 5,643,745 (concerning
antibodies expression); and 5,695,965 (Neuros-
pora expression system)
Licensing information: Contact Neugenesis
regarding commercial use, licensing, custom ser-
vices and collaborations.
Neugenesis is working with Novozymes in
expression systems development. Novozymes
has further initiated a research collaboration with
a university partner for evaluation of technology
that will allow for in-vivo modification of fungal
glycosylation patterns, addressing questions con-
cerning post-translation modifications. The part-
nership is currently looking for additional partners
who have an interest in securing access to future
MAb manufacturing capacity and technology.
Broad/Platform Technologies
109
Availability: Invitrogen markets DES and com-
ponents for non-commercial use.
Licensing information: Invitrogen states, Pur-
chase of DES products from Invitogen grants you
a limited, non-exclusive license to use the product
for research purposes only. For more
information, contact Invitrogen... It is unstated
whether Invitrogen or GSK handles commercial
licensing.
197
Streptomyces lividans
Organizations involved:
Centro Informtico Cientfico de Andaluca
(Spanish Center on Biotechnology) - Licensor,
primary
Description: Streptomyces lividans bacteria, e.g,
Y62 strain, with extracellular overproduction of
desired protein and decreased signal petidases
type I (protease) activity, are useful for protein
manufacture. Genes coding for signal petidases
type I are used to obtain Streptomyces deficient in
extracellular protease activity. Secreted proteins
remain more stable extracellularly over time.
Use with: Streptomyces lividans
Use to make: proteins
Benefits: Streptomyces lividans is easy to culti-
vate.
Patents: Exemplary patent applications include
WO/2004/016730, BACTERIA THAT CAN
OVERPRODUCE AND SECRETE PROTEINS,
MELLADO, et. al., assigned to CONSEJO SUPE-
RIOR DE INVESTIGACIONES CIENTFICAS.
Licensing information: Centro Informtico
Cientfico de Andaluca. Collaborators and licens-
ees are sought.
Further info.: FEMS Microbiology Letters, An
inducible expression system of histidine-tagged
proteins in Streptomyces lividans for one-step
purification by Ni2+ affinity chromatography,
Volume 137 Issue 2-3 Page 135 - April 1996.
Eukaryotes
198
Antibiotic inducible promoters
- eukaryotes
Organizations involved:
Cistronics Cell Technology GmbH - Licensor,
primary
Institute for Chemical and Bio-Engineering,
ETH - Licensor, secondary
Description: Actinomycetes antibiotic resistance
promoters in expressed fusion proteins are useful
for regulated expression in eukaryotic cells, and
provide a new system for antibiotic-regulated
gene expression in eukaryotic cells. Proteins are
provided that can be used to activate or repress
transcription from a desired nucleotide sequence
operatively linked to a Pabr sequence. This sys-
tem is based on sequences from an Actinomycetes
antibiotic responsive operon (resistance pro-
moter), the Pabr sequence, operatively linked to a
second polypeptide which activates or represses
transcription in eukaryotic cells, with activation
or repression using polypeptides that bind to Pabr
(without use of antibiotics). Vectors contain the
Pabr-bind sequence along with the desired protein
for expression of fusion proteins which regulate
transcription.
Use with: eukaryotic cells
Use to make: proteins
Patents: Exemplary patents include U.S.
6,287,813, Antibiotic-based gene regulation
system, Fussenegger, et al., Sept. 11, 2001, as-
signed to Cistronics Cell Technology GmbH. The
abstract states: The invention relates to a novel
system for gene regulation in eukaryotic cells, and
methods of using the same for protein production,
tissue engineering and gene therapy. In particular,
the invention provides a new system for antibi-
otic-regulated gene expression in eukaryotic cells
based on sequences from Actinomycetes antibiot-
ic resistance promoters, polypeptides that bind to
the same in an antibiotic responsive manner, and
nucleotides encoding such polypeptides. Further,
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
110
the invention provides novel and sensitive meth-
ods of screening for candidate antibiotics.
Licensing information: Contact Cistronics
Cell Technology GmbH, which has licensed this
technology from the Institute for Chemical and
Bio-Engineering, ETH.
Further info.: Animal Cell Technology Meets
Genomics, Proceedings of the 18th ESACT
Meeting Granada, Spain, May 11-14, 2003.
199
AttSite recombinases - precise
gene insertion; eukaryotes
Organizations involved:
RheoGene, Inc. - Licensor, primary
University of Pittsburgh Medical Center
(UPMC) - Assignee, patent
Description: AttSite recombinase enzyme-
based technology is useful to guide gene inser-
tion into specific hot spots within the genome
to ease the exchange of DNA and development
of new genetic sequences and eukaryotic cell
lines (plants, and animals). Each AttSite enzyme
recognizes a different DNA sequence. Different
enzyme combinations can be used to reproducibly
introduce multiple genes into the same site in the
genome, or into different sites in the same cell.
Only RheoGenes AttSite platform has this level
of precision and flexibility.
The methods involve providing a eukaryotic
cell that has a first recombination attachment
site and a second recombination attachment site;
contacting the first and second recombination
attachment sites with prokaryotic recombinase
polypeptide, resulting in recombination between
the recombination attachment sites, in which the
recombinase polypeptide can mediate recombi-
nation between the first and second recombina-
tion attachment sites. The first recombination
attachment site is a phage genomic recombina-
tion attachment site (attP) or a bacterial genomic
recombination attachment site (attB), the second
recombination site is attB or attP, and the recom-
binase is selected from Listeria monocytogenes
phage recombinase, a Streptococcus pyogenes
phage recombinase, a Bacillus subtilis phage re-
combinase, a Mycobacterium tuberculosis phage
recombinase and a Mycobacterium smegmatis
phage recombinase,
Use with: eukaryotic cells
Use to make: proteins; glycoproteins
Patents: Exemplary U.S. applications include
20060172377, Site-specific serine recombinases
and methods of their use,Padidam, M., assigned
to RheoGene
Licensing information: Nonexclusive research
and commercial licenses for AttSite(TM) Recom-
binases are available from RheoGen.
Reported licensees have been reported to include
Centocor/J&J and Symphogen.
200
Bovine growth hormone (bGH)
polyadenylation sequence
- eukaryotes; expression
enhancement
Organizations involved:
Research Corporation Technologies Inc. (RCT)
- Licensor, primary
Upjohn Co. - Assignee, patent
Pfizer, Inc. - Parent co.
Description: The bovine growth hormone poly-
adenylation (bgh-PolyA) signal from the gene for
bovine growth hormone is a specialized termina-
tion sequence for protein expression in eukaryotic
cells. This DNA sequence is useful for producing
proteins and peptides in culture, and increasing
gene expression in gene therapy and protein pro-
duction in transgenic animals.
Use with: eukaryotes
Use to make: proteins; glycoproteins
Patents: The bovine growth hormone (bgh)
polyadenylation signal is patented under U.S.
5,122,458, European Patent 0 173 552, and Japa-
nese Patent 6-83669. U.S. 5,122,458, Use of a
bGH gDNA polyadenylation signal in expression
of non-bGH polypeptides in higher eukaryotic
cells, Post, et al., June 16, 1992, concerns use of
Broad/Platform Technologies
111
the polyadenylation signal to achieve a high level
of expression of peptides in eukaryotic cells. This
is assigned to Upjohn (subsequently acquired by
Pfizer).
Licensing information: RCT is now the licens-
ing agent for this technology, probably on behalf
of Pfizer, unless it fully acquired the technology
itself.
Products made using this tech.: As reported
in Biopharmaceutical Products in the U.S. and
European Markets, a bovine polyadenylation
signal is used in the expression vector for CHO
cell manufacture of Aldurazyme [Laronidase -I2S;
alpha-L-iduronidase] by BioMarin, marketed by
Genzyme.
201
Expression enhancers; Copy
number increase - eukaryotic
cells
Organizations involved:
RepliGen Corp. - Licensor, primary
Abbott Laboratories, Inc. - Licensor, secondary
Damon Biotech, Inc. - R&D
Massachusetts Institute of Technology (MIT)
- Assignee, patent
Description: Vectors with an a cellular enhancer
element achieve higher expression levels in
eukaryotic cells. The cellular enhancer element
increases the level of transcription of genes on its
3 and 5 sides. A blocking element is interposed
between the enhancer element and the marker
gene which shields the promoter of the marker
gene from the transcription-stimulating function
of the enhancer, limiting the effect of the enhancer
to transcriptions of the DNA encoding the protein
product of interest. Use of the vectors permits
isolation of viable clones characterized by a very
high level of expression of the protein of interest.
The resulting transformants express the exons
of the transcription unit at high levels as the
enhancer element increases the copy number of
mRNA. The enhancer element operates to in-
crease transcription independent of its orientation
and position provided it is located within an active
region on the DNA, generally between about 1-10
kilobases (kb) from the 3 or 5 end of the tran-
scription unit.
Use with: eukaryotic cells
Use to make: proteins; glycoproteins
Patents: Exemplary patents include U.S.
4,663,281, Enhanced production of proteinaceous
materials in eucaryotic cells, Gillies, et al., May
5, 1987, assigned to MIT, originally exclusively
licensed to Damon Biotech, then to Repligen,
which acquired certain Damon Biotech assets.
U.S., 5,665,578, Vector and method for
achieving high level of expression in eukaryotic
cells, Gillies, et al., Sept. 9, 1997, with similar
claims, is assigned to Abbott Labs., with Repligen
having sublicensing rights (apparently as a result
of a cross-licensing settlement between the two
companies).
On May 4, 2004, RepliGen filed a suit against
ImClone alleging use of a cell line infringing
U.S. 4,663,281 in connection with manufacture
of Erbitux (see below). In Sept. 2007, the dispute
was setted, with ImClone paying a total of $65.0
million in cash for full and final settlement of the
claims against ImClone in the matter, as well as
for a royalty-free, irrevocable worldwide subli-
cense to technology patented under U.S. Patent
No. 4,663,281. Repligen paid MIT with a portion
of the settlement payment. Repligen had original-
ly approached ImClone about licensing its patent
in 1996, seeking $200,000 plus 1% royalties, but
was denied.
Repligen also granted to ImClone a royalty-
free, irrevocable worldwide sublicense for the
future use of other patented technology, includ-
ing U.S. 5,665,578, which is owned by Abbott
Laboratories, but to which Repligen has the power
to sublicense under an agreement between Abbott
Laboratories and Repligen. Abbott had filed suit
aganst ImClone alleging infringement of U.S. Pat-
ent No. 5,665,578 in early 2007.
Licensing information: Contact Repligen regard-
ing commercial use and licensing.
Products made using this tech.: A transformed
murine cell line (M225) using this enhancer is
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
112
used by ImClone for manufacture of Erbitux
[Cetuximab - IMC-C225; epidermal growth fac-
tor receptor monoclonal antibody, recombinant]
marketed by Bristol-Myers Squibb.
202
Homologous Recombination;
Knock-out/knock-in animals
and cells
Organizations involved:
Cellectis SA - Licensor, primary
Institut Pasteur - Assignee, patent
Description: Homologous Recombination
enables specific replacement of a copy of a gene
present in the genome of a recipient eukaryotic or-
ganism by the integration of a gene different from
the inactivated gene. This is useful for the genera-
tion of gene knock-out/knock-in animals and cells
as models for screening and protein production.
Preferably, the recipient gene is present in at least
2 copies in the transfected host cell. The recipient
gene is defined as the gene where the insertion of
the different gene is made.
Use with: eukaryotes
Use to make: proteins; glycoproteins
Patents: Related patents assigned to Institute
Pasteur and exclusively licensed to Cellectis
include U.S. 6,528,313 and 6,528,314, both with
the title, Procedure for specific replacement of a
copy of a gene present in the recipient genome by
the integration of a gene different from that where
the integration is made. These broadly claim the
insertion by homologous recombination in mam-
malian cells of a coding sequence and a selection
gene and a promoter with a coding sequence and
a selection gene and the conditions to obtain such
recombined cells.
Licensing information: Contact Cellectis. Li-
censees include GlaxoSmithKline.
203
Internal Ribosome Entry
Sequences (IRES) RNA
translation enhancers; pCITE
vectors; Cardiovirus 2A IRES;
pIRES - eukaryotes
Organizations involved:
Wisconsin Alumni Research Foundation
(WARF) - Licensor, primary
University of Wisconsin - R&D
Description: The cardiovirus internal ribosomal
entry site (IRES), particularly encephalomyocar-
ditis (EMCV IRES), or fragments are useful for
enhancing RNA translation (RNA to protein) and
protein expression, making these broadly useful in
DNA-based vectors. The EMCV IRES is a popu-
lar RNA element used widely in experimental and
biopharmaceutical applications to express proteins
in eukaryotic cells and cell-free extracts. Inclu-
sion of the wild-type element in monocistronic or
bicistronic messenger RNAs (mRNAs) confers a
high level of cap-independent translation activity
to appropriately configured cistrons. The literature
now describes thousands of cDNA constructions
utilizing IRES technology. The encephalomyo-
carditis translational enhancer coding region is
included in a vector deposited as ATCC 67525.
In 1986, shortly after EMCV-R was sequenced,
the region responsible for IRES activity was local-
ized to a genome fragment about 430 bases long,
immediately 5 to the AUG, which begins the viral
polyprotein open reading frame (ORF). When
the enhancer element was excised and linked to
other portions of the virus ORF, the resulting T7
transcripts were highly active messenger RNAs
(mRNAs) in the absence of 5 capping reactions.
A related vector was commercialized in 1990 by
Novagen as pCITE-1. pCITE-1 provides high lev-
els of enhancer-conferred protein expression. This
vector allows easy linkage of exogenous cistrons
onto the cap-independent ranslation enhancer for
transcription of hybrid mRNAs and facile protein
expression in cell-free extracts. pCITE-2, a de-
rivative vector, was released by Novagen in 1990.
Broad/Platform Technologies
113
In 1995, Clontech released a new vector
(pIRES) that could be delivered directly into
mammalian cells as cDNA. Transfection with
IRES induced nuclear transcription of capped bi-
cistronic mRNAs is driven by a cytomegalovirus
(CMV) promoter (see related entry). Translation
of any upstream gene A
inserted into the 5-most A multiple cloning site
is (MCS) cap-dependent. Linked in tandem is a
modified EMCV IRES, a second MCS (B), and
a poly(A) signal sequence, protein synthesis for
gene B inserted at MCS-B was IRES-depen-
dent.
WARF is now offering a new IRES based on
EMCV 2A protein, particularly vectors encoding
the cardiovirus 2A polypeptide operably linked
to a promoter. When expressed in mammalian
cells, in the absence of any other viral proteins or
genes, protein 2A localizes to the nucleolus, binds
to ribosomes, and inhibits cap-dependent mRNA
translation, effectively preventing the translation
of anything that is not a viral protein. Because
cardiovirus protein 2A does not inhibit cap-in-
dependent IRES-driven mRNA translation, the
result is a net increase in IRES-dependent pro-
tein expression relative to cap-dependent protein
expression.
These vectors have a DNA transcriptional
promoter and a DNA enhancer coding sequence
capable of coding for an RNA translational
enhancer. The enhancer (IRES) has the charac-
teristics of a cardiovirus RNA enhancer sequence
that is located 5 of a cardiovirus AUG sequence.
The DNA enhancer sequence is positioned on the
vector so as to be subject to the transcriptional
promoter. There is also foreign DNA gene for
a protein of interest which is positioned on the
vector so as to be subject to the transcriptional
promoter. After transcription of the foreign DNA
gene and the DNA enhancer coding sequence to
their RNA variants, translation of the RNA variant
of the foreign DNA gene is subject to the con-
trol of the RNA variant of the enhancer coding
sequence (IRES).
Use with: eukaryotic cells
Use to make: proteins; glycoproteins
Background: Translation of the EMCV-driven
cistron is independent of the upstream gene, an
activity that could not be attributed to leaky scan-
ning or ribosome readthrough mechanisms. The
name, internal ribosome entry site or IRES was
used and has stuck with this element. IRES has
since been applied to many other virus or cellular
RNA fragments that also direct eukaryotic transla-
tion in a cap-independent manner.
Benefits: Claimed benefits for WARFs new
IRES include:
Introducing 2A into a cell doubles the ribo-
some content of that cell
May be used to enhance the yield of proteins
in cap-independent translation via the IRES
Acts independently-does not require other viral
proteins for its function
Can regulate cap-dependent or IRES-depen-
dent mRNA translation
Inhibits cellular mRNA transcription, but not
rRNA transcription, when combined with another
cardiovirus protein
Able to inhibit cellular mRNA transcription in
a virally-infected cell
Highly toxic-eventually kills every cell it
enters, and may be used therapeutically to selec-
tively kill cells, such as tumor cells
Patents: The primary exemplary U.S. patent
covering early IRESs is 4,937,190, Palmenberg,
et al., June 26, 1990, assigned to the Wisconsin
Alumni Research Foundation (Univ. of Wiscon-
sin). This likely has or will soon expire.
An exemplary application covering the new
WARF IRES is U.S. 20050019808, Composi-
tions and methods for regulating mRNA tran-
scription and translation, Palmenberg, A.C., et
al., Jan. 27, 2005. Claim 1 states: A nucleic acid
construct comprising: at least one DNA depen-
dent RNA polymerase promoter; a 2A cardiovirus
polynucleotide sequence encoding a 2A polypep-
tide, wherein the 2A polynucleotide is positioned
on the construct so as to be operably linked to at
least one promoter; and at least one foreign poly-
nucleotide coding for a detectable polypeptide,
Broad/Platform Technologies
127
for regulated expression in mammalian cells,
Mullick,A., et al., BMC Biotechnology 2006,
6:43.
221
Dihydrofolate reductase (DHFR)
System - selectable marker/
amplifcation; CHO and NS0
cells
Organizations involved:
Genentech, Inc. - Licensor, primary
Description: The Dihydrofolate Reductase
(DHFR) System is a selectable marker/amplifica-
tion widely used in CHO and NS0 cells and other
expression systems involving cells with inherent
low levels of DHFR expression. Use of the DHFR
as a selectable marker (see related entry) allows
selection of properly transformed cells that can
grow in absence of thymidine, glycine and hypo-
xanthine with methotrexate (MTX) positive selec-
tion/amplification, with the DHFR gene allowing
transformed cells to express DHFR, allowing
production of tetrahydrofolate and survival in the
presence of otherwise toxic methotrexate. Without
dhfr gene expression, cells are unable to produce
tetrahydrofolate, an essenatial metabolic cofactor.
Also, Dhfr- cells are only able to grow in media
containing thyrmidine, glycine and hypoxanthine,
which allow cells to overcome tetrahydrofolate
deficiency. Stable, DHFR+ transfected cells can
grown in unsupplemented media.
See also the entries for DHFR- CHO cell lines.
One of the disadvantages, compared to some
newer technologies, is that while the DHFR am-
plification system takes a longer time to develop
high-expressing clones through multiple rounds of
selection and amplification.
Use with: CHO cells; NS0 cells
Use to make: proteins; glcyoproteins
Background: Dihydrofolate reductase (DHFR;
5,6,7,8-tetrahydrofolate: NADP + oxidoreductase;
EC 1.5.1.3) catalyzes the NADPH-dependent
reduction of dihydrofolate to tetrahydrofolate, an
essential carrier of one-carbon units in the biosyn-
thesis of thymidylate, purine nucleotides, serine
and methyl compounds. It is an essential enzyme
in both eukaryotes and prokaryotes.
In selecting a preferred host cell for trans-
fection by DHFR vectors, it is appropriate to
select the host according to the type of DHFR
protein employed. If wild type DHFR protein
is employed, it is preferable to select a host cell
deficient in DHFR, permiting the use of the
DHFR coding sequence as a marker for success-
ful transfection in selective medium which lacks
hypoxanthine, glycine, and thymidine (e.g., see
related CHO cell line entries). On the other hand,
if DHFR protein with low binding affinity for
MTX is used as the controlling sequence, it is not
necesary to use DHFR resistant cells. Because
the mutant DHFR is resistant to methotrexate,
MTX containing media can be used as a means of
selection, provided that the host cells are them-
selves are methotrexate sensitive. Most eukaryotic
cells which are capable of absorbing MTX are
methotrexate sensitive, e.g., CHO-K1 (see related
entry).
Patents: Originally commercially developed by
Genentech, related patents have now expired and
the classic system is now in the public domain.
Exemplary patents include U.S. 4,713,339, Poly-
cistronic expression vector construction, Levin-
son, et al., Dec. 15, 1987, assigned to Genentech.
The abstract states: A polycistronic construction
of xenogeneic sequences expressible in eukary-
otic cells is described. DHFR encoding sequences
under control of the same promoter with another
foreign gene can be amplified with methotrex-
ate, and thus cause coamplification of the foreign
gene. Application of DHFR to the commercial
manufacture of Activase, recombinant tissue plas-
minogen activator (tPA), is claimed in 5,010,002
assigned to Genentech.
Licensing information: Now in public domain.
Products made using this tech.: Despite the
widespread promotion and use of the GS System
(see related entry), DHFR is still used for com-
mercial biopharmaceutical manufacture. For
example, products known to use the system (as
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
128
reported in Biopharmaceutical Products in the
U.S. and European Markets; see www.biopharma.
com) include
a) Myozyme [alglucosidase alfa; Pompase; alpha
glucosidase; glucosidase alpha (rhGAA) [recom-
binant] from Genzyme.
b) Activase, recombinant tissue plasminogen acti-
vator (tPA) from Genentech.
c) Kogenate FS [Antihemophilic Factor (Recom-
binant), Formulated with
Sucrose; Factor VIII, recombinant] from Bayer;
and relabeled as Advate by Baxter
d) Epogen (Erythropoietin, recombinant; EPO)
from Amgen and equivalent - Procrit from Ortho/
J&J.
e) Cerezyme (Imiglucerase -- beta-Glucocerebro-
sidase, recombinant) from Genzyme
f) Rebif (Interferon beta-1a) from Merck Serono
g) Enbrel (Etanercept - tumor necrosis factor
receptor2-immune globlulin G1 Fc fusion protein,
recombinant) from Wyeth
h) TNKase (Tenecteplase - TNK-tPA; tissue
plasminogen activator, TNK-, recombinant) from
Genentech
i) Luveris (Lutropin alfa - human luteinizing hor-
mone, recombinant) from Merck Serono.
222
Flp-In expression system; FLP-
Mediated Gene Modifcation
in Mammalian Cells; FLP
recombinase - mammalian
cells
Organizations involved:
Salk Institute for Biological Studies - Licensor,
primary
Life Technologies, Inc. - Retail seller
Description: A gene activation/inactivation and
site-specific integration system has been de-
veloped for mammalian cells. This is based on
recombination of transfected sequences by FLP,
a recombinase derived from Saccharomyces. In
several cell lines, FLP has been shown to rapidly
and precisely recombine copies of its specific tar-
get sequence. A chromosomally integrated, silent
reporter gene can be activated for expression by
FLP-mediated removal of intervening sequences
to generate clones of marked cells. Or, FLP can be
used to target transfected DNA to specific chro-
mosomal sites.
Thje Flp-In system provides efficient targeted
gene integration and saves time when generating
stable cell lines by eliminating the need to isolate
single clones. The Flp-In System integrates a
single copy of the gene of interest at a particular
genomic locus in every transfected cell. The time-
consuming process of isolation
and characterization of single cell clones is no
longer necessary, as a single Flp-In transfection
will yield an isogenic, homogenous population of
stably expressing cells. This integration of a gene
of interest into a single site limits chromosomal
position effects on gene expression, and allows
for reproducible analysis of multiple
genes (or variants of a single gene) without the
variability introduced by multiple integrated
copies or single integration events at different
chromosomal locations.
The Flp-In System, as sold by Invitrogen,
consists of three plasmids: the target site vector
(pFRT/lacZeo or pFRT/lacZeo2), an expression
vector (pEF5/FRT/V5-TOPO, pcDNA5/FRT/V5-
His or pSecTag/FRT/V5-His-TOPO), and the
vector that transiently expresses Flp recombinase
(pOG44). In a straightforward two-step process,
the gene of interest is inserted into the same
genetic locus in every cell (Figure 2). Since every
transfected cell is the same, one can
quickly select stable cells as a population rather
than isolating single clones.
Use with: mammalian cells
Use to make: proteins; glycoproteins
Patents: Exemplary patents include 5,654,182,
FLP-mediated gene modification in mammalian
cells, and compositions and cells useful therefor,
Wahl, et al., August 5, 1997; and 5,677,177.
Availability: Invitrogen markets the FLP system
for research use only.
Broad/Platform Technologies
129
Licensing information: Contact the Salk Institute
for Biological Studies.
223
Gene-Activated (GA)
expression, in vivo -
mammalian cells
Organizations involved:
Transkaryotic Therapies, Inc. (TKT) - Licensor,
primary
Shire Pharmaceuticals Group plc - Parent co.
Cell Genesys, Inc. - R&D
Description: Methods and vectors are provided
enabling specific activation of expression of a
human gene within human cells, i.e., the genome
is altered to significantly increase expression of
the desired preexisting human gene. This method
is used by Transkaryotic Therapies, Inc. (TKT),
Shire Pharmaceuticals Group plc, for manufac-
ture of Dynepo (Gene-Activated Erythropoietin).
Exogenous DNA is stably integrated into the cell
genome or is expressed in the cells episomally.
The method may be used with primary and sec-
ondary somatic cells, such as fibroblasts, keratino-
cytes, epithelial cells, endothelial cells, glial cells,
neural cells, formed elements of the blood, muscle
cells, other somatic cells which can be cultured
and somatic cell precursors, which have already
been transfected with exogenous DNA encoding a
desired protein.
Use with: mammalian cells
Use to make: proteins; glycoproteins
Patents: U.S. 5,994,127, In vivo production and
delivery of erythropoietin or insulinotropin for
gene therapy, Selden, et al., Nov. 30, 199, as-
signed to Transkaryotic Therapies, Inc. describes
Gene-Activated (GA) production of EPO and in-
sulinotropin. The abstract states, The present in-
vention relates to transfected primary and second-
ary somatic cells of vertebrate origin, particularly
mammalian origin, transfected with exogenous
genetic material (DNA) which encodes erythro-
poietin or an insulinotropin [e.g., derivatives of
glucagon-like peptide 1(GLP-1)], methods by
which primary and secondary cells are transfected
to include exogenous genetic material encod-
ing erythropoietin or an insulinotropin, methods
of producing clonal cell strains or heterogenous
cell strains which express eruthropoietin or an
insulinotropin, methods of gene therapy in which
the transfected primary or secondary cells are
used, and methods of producing antibodies using
the transfected primary or secondary cells. The
present invention includes primary and secondary
somatic cells, such as fibroblasts, keratinocytes,
epithelial cells, endothelial cells, glial cells, neural
cells, formed elements of the blood, muscle cells,
other somatic cells which can be cultured and
somatic cell precursors, which have been trans-
fected with exogenous DNA encoding EPO or
an insulinotropin, which is stably integrated into
their genomes or is expressed in the cells episom-
ally.
Licensing information: Contact TKT/Shire re-
garding commercial use and licensing.
Cell Genesys (now Abgenix, Inc.) originally
developed gene activation methods for recom-
binant expression of human therapeutic proteins.
In June 2002, Cell Genesys exclusively licensed
(divested) this technology for certain therapeutic
proteins to Transkaryotic Therapies, Inc. (TKT).
Cell Genesys received an up-front license fee of
$26 million, and was eligible to receive payments
up to $17 million upon the completion of certain
patent-related milestones. The agreement did not
include any royalty payments.
In Aug. 2004, Serono S.A. (now Merck Serono
S.A.) filed suit in U.S. District Court alleging that
TKT and Cell Genesys Inc. were infringing its
U.S. patent 5,272,071, assigned to Applied Re-
search Systems Ars Holding N.V., a subsidiary of
Serono S.A., concerning gene activation. The U.S.
patent office had previously declared an interfer-
ence brought by Cell Genesys between 5,272,071
(U.S. equivalent of EP 0505500) and Cell Gene-
sys gene activation patent application to which
TKT had a license. TKT reported that the patent
office had determined that both Serono and Cell
Genesys (TKT) were entitled to some claims in
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
130
their respective patent and application, and both
parties appealed this decision. [Final resolution of
this dispute was not retrieved, but TKT continues
to manufacture its GA-EPO product for marketing
where lack of Amgen patents covering EPO allow
this. In April 2005, Shire Pharmaceuticals Group
plc initiated acquisition of TKT for ~$1.6 billion.
Products made using this tech.: TKT uses this
method for manufacture of Dynepo [Erythropoi-
etin - epoetin delta; Gene-Activated Erythropoie-
tin; GA-EPO; EPO, gene activated, recombinant].
224
Glutamine synthetase
(GS) System - NS0, CHO,
mammalian cells
Organizations involved:
Celltech Biologics plc - R&D
Lonza Biologics plc - Licensor, primary
Description: The GS system allows selection and
amplification of successfully transformed cells.
Glutamine synthetase (GS) catalyzes formation
of glutamine from glutamate and ammonium
ion. This enzymatic reaction provides the only
pathway for glutamine formation in a mamma-
lian cells. Mammalian cells deficient in the GS
gene/protein require glutamine supplementation
to grow and survive. With these GS- cell lines, a
transfected GS gene can function as a selectable
marker by permitting growth in a glutamine-free
medium. The GS gene is used in recombinant vec-
tors as a selectable marker along with the desired
gene(s) for expression in cells deficient in GS,
e.g., NS0. Only successfully transformed cells,
i.e., carrying both the GS and desired gene(s), are
capable of producing their own GS enzyme and
surviving in glutamine-deficient culture media,
with this allowing easy, reliable selection of
transformed cells. Other cell lines, such as CHO,
express sufficient GS to survive without exog-
enous glutamine. With these cells, the specific GS
inhibitor, methionine sulphoximine (MSX), can
be used to inhibit endogenous GS activity such
that only transfectants with additional GS activity
can survive. This can be used to amplify the target
gene (also amplifying the GS gene, so success-
fully transformed cells overproduces glutamine).
Use of a weak promoter on the GS gene and
strong promoter on product gene selects for rare
integration into transcriptionally efficient sites
in the cells genome. Using appropriate screen-
ing techniques, amplification is often not found
to be necessary. Particularly with constitutively
GS-minus cell types, high yielding cell lines can
often be identified on first selection. However,
yield improvements may be made by the addition
of MSX, particularly for GS-plus cell lines such
as CHO. Hybridoma cells expressing GS produce
less ammonia/ammonium byproducts, can be cul-
tured in media with glutamine, increasing produc-
tion of monoclonal antibodies.
In practice, the most commonly used cell
lines are reported to be NS0 and CHO. The GS-
NS0 cell line has been used most commonly for
antibody production. GS-CHO has been used to
express a large range of other protein types.
Maximum expression levels attainable depend
on the product, but cell lines have been created
that are capable of producing up to 3.6g/L of
recombinant antibody, with specific production
rates of 15-65pg/cell/day.
Use with: mammalian cells; CHO cells; NS0
cells
Use to make: proteins; glycoproteins; antibod-
ies
Benefits: Claimed benefits include:
speed (DNA to IND in 12 months possible)
ease of use (support package available to licens-
ees)
high yielding cell lines (>5g/L for GS-CHO, in
chemically-defined, protein-free media)
regulatory acceptance, with multiple approved
products
predictable scale Up (1L to 5000L or more)
Patents: The glutamine synthetase (GS) gene
amplification/expression system used to select
and amplify transformed cells is based on patents
issued to Celltech Biologics plc, now assigned to
Lonza Group plc, subsidiary of Alusuise-Lonza
Group. Related patents include U.S. 5,770,359
Broad/Platform Technologies
131
and 5,747,308 (coassigned to the University
of Glasgow). These patents describe dominant
selectable markers for use in co-amplification of
non-selected genes and transformation of host cell
lines to glutamine independence. The GS gene
is used in recombinant vectors as a selectable
marker along with (an)other gene(s) for expres-
sion in cells deficient in GS, e.g., NS0 cells. Only
successfully transformed cells are capable of
producing their own GS enzyme and surviving
in glutamine-deficient culture media, with this
allowing easy, reliable selection and amplification
of transformed cells.
Licensing information: The system including
vectors, GMP host cells, and technical manuals
can be supplied under Research Evaluation or
License Agreements. Contact Lonza.
Products made using this tech.: Lonza reports,
Over 70 biotechnology and pharmaceutical
companies have successfully used this system
[presumably, licensed], the GS gene expression
system, worldwide. Lonza Biologics has itself
created over 250 cell lines using the GS System.
Marketed products reported by Lonza as manu-
factured using the GS Gene Expression System
include:
a) Zenapax [Daclizumab - interleukin-2 alpha re-
ceptor monoclonal antibody, recombinant], manu-
factured and marketed by Hoffmann-La Roche
Inc., originally developed by Protein Design Labs.
(Protein Biosciences).
b) Synagis [Palivizumab - MEDI-493; respira-
tory syncytial virus (RSV) monoclonal antibody,
recombinant] developed, manufactured and mar-
keted by Medimmune, now part of AstraZeneca.
Other marketed products, as reported by Bio-
pharmaceutical Products in the U.S. and European
Markets (www.biopharma.com), include:
a) ReoPro [Abciximab - Platelet monoclonal anti-
body, recombinant chimeric] from Centocor/J&J
b) Saizen; Serostim; Zorbtive (multiple trade
names for Somatropin (rDNA origin) from EMD
Serono, Inc., Merck Serono S.A.
c) Zenapax [Daclizumab - Interleukin-2 alpha
receptor monoclonal antibody, recombinant] from
Hoffmann-La Roche AG, with Lonza a contract
manufacturer
d) Remicade [Infliximab - cA2; Avakine; tumor
necrosis factor monoclonal antibody, recombi-
nant] from Centocor/J&J
e) Campath [Alemtuzumab - MabCampath; CD52
monoclonal antibody, recombinant humanized rat]
from Genzyme Corp. and Schering-Plough
f) Mylotarg [Gemtuzumab Ozogamicin - CMA-
676; recombinant humanized CD33 monoclonal
antibody-calicheamicin cytotoxin conjugate (im-
munotoxin)] from Wyeth
g) Erbitux [Cetuximab - IMC-C225; epidermal
growth factor receptor monoclonal antibody,
recombinant] from Bristol-Myers Squibb and
ImClone
h) Xigris [Drotrecogin alfa (activated) - recombi-
nant human activated protein C; rhAPC] from Eli
Lilly, and
i) Rituxan [Rituximab - IDEC-C2B8; MabThera;
CD20 monoclonal antibody, recombinant] from
Hoffmann-La Roche AG.
225
GPEx Gene Product Expression
Technology - mammalian; CHO
Organizations involved:
Catalent Pharma Solutions - Licensor, primary
Blackstone Group - Parent co.
Cardinal Health, Inc. (see Catalent Pharma
Solutions) - Former inv.
Gala Biotech - Assignee, patent
Description: GPEx retrovector technology is
based on a pseudotyped high-titer vector, which
ensures that stable transductions occur in virtually
100% of target cells (any mammalian cell line,
but usually CHO cells). Virtually any cDNA, in-
cluding monoclonal antiibodies, can be packaged
into the GPEx retrovector and used to transduce
diverse mammalian cell lines. Catalent claims,
The GPEx gene insertion technology produces
mammalian cell lines - typically CHO cells - that
deliver higher initial yields of proteins than any
conventional system based on transfection or elec-
troporation.
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
132
This technology typically produces a stable
clonal cell line in about four months for proof of
concept studies. High producing cell lines suitable
for Master Cell Banking (MCB) and subsequent
cGMP production are typically obtained in less
than five months after receipt of client cDNA.
Selected clones have consistent productivity from
25 to over 50 picograms per cell, per day. Process
development with minimal inputs can consistently
reach several gm/L in six months in batch-fed
bioreactor systems.
One reason for higher yields is that the GPEx
retrovector targets high-expressing chromosomal
matrix attachment sites (MARS) in the target cell
genome, leading to markedly higher expression of
the gene in transduced cells compared with other
systems. Another important reason is that the
optimization of GPEx cell lines employs an itera-
tive insertion process that drives up the inserted
gene copy number and proportionally increases
protein expression levels. The optimized cell line
may therefore have a dozen or more copies of the
desired gene, all stably inserted and expressing
the target protein.
Cardinal manufactured its first Mab for clinical
use (for Ludwig Institute for Cancer Research)
using GPex in 2005.
Use with: mammalian cells; CHO cells; BHK
cells
Use to make: proteins; glycoproteins; antibod-
ies
Benefits: GPEx eliminates the need for selectable
markers, saving time and cost. In comparison,
conventional transfection or electroporation meth-
ods can produce an insertion frequency as low as
1 out of every 100,000 cells, necessitating addi-
tional selection steps and use of selectable marker
genes. Other methods carry the risk of gene exci-
sion or silencing, making them relatively unstable.
Using conventional gene insertion and expres-
sion techniques, developing a stable production
cell line for a target protein can take as long as
18 months. GPEx overcomes the inefficiencies of
conventional systems and delivers a stable pro-
duction cell line for your target protein in as little
as 4.5 months.
Other benefits include:
Monoclonal antibody heavy- and light-chain
coexpression
Receptors, coexpressed with a secreted sur-
rogate
Inactive proteins, coexpressed with associated
processing enzymes
Sequential transduction can be used to intro-
duce required genes without antibiotic selection
Expressions can be enhanced by clonal selec-
tion or additional rounds of transduction without
antibiotic selection
Cell lines are developed in serum-free culture
media
No antibiotic selection requirement
Patents: Related patents include U.S. 7,332,333,
Host cells containing multiple integrating vec-
tors, Bremel, R.D. ,et al., Feb. 19, 2008, assigned
to Gala Design, Inc., and licensed to Chronos.
The abstract states, The present invention relates
to the production of proteins in host cells, and
more particularly to host cells containing multiple
integrated copies of an integrating vector. Suitable
integrating vectors for use in the present inven-
tion include retrovirus vectors, lentivirus vectors,
transposon vectors, and adeno-associated virus
vectors. Methods are provided in which the host
cells are prepared by using the integrating vectors
at a high multiplicity of infection. The host cells
are useful for producing pharmaceutical proteins,
variants of proteins for use in screening assays,
and for direct use in high throughput screening.
Availability: GPEx technology has been used to
generated over 150 cell lines for production of
recombinant proteins and antibodies. Cardinal/
Catalent has concluded agreements with multiple
companies for development of GPex cell lines,
including with Centocor/J&J and Trubion. The
company is developing a cell line for expression
of a prrostate specific membrane antigen (PSMA)
Mab for PSMA Development Company LLC
(PDC).
Licensing information: Cardinal/Catalent does
not actually licence GPEx to any firm, although,
its would consider doing so if we were presented
with the right size deal from a large company.
Broad/Platform Technologies
133
Rather, it offers in-house R&D and contract man-
ufacturing services, including strain development
and optimization, and full commercial manufac-
turing capabilities. Usually at the conclusion of a
feasibility study, the client can out-license the cell
line for production at the facility of your choice,
or continue to work with Catalent.
Cardinal (before it became Catalent in sp-
ing 2006) said it was facing a robust market
in terms of interest in its GPEx technology and
already had 30 various deals in place.
Products made using this tech.: Cardinal re-
ported that a biogeneric product is manufactured
using GPex.
226
MARtech; Matrix Attachment
Regions; Scaffold Attachment
Regions; SARs - mammalian
cells
Organizations involved:
Selexis - Licensor, primary
Ecole Polytechnique Fdrale de Lausanne
(EPFL) - R&D
Description: The MARtech expression system
provides increased recombinant protein expres-
sion in mammalian cells in suspension and in
serum-free media by more than 20 fold. MARtech
is based on human Matrix Attachment Regions
(MARs) genetic elements which control the
dynamic organization of chromatin. MARs func-
tion by insulating nearby genes from the effect of
surrounding chromatin, thereby increasing copy
number dependent, position-independent, expres-
sion of genes. MARtech increases the number
of independently transformed cells that express
the desired protein and promotes higher amounts
of recombinant protein produced by these cells.
MAR-binding proteins may also recruit histone
acetyltransferases and ATP-dependent chromatin
remodeling complexes, and thereby increase gene
expression.
MARs (Matrix Attachment Regions), also
called SARs (Scaffold Attachment Regions), are
300-3000 bp long DNA elements proposed to play
a role in nuclear and chromosomal architecture.
They attach chromatin to proteins of the nuclear
matrix and thereby partition the eukaryotic ge-
nomes into independent chromatin loops. Because
of their co-localization with transcription units
and regulatory elements in genomes, MARs have
been implicated in the regulation of mammalian
gene expression.
Use with: mammalian cells
Use to make: proteins; glycoproteins
Benefits: Gene transfer in eukaryotic cells and
organisms suffers from epigenetic effects that
result in low or unstable transgene expression and
high clonal variability. Use of epigenetic regula-
tors such as matrix attachment regions (MARs) is
an approach to alleviate these unwanted effects.
Advantages over traditional approaches in-
clude:
High Yield : Predictable high expression levels at
rates typically ranging from 50 to 80 p/c/d/
Speed : High productivity clones approximately 5
weeks
Expression : High levels and long-term expression
of therapeutic proteins
Stability : Not associated with chromosomal rear-
rangements nor chromosomal breaks
Random integration a single site
Integration position sites known to be very stable
(sub-telomeric)
No chromosomal breaks
No multiple integration sites
Effective : Very effective in a variety of cell lines
Regulatory : Eeasily analyzed for regulatory ap-
proval.
Patents: Exemplary patents include U.S.
7,129,062, Matrix attachment regions and
methods for use thereof, Mermod, et al., Oct. 31,
2006, assigned to Selexis S.A.
Licensing information: Contact Selexis regard-
ing commercial use and licensing.
Further info.:
Use of scaffold/matrix-attachment regions for pro-
tein production, Florian M. Wurm, CHAPTER 10,
in Makrides (Ed.) Gene Transfer and Expression
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
134
in Mammalian Ce!!s, by Wiley, 2008.
227
RheoSwitch Mammalian
Inducible Expression System;
RheoSwitch Ligand RSL1
promoter ; Ecdysone receptor
induction - mammalian cells;
adjustable expression
Organizations involved:
RheoGene, Inc. - Licensor, primary
New England Biolabs - Retail seller
Description: The RheoSwitch Mammalian
Inducible Expression System permits maxi-
mum control of gene expression in mammalian
cells. Analogous to the operation of a rheostat,
RheoSwitch technology allows induction and
adjustable control of gene expression. Vectors and
steroid hormone 20-OH ecdysone (beta-ecdysone;
EcR) control elements from Drosophila enable
control of expression using a synthetic ligand of
an insect steroid receptor superfamily. The steroid
hormone 20-OH ecdysone controls timing of
development in many insects. Precise regulation
of gene expression is achieved through the highly
specific interaction of a synthetic inducer, Rheo-
Switch Ligand RSL1, with a chimeric bipartite
nuclear receptor. This receptor is activated in the
presence of RSL1 ligand, and the level of gene
expression can be regulated by adjusting the con-
centration of RSL1 ligand in the culture media.
The system is built on a two-hybrid switch
format that activates transcription in the presence
of inducer and represses gene expression in its
absence. The synthetic receptor is composed of
two proteins, RheoReceptor-1 and RheoActiva-
tor, that dimerize to make a holoreceptor. Both are
expressed from strong constitutive promoters on
plasmid pNEBR-R1. The RheoReceptor-1 protein
is a highly engineered ligand-binding domain
(LBD) of an insect EcR nuclear receptor fused to
the yeast GAL4 DNA binding domain. The Rheo-
Activator protein is an insect/mammalian RXR
hybrid LBD fused to the viral activation domain
VP16. RSL1 ligand is synthetic diacylhydrazine,
[N-(2-ethyl-3-methoxybenzoyl)-N-(3,5-dimeth-
ylbenzoyl)-N-tert-butylhydrazine]. It is one of a
family of compounds that have been found to act
as non-steroidal ecdysone agonists and can func-
tion as gene inducer.
The gene to be expressed is cloned into the
pNEBR-X1 plasmid under control of five tandem
repeats of the GAL4 response element (5XRE). In
the absence of RSL1 ligand, the receptor represses
transcription by binding to the GAL4 elements in
a transcriptionally inactive conformation. Upon
induction, the RSL1 ligand tightly binds and
changes the conformation of the RheoRecep-
tor-1 protein which stabilizes the holoreceptor
heterodimer on the 5XRE. The activated holore-
ceptor and the VP16 activation domain bind and
recruit to the promoter transcriptional coactivators
along with basal machinery, resulting in a highly
induced transcriptional state. This high induction
of transcription coupled with extremely low basal
expression results in extremely high induction
levels (greater than 10,000 fold induction), with a
degree of control that is superior to other systems.
Use with: mammalian cells
Use to make: proteins; glycoproteins
Benefits: Claimed benefits include:
Precise regulation of expression levels
Negligible basal expression
Synthetic inducer and engineered receptor elimi-
nate non-specific side effects
Greater than 1,000 fold induction levels routinely
obtained
No special culture and media requirements
The RheoSwitchs precise control is unrivaled
among mammalian expression systems, giv-
ing negligible levels of basal expression in the
absence of inducer and greater than 10,000 fold
induction when RSL1 ligand is present. Unlike
other systems, which rely on steroids or other
drugs, the synthetic ligand RSL1 shows no pleio-
tropic effects in mammalian cells and exhibits no
cross talk with endogenous transcription factors.
The RheoSwitch System has no special culture
media requirements.
Broad/Platform Technologies
135
Patents: Exemplary patents include 7,091,038,
Ecdysone receptor-based inducible gene ex-
pression system, Palli, et al., August 15, 2006,
assigned to RheoGene, Inc.
RHeoGene holds an exclusive license to Stan-
ford University patents U.S. 5,514,578, 6,245,531
and EP Patent 0517805 that cover certain ecdy-
sone-based products.
Availability: The system is available from Invit-
rogen, New England Biolabs and, perhaps, other
vendors for non-commercial uses.
Licensing information: Contact RheoGene re-
garding commercial use and licensing.
228
Selexis Genetic Elements
(SGEs) - mammalian cell lines
Organizations involved:
Selexis - Licensor, primary
Description: Selexis Genetic Elements (SGEs)
enable the rapid development of high performance
mammalian cell lines. SGEs can boost the number
of cells that actively produce recombinant pro-
tein after transfection up to 20-fold in serum-free
media. Selexis Genetic Elements (from 1.6Kb to
4Kb in length) are used to control the promoter
accessibility of the expression cassette. SGEs are
based on human DNA sequences which were dis-
covered using a novel bioinformatics tool and are
now applied to control the dynamic organization
of transgenes in chromatin. SGEs function by in-
sulating nearby genes from the effect of surround-
ing chromatin, thereby increasing copy number
dependent, position-independent expression of
genes. For this reason, SGEs increase the number
of independently transformed cells that express
desired protein and promote higher amounts of
recombinant protein produced by these cells.
Selexis Genetic Elements can reduce tradi-
tional cell line development from 12 months to ~5
weeks. The increased number of clones that have
integrated the transgene cassette (post transfec-
tion) allows a fast isolation of high-yield express-
ing cell clones.
Selexis Genetic Elements has been tested in a
variety of cell types including: CHO, HEK 293,
BHK, C2C12, and a B cell derivative. Selexis
with its partners have tested over 30 cell lines.
These include cell lines expressing green fluores-
cent protein (GFP), various monoclonal antibod-
ies, a clotting factor, the EGF receptor, EPO as
well as membrane bound receptors. Selexis has
tested different versions of the CMV promoter as
well as the ubiquitin promoter and the SV40 early
promoter.
Over 10 cell lines have been scaled up and
tested in bioreactors. The Selexis Genetic Ele-
ments cell lines were compared with cell lines
made the traditional manner. The cell lines made
with Selexis Genetic Elements were found to
maintain the high level of expression and yield
stable production at this high level during optimi-
zation in the bioreactor. The yield improvement
seen during the creation of the cell lines translated
into increased yields in the bioreactor.
Use with: mammalian cells; CHO; HEK-293;
BHK
Use to make: proteins; glycoproteins; antibod-
ies
Background: It is believed SGEs function by
enabling the formation of highly transcribed loops
within the chromatin structure. When combined
with optimal cell line development procedure (the
Selexis SURE process; see related entry) a higher
number of high performance cells are generated.
Benefits: Aadvantages over traditional approach-
es include:
High Yield : Predictable high expression levels at
rates typically ranging from 50 to 80 p/c/d/
Speed : High productivity clones approximately 5
weeks
Expression : High levels and long-term expression
of therapeutic proteins
Stability : Not associated with chromosomal rear-
rangements nor chromosomal breaks; Random
integration a single site; Integration position sites
known to be very stable (sub-telomeric); No chro-
mosomal breaks; No multiple integration sites
Effective : Very effective in a variety of cell lines.
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
136
Regulatory : Selexis Genetic Elements cell lines
can be easily analyzed for regulatory approval.
Selexis provides a step-by-step description of
the assembly of all gene fragments present in its
vectors, including the Selexis genetic elements,
to form the final gene constructs. The entire
sequences of the expression vectors, including
Selexis genetic elements, are defined, satisfying
the requirements of the points to consider docu-
ment from the FDA.
Patents: No specific yet published patents/ap-
plications covering this technology were retrieved
from simple searching.
Licensing information: Contact Selexis.
As of the end of 2007, two companies had
taken cell lines developed with the Selexis tech-
nologies forward to produce cGMP material for
use in human clinical trials.
229
STabilizing Anti-Repression;
STAR elements - expression
enhancement; mamalian cells
Organizations involved:
Chromagenics BV - Former inv.
Crucell Holland B.V. - Licensor, primary
Description: STAR technology involves genetic
elements, called STAR elements, that enable
stable and high-yield gene expression important
to recombinant antibody and protein production in
mammalian cells. The technology has the poten-
tial to increase production yields, thereby reduc-
ing production costs. STAR is effective for pro-
duction of antibodies and proteins in mammalian
cell lines, such as Crucells PER.C6 human cell
technology and the widely used Chinese hamster
ovary (CHO) cell lines.
STAR sequences are nucleic acid sequences
that comprise a capacity to influence transcription
of genes in cis. Typically, although not necessar-
ily, the STAR sequences do not code by them-
selves for a functional protein. STAR technology
was discovered by Dr. Arie Otte (Nature Biotech-
nology 2003 May, 21 (5)) who founded Chroma-
genics B.V., a spin-off company of the University
of Amsterdam acquired by Crucell in March 2004.
Use with: CHO cells; PER.C6 cells; HEK-293
cells; NS0 cells; mammalian cells
Use to make: proteins; glycoproteins; antibod-
ies
Patents: Exemplary patents include 7,267,965,
Means and methods for regulating gene ex-
pression, Otte, et al., Sept. 11, 2007 assigned
to Chromagenics, with claim 1, A method for
producing a proteinaceous molecule in a cell
comprising: providing a cell selected from the
group consisting of a cell having an adenovirus
Early Region 1 (E1) sequence, a HuNS-1 my-
eloma cell, a 293 cell, a CHO cell, a Vero cell,
a WERI-Rb-1retinoblastoma cell, a BHK cell,
a non-secreting mouse myeloma Sp2/0-Ag 14
cell, a non-secreting mouse myeloma NSO cell,
and an NCI-H295R adrenal gland carcinoma
cell; wherein said cell comprises an anti-repres-
sor activity sequence operably linked to a nucleic
acid sequence encoding a proteinaceous molecule
of interest, wherein said anti-repressor activity
sequence comprises SEQ ID NO:7; expressing the
proteinaceous molecule in said cell; and isolating
said proteinaceous molecule.
Licensing information: Contact Crucell.
Genentech and Medarex have been reported to
be testing STAR.
230
Ubiquitous chromatin opening
element (UCOE) expression -
mammalian cells
Organizations involved:
Celliance, Serologicals Ltd., now Millipore
- Licensor, primary
Innovata plc - Assignee, patent
ML Laboratories - R&D
Kings College - R&D
Description: The Ubiquitous chromatin opening
element (UCOE) system is an efficient expression
system for the rapid production of recombinant
proteins in mammalian cells, with rapid and high-
Broad/Platform Technologies
137
yielding production of correctly folded and gly-
cosylated proteins. Over 50% of UCOE-derived
clones have higher expression than the best non-
UCOE-derived clones, UCOE-derived vectors
produce substantially more expressing clones than
non-UCOE-derived vectors. High-yielding cell
lines can be derived in less than 60 days, without
amplification. When selected clones are grown
in bioreactors, yields of 1.5-2.0g/L can easily be
achieved in non-optimized conditions.
UCOE gives major improvements in gene
expression in stably-transfected mammalian cells
through effects on the structure of chromatin.
UCOE prevents transgene silencing and gives
consistent, stable and high-level gene expres-
sion irrespective of the chromosomal integration
site. UCOE expression elements are small DNA
elements (isolated from the area around house-
keeping genes, which need to be active most of
the time) that create a transcriptionally active,
open chromatin environment around an integrated
transgene, maximizing its potential to be tran-
scribed into protein, irrespective of the position
of the transgene in the chromosome. This has
potential advantages over transient transfection,
minimizing the quantities of DNA and costly
transfection agent, as well as allowing aliquots of
the pool to be stored frozen for future use if the
protein is required at a later stage.
UCOE improves the yield, consistency and
stability of protein production in cultured mam-
malian cells, allowing simpler and quicker
generation of stable, highly productive cell lines
suitable for larger-scale manufacture of protein
therapeutics. Ubiquitous Chromatin Opening Ele-
ments (UCOEs) have been isolated which ensure
efficient expression in a wide range of cell types
including CHO-K1, CHO-S, HeLa, and NS0 cells.
UCOE containing vectors have been shown to
markedly enhance a wide variety of intracellular,
and secreted proteins. Inclusion of a UCOE in
an expression vector permits efficient expression
in the vast majority of stable clones, while with
conventional vectors, only a minor proportion of
transfectants show high-level expression. There is
no need for gene amplification and as the vectors
integrate at low copy number, they are stable ge-
netically and expression has been demonstrated to
be maintained over 130 generations. The size of
the latest generation, optimized UCOEs has been
reduced to >2kb.
UCOEs allows the rapid isolation of high-
yielding, stably expressing clones and have
been evaluated with numerous therapeutic genes
including erythroprotein (EPO) and antibodies.
Typically a screen of 96 antibody expressing
clones gives 5-10 clones yielding greater than
1g/L in shake flasks within 8 weeks of transfec-
tion. Expression of antibody has been shown to be
stable for at least 50 generations.
Use with: mammalian cells; CHO cells; NS0
cells
Use to make: proteins; glycoproteins; antibod-
ies
Background: When transfected genes integrate
into mammalian chromosomes, the structure
of the chromatin at the site of integration has a
profound effect on expression of the transgene.
Consequently, only relatively rare transfected
clones where the expression vector has integrated
into open chromatin show efficient expression
of the transgene. The successful integration of a
transgene into mammalian cells is largely de-
pendent upon the chromatin structure around the
integration site. Most mammalian chromatin ex-
ists in the closed (heterochromatin) form, which is
transcriptionally silent. With conventional vectors,
most integrations suffer from position effects and
chromatin shutdown, which leads to gene-silenc-
ing and unproductive integration events. As a
result, pools of stably-transfected cells generally
show low productivity and stability, as only a few
cells might be good producers, and gene silencing
and overgrowth of non-producers rapidly dimin-
ish the overall productivity of the pool. This also
makes it very difficult and time consuming to find
high-expressing stable clones needed for large-
scale manufacturing.
Benefits: UCOE benefits include:
Rapid production of gram quantities of pro-
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
138
teins using stable pools
Ensures high-level expression, independent of
the site of chromosomal integration
Evaluated for recombinant antibodies, secreted
and intracellular proteins
Rapid isolation of stably transfected clones
Unique mechanism that prevents gene silenc-
ing through effects on chromatin structure
Majority of clones show high-level expression
No extensive cell line screening
Cell lines are stable over 130 generations
Small (4-8kb) DNA elements are capable
of making two protein chains per plasmid, e.g.,
antibodies
Another advantage of this system is the abil-
ity to produce frozen stable pools, which can be
used in subsequent fermentations. The technology
represents a significant cost saving both in the
production of research grade quantities of product
and in the selection of high expressing clones for
large-scale manufacturing.
Patents: Exemplary patents include 6,689,606,
Polynucleotide, Antoniou, et al. (Kings Col-
lege, London), Feb. 10, 2004, assigned to ML
Labs., with its abstract stating, The present
invention relates to a polynucleotide comprising
a ubiquitous chromatin opening element (UCOE)
which is not derived from an LCR. The present
invention also relates to a vector comprising the
polynucleotide sequence, a host cell comprising
the vector, use of the polynucleotide, vector or
host cell in therapy and in an assay, and a method
of identifying UCOEs. The UCOE opens chro-
matin or maintains chromatin in an open state and
facilitates reproducible expression of an operably-
linked gene in cells of at least two different tissue
types.
Other UCOE patents assigned to ML Labs.
include U.S. 6,881,556 and 6,689,606
Licensing information: Contact Celliance/Mil-
lipore. UCOE was originally commercially
developed by Innovata plc (formerly ML Labs.
and Quadrant), and has been exclusively licensed
in 2005 to Celliance Corp., now part of Millipore
Corp.
278
Plant expression - glycosylation
Organizations involved:
Washington State University Research Founda-
tion (WSURF)
Description: Methods based on preventing
acquisition of golgi-mediated modifications to
asparagine-linked glycans (preventing undesirable
glycosylation of proteins in plants) by retaining
proteins in the endoplastic reticulum (ER) of plant
cells enable expression of pharmaceutically active
glycoproteins with human-like glycosylation in
plants.
Use with: plant cells; plants
Use to make: proteins; glycoproteins
Background: Most proteins have to be put into
the secretory pathway before they acquire the
proper structure to be functionally active. A
complication is that these proteins almost always
have sugar chains attached to them when they are
first expressed and put into the lumen of the ER.
At this stage, the sugar chains of plant-expressed
(glyco)proteins are not different from those on
human proteins. It is only when the newly syn-
thesized protein moves to the golgi apparatus
that plant-specific changes which are very im-
munogenic in certain animals occur. By trapping
the desired protein in the ER, the changes can
be prevented. For many reasons, this goal has
been quite difficult to achieve. The discovery of a
unique function of a protein by Washington State
University researchers appears to accomplish this
goal and be useful in plant biotechnology applica-
tions where it is important to prevent acquisition
of golgi-mediated modifications to recombinant
proteins. Directing a recombinant protein into the
ER to PAC vesicle pathway where it can accu-
mulate without undergoing glycan modifications
or proteolytic cleavage seems to accomplish the
goal.
Patents: Application(s) reported filed, but no
published applications retrieved from several
simple searches.
Licensing information: Contact Washington
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
178
State University Research Foundation (WSURF).
279
pPIPRA vectors - plants; public
domain
Organizations involved:
Public Intellectual Property Resource for Agri-
culture (PIPRA) - Licensor, primary
Description: The Public Intellectual Property Re-
source for Agriculture (PIPRA) is facilitating the
design, construction, and testing of plant transfor-
mation vectors with maximal freedom-to-operate
(no or minimal patent licensing). PIPRA staff, a
working group of leading plant transformation
scientists, and PIPRAs pro bono attorneys are
working together to create vectors with as many
components as possible from the public domain or
owned by PIPRA members with known licensing
terms. The vectors will be distributed on a roy-
alty-free basis for humanitarian uses.
The pPIPRA plant transformation vector
system will incorporate technically proven, plant-
derived components and marker-free strategies.
PIPRA will design, develop and test the transfor-
mation system in model monocot and dicot plant
systems before making the transformation tools
available. The pPIPRA transformation system is
funded by a $600,000 grant from the Rockefeller
Foundation.
Use with: plants
Use to make: proteins
Licensing information: Contact PIPRA.
280
ProCellEx Plant Expression -
plant cells; glycosylation
Organizations involved:
Protalix BioTherapeutics, Inc - Licensor, pri-
mary
Description: ProCellEx is an expression system
based on plant cell, such as carrot and tobacco
cells, culture for the development, expression and
manufacture of recombinant proteins, including
expression of enzymes with human-like glyco-
sylation. The system facilitates the creation and
selection of high expressing, genetically stable
plant cell lines capable of expressing recombinant
proteins. Large-scale production, takes place in
flexible, sterile, polyethylene bioreactors which
are confined to a clean-room environment. Plant
cell cultures are grown on aqueous media consist-
ing of highly purified water and defined inorganic
nutrients in a completely closed and controlled
environment using simple polyethylene bioreac-
tors that are able to be maintained at the room
temperature of the clean-room in which they are
placed. Agrobacterium tumefaciens is cell used
for transfection (see relate entry).
ProCellEx produces enzymes which have uni-
form glycosylation patterns and therefore do not
require the lengthy and expensive post-expression
modifications that are required for certain proteins
produced by mammalian cell-based systems, in-
cluding the proteins for the treatment of Gaucher
disease (e.g., Cerezyme or recombinant gluco-
cerebrosidase manufactured and marketed by
Genzyme, which is glycosylated using enzymes
after expression of the protein). Such post-expres-
sion modifications in mammalian cell-produced
proteins are made in order to expose the terminal
mannose sugar residues. In the production of Cer-
ezyme, exposing these terminal mannose sugar
residues involves a multitude of highly technical
steps using enzymes which add time and cost to
the production process. In addition, these steps do
not guarantee the exposure of all of the required
terminal mannose sugar residues, resulting in
potentially lower effective yields and inconsis-
tency in potency from batch to batch. ProCellEx
protein expression system, by contrast, produces
glucocerebrosidase in a ready to use form that
does not require additional glycosylation or other
modifications to make it suitable for use in en-
zyme replacement therapy for Gaucher disease.
Use with: plants cells
Use to make: proteins; glycoproteins
Patents: Exemplary applications include U.S.
Broad/Platform Technologies
179
20080038232, Production of high mannose pro-
teins in plant culture, Shaaltiel, Y., et al., Feb. 14,
2008, assigned to Protalix Ltd., with its abstact
stating, A device, system and method for produc-
ing glycosylated proteins in plant culture, particu-
larly proteins having a high mannose glycosyl-
ation, while targeting such proteins with an ER
signal and/or by-passing the Golgi. The invention
further relates to vectors and methods for expres-
sion and production of enzymatically active high
mannose lysosomal enzymes using transgenic
plant root, particularly carrot cells. More particu-
larly, the invention relates to host cells, particular-
ly transgenic suspended carrot cells, vectors and
methods for high yield expression and production
of biologically active high mannose Glucocer-
ebrosidase (GCD). The invention further provides
for compositions and methods for the treatment of
lysosomal storage diseases.
Licensing information: Contact Protalix.
Products made using this tech.: Protalix is con-
ducting clinical trials with carrot cell-expressed
glucocerebrosidase. A Phase III U.S. trial started
in late 2007.
281
Profcia expression - transient
expression, plants
Organizations involved:
Medicago, Inc. - Licensor, primary
Description: The Proficia expression system for
transient expression enables recombinant protein
manufacture in dicotyledonous (higher) plants. A
gene of interest under the transcriptional con-
trol of promoting sequences is regulated by the
presence of nitrogen sources. Very high level of
expression can be achieved (about 10X more than
transgenic plants) with production starting in five
days. This technology may be used with Lemna
(duckweed; see related entry).
In a preferred embodiment, the method in-
volves: a) introducing the vector into a suitable
Agrobacterium tumefaciens strain; b) using the
Agrobacterium strain to transfer T-DNA into a
plant cell; c) selecting for transgenicity of plant
cells on a suitable medium; d) regenerating
embryos or plantlets from transgenic cells; and e)
growing mature plants from regenerated embryos.
Use with: plants
Use to make: proteins; glycoproteins
Patents: Exemplary patents include U.S.
7,125,978, Promoter for regulating expression
of foreign genes, Vezina, et al., Oct. 24, 2006,
including a promoter with expression induced
by light; and 6,420,548, Method for regulating
transcription of foreign genes, both assigned to
Medicago.
Licensing information: Contact Medicago.
Acambis plc (becoming part of Sanofi Pasteur
S.A.) has been reported to have evaluated this
technology.
282
RNA-dependent RNA
polymerase (RdRP) - universal
Organizations involved:
Plant Bioscience Ltd. - Licensor, primary
Description: RNA-dependent RNA polymerase
(RdRP; EC 2.7.7.48) gene sequences encode
polypeptides having the enzymatic activity of an
RNA-directed RNA polymerase (RdRP). RdRP
is capable of RNA-directed RNA synthesis, using
RNA as a template for synthesizing complemen-
tary RNA molecules. RdRP is also capable of
accepting single-stranded DNA molecules as tem-
plates for RNA transcription. Vectors containing
RdRP operatively linked to regulatory elements
allow expression in prokaryotic and/or eukaryotic
host cells. RdRP and antagonists/inhibitors are
particuarly useful for transgenic plant cells and
plants.
Use with: universal (generic); plant cells;
plants
Use to make: proteins
Patents: Related patent include U.S. 6,218,142
and 7,09,1397, Nucleic acid molecules encoding
polypeptides having the enzymatic activity of an
RNA-directed RNA polymerase (RdRP), as-
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
180
signed to Plant Bioscience Ltd.
Licensing information: Contact Plant Bioscience
Ltd.
283
Stratosome Biologics System;
Oilbody expression; Saffower
plant seed expression
Organizations involved:
SemBioSys Genetics Inc. - Licensor, primary
Description: Stratosome is a system for expres-
sion of proteins in safflower (Carthamus tinctorius
L.) plant seeds, attaching them to oily molecules
naturally occurring in safflower seeds, greatly
facilitating purification. Proteins are stable in
this form more or less indefinitely. Purification is
simplified by the separation of the oily structures
from homogenized seeds. Similar oil-protein
structures may be used for oral deliver of proteins.
SemBioSys is developing recombinant safflow-
er expressed insulin, which could could reduce
capital costs for an insulin facility by 70%, and
cost-of-goods by more than 40% compared with
current production methods. In 2006, the compa-
ny reported accumulation in safflower with 1.2%
of total seed protein, a level considered com-
mercially viable. For manufacture of Apo A1,
SemBioSys estimates the cost to grow a ton of
seed, about an acre of safflower which would then
produce about 2 kg of Apo A1, would be about
$800, compared to the current cost which is in the
range of about $400 per gram of Apo A1.
The Stratosome Biologics System is based
on the discovery that recombinant proteins can
be targeted to, or captured on, naturally occur-
ring oilbodies in seeds. A transgene encoding an
oleosin/proteinX fusion protein is transferred into
an oilseed plant; where proteinX is a protein
of interest (e.g. recombinant human insulin). As
the plant grows the oleosin/proteinX fusion is
naturally targeted to and captured on oilbodies in
the seed. Based on the principle that oil is lighter
than water, harvested seed is milled in an aque-
ous buffer and the oilbodies are purified from
seed-derived impurities through a series of simple
centrifugation and wash processes. An enzyme
or chemical that recognizes an engineered cleav-
age site between the oleosin/proteinX fusion is
added to the purified oilbodies cleaving proteinX
from the oilbody-associated oleosin. The oilbod-
ies are removed via centrifugation and proteinX,
now in the aqueous phase, is purified simply and
economically using conventional downstream
processing.
Stratosome Biologics System is the only trans-
genic system to offer comprehensive enabling
advantages in the low cost production of high vol-
ume recombinant proteins. High levels of expres-
sion coupled with an intrinsic, unique purification
process allows the system to offer both upstream
and downstream advantages in the production of
recombinant proteins over conventional (mi-
crobial, yeast and animal cell culture) and other
transgenic (plant and animal) approaches. includ-
ing lower production costs (up to 75% reduction
in unit costs); lower capital costs for production/
purification facilities (up to a 70% reduction); and
enhanced flexibility and scalability of production.
A large number of simple and complex pro-
teins, ranging from approximately one kilodalton
(nine amino acids) to over 100 kilodaltons, have
been produced at high levels using the Strato-
some Biologics System. From one ton of seed,
it is possible to recover more than one kilogram
of purified recombinant protein. The system can
be used with recombinant polypeptides, mul-
timeric-protein-complexes, heteromultimeric-
protein-complexes, multimeric-fusion-proteins,
heteromultimeric-fusion-proteins, immunoglobu-
lin-polypeptide-chains, immunoglobulins, redox-
fusion-polypeptides, and/or thioredoxin-related
proteins; in association with oil bodies.
Use with: safflower plants
Use to make: proteins; glycoproteins; antibod-
ies
Benefits: Key advantages in oilbody/protein
manufacture, include:
Physiology - safflower is a highly productive
oilseed crop whose seed offers superior inventory
Broad/Platform Technologies
181
management advantages;
Regulatory - safflower is a low acreage crop
that is predominantly self-pollinating and can be
easily segregated from other safflower production.
SemBioSys is the only company to have pro-
duced transgenic safflower in field conditions;
having done so in Canada, the United States and
Mexico, and counter-seasonally in Chile for at
least four years. Current production results in 200
tons of seed harvested from 280 acres.
Patents: Related patents include 7,098,383,
Methods for the production of multimeric immu-
noglobulins, and related compositions, Szarka, et
al., Aug. 29, 2006, with its abstract stating, Im-
proved methods for the production of multimeric-
protein-complexes, such as redox proteins and
immunoglobulins, in association with oil bodies
are described. The redox protein is enzymatically
active when prepared in association with the oil
bodies. Also provided are related nucleic acids,
proteins, cells, plants, and compositions.
Other related patents include SemBiosSy US
Patents: 7,091,401 - Expression of epidermal
growth factor in plant seeds; 6,924,363 - Oil
bodies and associated proteins as affinity matri-
ces; 6,777,591 - Legume-like storage protein
promoter isolated from flax and methods of
expressing proteins in plant seeds using the pro-
moter; 6,761,914 - Immunogenic formulations
comprising oil bodies; 6,753,167 - Preparation
of heterologous proteins on oil bodies; 6,750,046
- Preparation of thioredoxin and thioredoxin
reductase proteins on oil bodies; 6,599,513
- Products for topical applications comprising
oil bodies; 6,509,453 - Oil bodies and associ-
ated proteins as affinity matrices; 6,288,304
- Expression of somatotropin in plant seeds;
5,948,682 - Preparation of heterologous proteins
on oil bodies; 5,856,452 - Oil bodies and as-
sociated proteins as affinity matrices; 5,792,922
- Oil body protein cis-elements as regulatory
signals; 5,650,554 - Oil body proteins as carri-
ers of high-value peptides in plants.
Licensing information: Contact SemBioSys,
which offers CMO services.