U.S. patent application number 12/817382 was filed with the patent office on 2010-10-14 for recombinant production of heparin binding proteins.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Michelle D. Butler, Jeffrey L. Cleland, David W. Kahn, Shelly Pizarro, Charles H. Schmelzer, Marjorie E. Winkler.
Application Number | 20100261888 12/817382 |
Document ID | / |
Family ID | 38668202 |
Filed Date | 2010-10-14 |
United States Patent
Application |
20100261888 |
Kind Code |
A1 |
Butler; Michelle D. ; et
al. |
October 14, 2010 |
Recombinant Production of Heparin Binding Proteins
Abstract
A process for recovering and purifying refolded heparin binding
proteins produced in heterologous host cells includes the step of
incubation of the solubilized protein with a polyanionic species
such as dextran sulfate.
Inventors: |
Butler; Michelle D.; (San
Francisco, CA) ; Cleland; Jeffrey L.; (San Carlos,
CA) ; Kahn; David W.; (North Potomac, MD) ;
Pizarro; Shelly; (San Carlos, CA) ; Schmelzer;
Charles H.; (Burlingame, CA) ; Winkler; Marjorie
E.; (Burlingame, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
38668202 |
Appl. No.: |
12/817382 |
Filed: |
June 17, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11613012 |
Dec 19, 2006 |
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12817382 |
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60807432 |
Jul 14, 2006 |
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60753615 |
Dec 22, 2005 |
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Current U.S.
Class: |
530/399 ;
530/412 |
Current CPC
Class: |
C07K 1/14 20130101; C07K
14/52 20130101; C07K 14/47 20130101 |
Class at
Publication: |
530/399 ;
530/412 |
International
Class: |
C07K 1/14 20060101
C07K001/14 |
Claims
1. A process for recovering a heparin binding protein from a
prokaryotic cell culture, the process comprising the steps of (a)
isolating said heparin binding protein from the periplasm of said
prokaryotic cell culture; (b) denaturing said isolated heparin
binding protein in a first buffered solution comprising a
chaotropic agent and a reducing agent; (c) incubating said
denatured heparin binding protein in a second buffered solution
comprising a chaotropic agent and a sulfated polyanionic agent for
such a time and under such conditions that refolding of the heparin
binding protein occurs; and (d) recovering said refolded heparin
binding protein, wherein there is about a 2 to 5-fold increase in
refolded heparin binding protein recovered compared to incubating
with no sulfated polyanionic agent.
2. The process of claim 1, wherein the heparin binding protein is a
heparin binding growth factor.
3. The process of claim 2, wherein the heparin binding growth
factor is vascular endothelial growth factor (VEGF).
4. The process of claim 3, wherein the VEGF is VEGF.sub.165.
5. The process of claim 1, wherein the sulfated polyanionic agent
is between about 3,000 daltons and 10,000 daltons.
6. The process of claim 3, wherein said second buffered solution
comprises a dextran sulfate.
7. The process of claim 3, wherein said second buffered solution
comprises sodium sulfate.
8. The process of claim 3, wherein said second buffered solution
comprises heparin.
9. The process of claim 6, wherein the dextran sulfate is between
about 8,000 and 10,000 daltons.
10. The process of claim 3, wherein said first and second buffered
solutions comprise HEPPS pH 8.0.
11. The process of claim 1, wherein said second buffered solution
further comprises a reducing agent.
12. The process of claim 11, wherein the reducing agent of the
second buffered solution comprises a combination of cysteine and
DTT.
13. The process of claim 1, wherein said second buffered solution
further comprises a nonionic detergent.
14. The process of claim 1, wherein said second buffered solution
further comprises arginine and/or lysine.
15. The process of claim 1, wherein said recovery step (d)
comprises sequentially contacting said refolded heparin binding
protein to a hydroxyapatite chromatographic support, a first
hydrophobic interaction chromatographic support; a cationic
chromatographic support, and a second hydrophobic interaction
chromatographic support, and selectively eluting the heparin
binding protein from each support.
16. The process of claim 15, wherein said first and second
hydrophobic interaction chromatographic support is selected from
the group consisting of butyl-, propyl-, octyl- and aryl-agarose
resins.
17. The process of claim 15, wherein said first hydrophobic
interaction chromatographic support is a butyl-agarose support and
said second hydrophobic interaction chromatographic support is a
phenyl-agarose support resin.
18. The process of claim 1, wherein said recovery step (d)
comprises sequentially contacting said refolded heparin binding
protein to a cation exchange support; a hydrophobic interaction
chromatographic support, and an ion exchange chromatographic
support, and selectively eluting the heparin binding protein from
each support.
19. A method for recovering a heparin binding protein from a
prokaryotic cell culture, the method comprising the steps of (a)
isolating said heparin binding protein from the periplasm of said
prokaryotic cell culture; (b) denaturing said isolated heparin
binding protein in a first buffered solution comprising a
chaotropic agent and a reducing agent; (c) incubating said
denatured heparin binding protein in a second buffered solution
comprising a chaotropic agent and a sulfated polyanionic agent for
such a time and under such conditions that refolding of the heparin
binding protein occurs, wherein there is about a 2 to 5-fold
increase in refolded heparin binding protein recovered compared to
incubating with no sulfated polyanionic agent; and (d) sequentially
contacting said refolded heparin binding protein with a
hydroxyapatite chromatographic support, a first hydrophobic
interaction chromatographic support, a cationic chromatographic
support, and a second hydrophobic interaction chromatographic
support, and selectively eluting the heparin binding protein from
each support.
20. A method for purifying a heparin binding protein, the method
comprising the steps of sequentially contacting a refolded heparin
binding protein with a hydroxyapatite chromatographic support, a
first hydrophobic interaction chromatographic support, a cationic
chromatographic support, and a second hydrophobic interaction
chromatographic support, and selectively eluting the heparin
binding protein from each support.
21. A method for recovering a heparin binding protein from a
prokaryotic cell culture, the method comprising the steps of (a)
isolating said heparin binding protein from the periplasm of said
prokaryotic cell culture; (b) denaturing said isolated heparin
binding protein in a first buffered solution comprising a
chaotropic agent and a reducing agent; (c) incubating said
denatured heparin binding protein in a second buffered solution
comprising a chaotropic agent and a sulfated polyanionic agent for
such a time and under such conditions that refolding of the heparin
binding protein occurs, wherein there is about a 2 to 3-fold
increase in refolded heparin binding protein recovered compared to
incubating with no sulfated polyanionic agent; and, (d)
sequentially contacting said refolded heparin binding protein with
a cation exchange support; a hydrophobic interaction
chromatographic support, and an ion exchange chromatographic
support, and selectively eluting the heparin binding protein from
each support.
22. The method of claim 19 or 21, wherein the polyanionic agent is
between about 3,000 daltons and 10,000 daltons.
23. A method for purifying a heparin binding protein, the method
comprising the steps of sequentially contacting a refolded heparin
binding protein with a cation exchange support; a hydrophobic
interaction chromatographic support, and an ion exchange
chromatographic support, and selectively eluting the heparin
binding protein from each support.
Description
RELATED APPLICATION
[0001] This application is a continuation of U.S. Ser. No.
11/613,012, filed on Dec. 19, 2006 which claims priority to and the
benefit of U.S. Provisional Application Ser. No. 60/753,615, filed
Dec. 22, 2005, and U.S. Provisional Application Ser. No.
60/807,432, filed Jul. 14, 2006, the specifications of which are
incorporated herein in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to methods for obtaining
heparin-binding proteins produced in cell culture. The invention
includes methods for recovering and purifying refolded heparin
binding proteins that have been produced in prokaryotic host cells
and are present in these cells, typically in the periplasmic or
intracellular space. The heparin binding proteins produced in
prokaryotic host cells can also be found as soluble proteins or a
mixture of soluble and insoluble proteins.
BACKGROUND
[0003] It is known that a large variety of naturally occurring,
biologically active polypeptides bind heparin. Such heparin-binding
polypeptides include cytokines, such as platelet factor 4 and IL-8
(Barber et al., (1972) Biochim. Biophys. Acta, 286:312-329; Handin
et al., (1976) J. Biol. Chem., 251:4273-422; Loscalzo et al.,
(1985) Arch. Biochem. Biophys. 240:446-455; Zucker et al., (1989)
Proc. Natl. Acad. Sci. USA, 86:7571-7574; Talpas et al., (1991)
Biochim. Biophys. Acta, 1078:208-218; Webb et al., (1993) Proc.
Natl. Acad. Sci. USA, 90:7158-7162) heparin-binding growth factors
(Burgess and Maciag, (1989) Annu. Rev. Biochem., 58:576-606;
Klagsbrun, (1989) Prog. Growth Factor Res., 1:207-235), such as
epidermal growth factor (EGF); platelet-derived growth factor
(PDGF); basic fibroblast growth factor (bFGF); acidic fibroblast
growth factor (aFGF); vascular endothelial growth factor (VEGF);
and hepatocyte growth factor (HGF) (Liu et al., (1992)
Gastrointest. Liver Physiol. 26:G642-G649); and selectins, such as
L-selectin, E-selectin and P-selectin (Norgard-Sumnicht et al.,
(1993) Science, 261:480-483). See also, Munoz and Linhardt., (2004)
Arterioscler Thromb Vasc Biol., 24:1549-1557.
[0004] International Publication No. WO 95/07097 describes
formulations of heparin binding proteins including heparin binding
growth factors such as VEGF, with purified native heparin or other
polyanionic compounds for therapeutic use. Heparin derived
oligosaccharides and various other polyanionic compounds have been
shown to stabilize the active conformation for heparin binding
growth factors (Barzu et al., (1989) J. Cell. Physiol. 140:538-548;
Dabora et al., (1991) J. Biol. Chem. 266:23627-23640) and heparin
affinity chromatography has been employed in various purification
schemes (see generally, International Publication No. WO
96/02562).
[0005] Many of the heparin binding proteins of mammalian origin
have been produced by recombinant technology and are clinically
relevant (Munoz and Linhardt, (2004) Arterioscler Thromb Vasc
Biol., 24:1549-1557; Favard et al. (1996) Diabetes and Metabolism
22(4):268-73; Matsuda et al., (1995) J. Biochem. 118(3):643-9;
Roberts et al., (1995) Brain Research 699(1):51-61). For example,
VEGF is a potent mitogen for vascular endothelial cells. It is also
known as vascular permeability factor (VPF). See, Dvorak et al.,
(1995) Am. J. Pathol. 146:1029-39. VEGF play important roles in
both vasculogenesis, the development of the embryonic vasculature,
and angiogenesis, the process of forming new blood vessels from
pre-existing ones. See, e.g., Ferrara, (2004) Endocrine Reviews
25(4):581-611; Risau et al., (1988) Dev. Biol., 125:441-450;
Zachary, (1998) Intl. J. Biochem Cell Bio 30:1169-1174; Neufeld et
al., (1999) FASEB J. 13:9-22; Ferrara (1999) J. Mol. Med.
77:527-543; and, Ferrara and Davis-Smyth, (1997) Endocri. Rev.
18:4-25 Clinical applications for VEGF include those where the
growth of new capillary beds is indicated as, for example, in
promoting wound healing (see, for example, International
Publication No. WO 91/02058; and, Attorney Docket No. P2358R1,
entitled "Wound Healing" filed on Jun. 16, 2006), in promoting
tissue growth and repair, e.g., liver (see, e.g., WO2003/0103581),
bone (see, e.g., WO2003/094617), etc. See also, Ferrara, (2004)
Endocrine Reviews 25(4):581-611.
[0006] Typically, therapeutically relevant recombinant proteins are
produced in a variety of host organisms. Most proteins can be
expressed in their native form in eukaryotic hosts such as CHO
cells. Animal cell culture generally requires prolonged growing
times to achieve maximum cell density and ultimately achieves lower
cell density than prokaryotic cell cultures (Cleland, J. (1993) ACS
Symposium Series 526, Protein Folding: In Vivo and In Vitro,
American Chemical Society). Additionally, animal cell cultures
often require expensive media containing growth components that may
interfere with the recovery of the desired protein. Bacterial host
expression systems provide a cost-effective alternative to the
manufacturing scale production of recombinant proteins. Numerous
U.S. patents on general bacterial expression of recombinant
proteins exist, including U.S. Pat. Nos. 4,565,785; 4,673,641;
4,795,706; and 4,710,473. A major advantage of the production
method is the ability to easily isolate the product from the
cellular components by centrifugation or microfiltration. See,
e.g., Kipriyanov and Little, (1999) Molecular Biotechnology, 12:
173-201; and, Skerra and Pluckthun, (1988) Science, 240:
1038-1040.
[0007] Recombinant heparin binding growth factors such as acidic
fibroblast growth factor, basic fibroblast growth factor and
vascular endothelial growth factor have been recovered and purified
from a number of sources including bacteria (Salter D. H. et al.,
(1996) Labor. Invest. 74(2):546-556 (VEGF); Siemeister et al.,
(1996) Biochem. Biophys. Res. Commun. 222(2):249-55 (VEGF); Cao et
al., (1996) J. Biol. Chem. 261(6):3154-62 (VEGF); Yang et al.,
(1994) Gaojishu Tongxun, 4:28-31 (VEGF); Anspach et al., (1995) J.
Chromatogr. A 711(1):129-139 (aFGF and bFGF); Gaulandris (1994) J.
Cell. Physiol. 161(1):149-59 (bFGF); Estape and Rinas (1996)
Biotech. Tech. 10(7):481-484 (bFGF); McDonald et al., (1995) FASEB
J. 9(3):A410 (bFGF)). However, bacterial expression systems such as
E. coli lack the cellular machinery to facilitate proper refolding
of the proteins and generally do not result in the secretion of
large proteins into the culture media. Recombinant proteins
expressed in bacterial host cells are often found as inclusion
bodies consisting of dense masses of partially folded and misfolded
reduced protein. In this form, the recombinant protein is generally
inactive. For example, the predominant active form of VEGF is a
homodimer of two 165-amino acid polypeptides (VEGF-165). In this
structure, each subunit contains 7 pairs of intrachain disulfide
bonds and two additional pairs which effect the covalent linkage of
the two subunits (Ferrara et al., (1991) J. Cell. Biochem.
47:211-218). The native conformation includes a strongly basic
domain which has been shown to readily bind heparin (Ferrara et al
(1991) supra). Covalent dimerization of VEGF is needed for
effective receptor binding and biological activity (Potgens et al.,
(1994) J. Biol. Chem. 269:32879-32885; Claffey et al., (1995)
Biochim. et Biophys. Acta 1246:1-9). The bacterial product
potentially contains several misfolded and disulfide scrambled
intermediates.
[0008] Additionally, refolding often produces misfolded and
disulfide-linked dimers, trimers, and multimers. (Morris et al.,
(1990) Biochem. J., 268:803-806; Toren et al., (1988) Anal.
Biochem., 169:287-299). This association phenomenon is very common
during protein refolding, particularly at higher protein
concentrations, and appears often to involve association through
hydrophobic interaction of partially folded intermediates (Cleland
and Wang, (1990) Biochemistry, 29:11072-11078).
[0009] Misfolding occurs either in the cell during fermentation or
during the isolation procedure. Proteins recovered from periplasmic
or intracellular space must be solubilized and the soluble is
protein refolded into the native state. In vitro methods for
refolding the proteins into the correct, biologically active
conformation are essential for obtaining functional proteins.
Typical downstream processing of proteins recovered from inclusion
bodies includes the dissolution of the inclusion body at high
concentration of a denaturant such as urea followed by dilution of
the denaturant to permit refolding to occur (see, U.S. Pat. Nos.
4,512,922; 4,511,502; and 4,511,503). See also, e.g., Rudolph and
Lilie, (1996) FASEB J. 10:49-56; and, Fischer et al., (1993),
Biotechnology and Bioengineering, 41:3-13. Such recovery methods
are regarded as being universally applicable, with minor
modifications, to the recovery of biologically active recombinant
proteins from inclusion bodies. These methods have been applied to
heparin binding protein such as VEGF (Siemeister et al. (1996)
supra). These methods seek to eliminate random disulfide bonding
prior to coaxing the recombinant protein into its biologically
active conformation through its other stabilizing forces and may
not eliminate improperly folded intermediates or provide homogenous
populations of properly folded product.
[0010] Reversed micelles or ion exchange chromatography have been
used to assist refolding of denatured proteins by enclosing a
single protein within micelles or isolating them on a resin and
then removing the denaturant (Hagen et al., (1990) Biotechnol.
Bioeng. 35:966-975; Creighton (1985) in Protein Structure Folding
and Design (Oxender, D. L. Ed.) pp. 249-251, New York: Alan R.
Liss, Inc.). These methods have been useful in preventing protein
aggregation and facilitating proper refolding. To alter the rate or
extent of refolding, conformation-specific refolding has been
performed with ligands and antibodies to the native structure of
the protein (Cleland and Wang, (1993), in Biotechnology, (Rehm
H.-J., and Reed G. Eds.) pp 528-555, New York, VCH). For example,
creatine kinase was refolded in the presence of antibodies to the
native structure (Morris et al., (1987) Biochem. J. 248:53-57). In
addition to antibodies, ligands and cofactors have been used to
enhance refolding. These molecules would be more likely to interact
with the folding protein after formation of the native protein.
Therefore, the folding equilibrium could be "driven" to the native
state. For example, the rate of refolding of ferricytochrome c was
enhanced by the extrinsic ligand for the axial position of the heme
iron (Brems and Stellwagon, (1983) J. Biol. Chem. 258:3655-3661).
Chaperone proteins have also been used to assist with protein
folding. See, e.g., Baneyx, (1999) Current Opinion in
Biotechnology, 10:411-421.
[0011] There is a need for new and more effective methods of
folding and/or recovering heparin binding proteins from a host cell
culture, e.g., for the efficient and economical production of
heparin binding proteins in bacterial cell culture that provides
for elimination or reduction of biologically inactive intermediates
and improved recovery of a highly purified biologically active
properly refolded protein and that is generally applicable to
manufacturing scale production of the proteins. The invention
addresses these and other needs, as will be apparent upon review of
the following disclosure.
SUMMARY OF THE INVENTION
[0012] The invention provides a method for recovering and purifying
refolded heparin binding proteins from cell culture. In particular
the invention provides a method of recovering a heparin binding
protein from prokaryotic host cells, e.g., bacterial cells. For
example, a method comprises the steps of (a) isolating insoluble
heparin binding protein from the periplasmic or intracellular space
of said bacterial cells; (b) solubilizing said isolated insoluble
heparin binding protein in a first buffered solution comprising a
chaotropic agent and a reducing agent, and c) incubating said
solubilized heparin binding protein in a second buffered solution
comprising a chaotropic agent and a sulfated polyanionic agent for
such a time and under such conditions that refolding of the heparin
binding protein occurs; and (d) recovering said refolded heparin
binding protein, wherein there is a 2 to 10 fold increase in
protein concentration recovered by incubating with a sulfated
polyanionic agent compared to a control. In one embodiment, the
second buffered solution further comprises arginine. In one
embodiment, the second buffered solution further comprises cysteine
or a mild reducing agent.
[0013] In one embodiment of the invention, there is a, e.g., 2-8
fold increase in protein concentration of recovered biologically
active refolded protein, or 2-5 fold increase in protein
concentration of recovered biologically active refolded protein, or
3-5 fold increase in protein concentration of recovered
biologically active refolded protein, or a 2-3 fold increase in
protein concentration of recovered biologically active refolded
protein. In another embodiment of the invention, there is a, e.g.,
greater than a 2.0 fold, a 2.5 fold, a 2.8 fold, a 3.0 fold, a
5-fold, a 6 fold, a 7.0 fold, an 8 fold, a 9 fold, etc., increase
in protein concentration recovered of biologically active refolded
protein. In one embodiment of the invention, there is a 3 to 5-fold
increase in protein concentration of biologically active refolded
VEGF.
[0014] The processes of the invention are broadly applicable to
heparin binding proteins and especially to heparin binding growth
factors and in particular, vascular endothelial growth factor
(VEGF). In certain embodiments of the invention, the sulfated
polyanionic agent is between about 3,000 and 10,000 daltons. In one
embodiment, the sulfated polyanionic agent utilized in the
production processes is a dextran sulfate, sodium sulfate or
heparin sulfate. In one aspect, the dextran sulfate is between
3,000 daltons and 10,000 daltons.
[0015] The invention additionally provides processes and methods
for purification of heparin binding proteins either alone or in
connection with the recovery of the heparin binding protein as
described herein. In a particular embodiment, purification methods
include contacting said refolded heparin binding protein with a
hydroxyapatite chromatographic support; a first hydrophobic
interaction chromatographic support, a cationic chromatographic
support and a second hydrophobic interaction chromatographic
support and selectively eluting the heparin binding protein from
each support. In another embodiment, a purification method
comprises contacting said refolded heparin binding protein with a
cation exchange support; a first hydrophobic interaction
chromatographic support, and an ion exchange or mixed-media
chromatographic support and selectively eluting the heparin binding
protein from each support. It is contemplated that the steps for
recovery steps can be performed in any order, e.g., sequentially or
altering the order of the chromatographic supports. In certain
embodiments of the invention, methods are provided for recovering
and purifying refolded heparin binding proteins from manufacturing
or industrial scale cell culture.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 illustrates a chromatograph from VEGF produced by
bacterial strain W3110 loaded on a POROS HE2/M column
(4.6.times.100 mm, PerSeptive BioResearch Products, Cambridge,
Mass.). For example, the POROS HE/2M column is equilibrated in 10
mM sodium phosphate, pH 7 containing 0.15 M sodium chloride. The
column is eluted using a linear gradient from 0.15-2 M sodium
chloride in, 10 mM sodium phosphate, pH 7 over 10 minutes. The
eluant is monitored at 280 nm. The protein recovered in each peak
corresponds to VEGF however only peak 3 corresponds to a
biologically active properly refolded VEGF.
[0017] FIG. 2 illustrates a graph depicting the stabilization of
native properly folded VEGF by heparin. The VEGF is suspended in 50
mM HEPES, pH 8, containing 5 mM EDTA, 0.2 M NaCl and 10 mM
cysteine.
[0018] FIGS. 3A-3D illustrates chromatographs from VEGF produced by
bacterial strain W3110 and incubated with 12 .mu.g/m' dextran
sulfate 5,000 daltons (FIG. 3A); 12 .mu.g/ml dextran sulfate 8,000
daltons (FIG. 3B); 12 .mu.g/ml dextran sulfate 10,000 daltons (FIG.
3C) or 25 .mu.g/ml heparin (FIG. 3D), 3,000 daltons and loaded on a
POROS HE2/M column (4.6.times.100 mm, PerSeptive BioResearch
Products, Cambridge, Mass.). For example, the column is
equilibrated in 10 mM sodium phosphate, pH 7 containing 0.15 M
sodium chloride. The column is eluted using a linear gradient from
0.15-2 M sodium chloride in, 10 mM sodium phosphate, pH 7 over 10
minutes. The eluant is monitored at 280 nm. The protein recovered
in each peak corresponds to VEGF however only peak 3 corresponds to
a biologically active properly refolded VEGF.
[0019] FIG. 4 illustrates the effect of scale on the refolding of
VEGF.
[0020] FIG. 5 illustrates the effect of heparin, low molecular
weight (MW) and high MW, and dextran sulfate, 10,000 daltons, on
VEGF refolding. Peak 3 corresponds to a biologically active
properly refolded VEGF.
[0021] FIG. 6 illustrates the effect of sodium sulfate on VEGF
refolding. Peak 3 corresponds to a biologically active properly
refolded VEGF.
[0022] FIG. 7 illustrates the effect of heparin, low molecular
weight (MW) and high MW, and dextran sulfate, 5,000 daltons, 8,000
daltons, and 10,000 daltons, on VEGF refolding. Peak 3 corresponds
to a biologically active properly refolded VEGF.
[0023] FIG. 8 illustrates the effect of heparin and dextran sulfate
on VEGF refolding. Peak 3 corresponds to a biologically active
properly refolded VEGF.
[0024] FIG. 9 illustrates an effect of urea and DTT on the
extraction of VEGF from bacterial inclusion bodies.
[0025] FIG. 10 illustrates an effect of urea and DTT concentration
on the refolding of VEGF.
[0026] FIG. 11 illustrates the amino acid sequence of VEGF.sub.165
with disulfide bonds indicated (SEQ ID NO.:1).
[0027] FIG. 12 illustrates the effect of the presence of charged
amino acids. At 0.75M concentration in the second buffered solution
both arginine and lysine are beneficial whereas histidine has
little additive effect as compared to the buffered solution without
it. Additionally arginine has been shown to have similar effect at
concentrations of 0.1 to 1M.
[0028] FIG. 13 illustrates the effect of dilution in the % refold
efficiency, where, although the total VEGF concentration is lower
as the dilution increases, the % refold efficiency is higher with
more dilution.
DETAILED DESCRIPTION
Definitions
[0029] "Heparin" (also referred to as heparinic acid) is a
heterogenous group of highly sulfated, straight-chain anionic
mucopolysaccharides, called glycosaminoglycans. Although others may
be present, the main sugars in heparin are: .alpha.-L-iduronic acid
2-sulfate, 2-deoxy-2-sulfamino-.alpha.-glucose 6-sulfate,
.beta.-D-glucuronic acid, 2-acetamido-2-deoxy-.alpha.-D-glucose,
and L-iduronic acid. These and optionally other sugars are joined
by glycosidic linkages, forming polymers of varying sizes. Due to
the presence of its covalently linked sulfate and carboxylic acid
groups, heparin is strongly acidic. The molecular weight of heparin
varies from about 3,000 to about 20,000 daltons depending on the
source and the method of determination.
[0030] Native heparin is a constituent of various tissues,
especially liver and lung, and mast cells in several mammalian
species. Heparin and heparin salts (heparin sodium) are
commercially available and are primarily used as anticoagulants in
various clinical situations.
[0031] "Dextran sulfate" is a sulfate of dextran whose principal
structure is a polymer of D-glucose. Glucose and optionally other
sugars are joined by .alpha.-D(1-6) glycosidic linkages, forming
polymers of varying sizes. Due to the presence of covalently linked
sulfate, dextran sulfate is strongly acidic. The sulfur content is
generally not less than 10%, and typically about 15%-20% with up to
3 sulfate groups per glucose molecule. The average molecular weight
of dextran sulfate is from about 1,000 to about 40,000,000 daltons.
Examples of dextran sulfate employable in the invention include the
sulfate of the dextrans produced from microorganisms such as
Leuconostoc mesenteroides and L. dextranicum.
[0032] "Polyanionic agent" as used within the scope of the
invention is meant to describe commercially available purified
native heparin preparations and compounds which are capable of
binding to heparin binding proteins including other "polyanionic
agents" such as sodium sulfate, heparin sulfate, heparan sulfate,
pentosan (poly) sulfate, dextran, dextran sulfate, hyaluronic acid,
chondroitin, chondroitin sulfate, dermatan sulfate, and keratan
sulfate. Particularly useful within the context of the invention is
a "sulfated polyanionic agent," such as for example, a sulfate
derivative of a polysaccharide, such as heparin sulfate, dextran
sulfate, the sulfates of the cyclodextrin produced by
microorganisms such as Bacillus macerans described in U.S. Pat. No.
5,314,872 as well as sulfates of other glucans such as .beta.-1,3
glucan sulfates, the .beta.-1,3 glucan being produced by
microorgansims belonging to the genus Alcaligenes or Agrobacterium,
and chondroitin sulfate as well as sulfated heparin fragments.
[0033] The above mentioned agents are generally available and
recognized by the skilled artisan. For example, sulfated heparin
fragments may be obtained from a library of heparin-derived
oligosaccharides that have been fractionated by gel-permeation
chromatography. The preparation of affinity-fractionated,
heparin-derived oligosaccharides was reported by Ishihara et al.,
(1993) J. Biol. Chem., 268:4675-4683. These oligosaccharides were
prepared from commercial porcine heparin following partial
depolymerization with nitrous acid, reduction with sodium
borohydride, and fractionation by gel permeation chromatography.
The resulting pools of di-, tetra-, hexa-, octa-, and
decasaccharides were sequentially applied to an affinity column of
human recombinant bFGF covalently attached to SEPHAROSE.TM. 4B, and
were further fractionated into subpools based on their elution from
this column in response to gradients of sodium chloride. This
resulted in five pools, designated Hexa-1 to Hexa-5, the structures
and biological activities of which were further evaluated. The
structure of Hexa-5C and its 500-MHz NMR spectrum are shown in FIG.
4 of Tyrell et al., (1993) J. Biol. Chem., 268:4684-4689. This
hexasaccharide has the structure
[IdoA(2-OSO.sub.3).alpha.1-4GlcNSO.sub.3(6-OSO.sub.3).alpha.1-4].sub.2Ido-
A(2-OSO.sub.3).alpha.1-4AMan.sub.R(6-OSO.sub.3). All
heparin-derived oligosaccharides discussed above, as well as other
heparin-like oligosaccharides are suitable for and can be used in
accordance with the invention. In one embodiment of the invention,
hexasaccharides and polysaccharides of heparin of higher unit size
(e.g. hepta-, octa-, nona- and decasaccharides) are used.
Furthermore, heparin-derived or heparin-like oligosaccharides with
a large net negative charge, e.g. due to a high degree of
sulfation, are used with advantage.
[0034] The term "heparin-binding protein" or "HPB" as used herein
refers to a polypeptide capable of binding heparin (as hereinabove
defined). The definition includes the mature, pre, pre-pro, and pro
forms of native and recombinantly produced heparin-binding
proteins. Typical examples of heparin-binding proteins are "heparin
binding growth factors," including but not limited to epidermal
growth factor (EGF), platelet derived growth factor (PDGF), basic
fibroblast growth factor (bFGF), acidic fibroblast growth factor
(aFGF), vascular endothelial growth factor (VEGF), hepatocyte
growth factor (HGF) (also known as scatter factor, SF), and nerve
growth factor (NGF), IL-8, etc.
[0035] As used herein, "vascular endothelial growth factor", or
"VEGF", refers to a mammalian growth factor derived originally from
bovine pituitary follicular cells having the amino acid sequence
disclosed in Castor, C. W., et al., (1991) Methods in Enzymol.
198:391-405, together with functional derivatives thereof having
the qualitative biological activity of a corresponding native VEGF,
including, but not limited to, the human VEGF amino acid sequence
as reported in Houck et al., (1991) Mol. Endocrin. 5:1806-1814. See
also, Leung et al. (1989) Science, 246:1306, and, Robinson &
Stringer, (2001) Journal of Cell Science, 144(5):853-865, U.S. Pat.
No. 5,332,671. The predominant form of VEGF is a 165 amino acid
homodimer having sixteen cysteine residues that form 7
intramolecular disulfide bonds and two intermolecular disulfide
bonds. Alternative splicing has been implicated in the formation of
multiple human VEGF polypeptides consisting of 121, 145, 165, 189
and 206 amino acids, however the VEGF.sub.121 variant lacks the
heparin binding domain of the other variants and therefore does not
fall within the definition of heparin binding protein set forth
herein. All isoforms of VEGF share a common amino-terminal domain,
but differ in the length of the carboxyl-terminal portion of the
molecule. The preferred active form of VEGF, VEGF.sub.165, has
disulfide bonds between amino acid residues Cys26-Cys68; Cys57-Cys
104; Cys61-Cys102; Cys117-Cys135; Cys 120-Cys 137; Cys 139-Cys;
158; Cys146-Cys160 in each monomer. See FIG. 11. See also, e.g.,
Keck et al., (1997) Archives of Biochemistry and Biophysics
344(1):103-113. The VEGF.sub.165 molecule is composed of two
domains: an amino-terminal receptor-binding domain (amino acids
1-110 disulfide linked homodimer) and a carboxyl-terminal
heparin-binding domain (residues 111-165). See, e.g., Keyt et al.,
(1996) J. Biol. Chem., 271(13):7788-7795. In certain embodiments of
the invention, the VEGF.sub.165 isolated and purified is not
glycosylated at residue 75 (Asn). See, e.g., Yang et al., (1998)
Journal of Pharm. & Experimental Therapeutics, 284:103-110. In
certain embodiments of the invention, the VEGF.sub.165 isolated and
purified is substantially undeamidated at residue Asn10. In certain
embodiments of the invention, the VEGF.sub.165 isolated and
purified is a mixture of deamidated (at residue Asn10) and
undeamidated protein, typically with majority of the protein being
undeamidated. Since VEGF.sub.165 is a homodimer, deamination can
occur on one or both polypeptide chains.
[0036] As used herein "properly folded" or "biologically active"
VEGF or other HBP and the like refers to a molecule with a
biologically active conformation. The skilled artisan will
recognize that misfolded and disulfide scrambled intermediates may
have biological activity. In such a case the properly folded or
biologically active VEGF or HBP corresponds to the native folding
pattern of the VEGF (described above) or other HBP. For example,
properly folded VEGF has the above noted disulfide pairs, in
addition to two intermolecular disulfide bonds in the dimeric
molecule however other intermediates may be produced by bacterial
cell culture (FIGS. 1 and 3A-3D). For properly folded VEGF the two
intermolecular disulfide bonds occur between the same residues,
Cys51 and Cys60, of each monomer. See, e.g., WO98/16551 patent.
Biological activities of VEGF include, but are not limited to,
e.g., promoting vascular permeability, promoting growth of vascular
endothelial cells, binding to a VEGF receptor, binding and
signaling through a VEGF receptor (see, e.g., Keyt et al., (1996)
Journal of Biological Chemistry, 271(10):5638-5646), inducing
angiogenesis, etc.
[0037] The terms "purified" or "pure HBP" and the like refer to a
material free from substances which normally accompany it as found
in its recombinant production and especially in prokaryotic or
bacterial cell culture. Thus the terms refer to a recombinant HBP
which is free of contaminanting DNA, host cell proteins or other
molecules associated with its in situ environment. The terms refer
to a degree of purity that is at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95% or
at least about 98% or more.
[0038] The terms "inclusion bodies" or "refractile bodies" refer to
dense intracellular masses of aggregated polypeptide of interest,
which constitute a significant portion of the total cell protein,
including all cell components. In some cases, but not all cases,
these aggregates of polypeptide may be recognized as bright spots
visible within the enclosure of the cells under a phase-contrast
microscope at magnifications down to 1,000 fold.
[0039] As used herein, the term "misfolded" protein refers to
precipitated or aggregated polypeptides that are contained within
refractile bodies. As used herein, "insoluble" or "misfolded" VEGF
or other HBP refers to precipitated or aggregated VEGF that is
contained within the periplasm or intracellular space of
prokaryotic host cells, or is otherwise prokaryotic host cell
associated, and assumes a biologically inactive conformation with
mismatched or unformed disulfide bonds. The insoluble HBP is
generally, but need not be, contained in refractile bodies, i.e.,
it may or may not be visible under a phase contrast microscope.
[0040] As used herein, "chaotropic agent" refers to a compound
that, in a suitable concentration in aqueous solution, is capable
of changing the spatial configuration or conformation of
polypeptides through alterations at the surface thereof so as to
render the polypeptide soluble in the aqueous medium. The
alterations may occur by changing, e.g., the state of hydration,
the solvent environment, or the solvent-surface interaction. The
concentration of chaotropic agent will directly affect its strength
and effectiveness. A strongly denaturing chaotropic solution
contains a chaotropic agent in large concentrations which, in
solution, will effectively unfold a polypeptide present in the
solution effectively eliminating the proteins secondary structure.
The unfolding will be relatively extensive, but reversible. A
moderately denaturing chaotropic solution contains a chaotropic
agent which, in sufficient concentrations in solution, permits
partial folding of a polypeptide from whatever contorted
conformation the polypeptide has assumed through intermediates
soluble in the solution, into the spatial conformation in which it
finds itself when operating in its active form under endogenous or
homologous physiological conditions. Examples of chaotropic agents
include guanidine hydrochloride, urea, and hydroxides such as
sodium or potassium hydroxide. Chaotropic agents include a
combination of these reagents, such as a mixture of a hydroxide
with urea or guanidine hydrochloride.
[0041] As used herein, "reducing agent" refers to a compound that,
in a suitable concentration in aqueous solution, maintains free
sulfhydryl groups so that the intra- or intermolecular disulfide
bonds are chemically disrupted. Representative examples of suitable
reducing agents include dithiothreitol (DTT), dithioerythritol
(DTE), beta-mercaptoethanol (BME), cysteine, cysteamine,
thioglycolate, glutathione, Tris[2-carboxyethyl]phosphine (TCEP),
and sodium borohydride.
[0042] As used herein, "buffered solution" refers to a solution
which resists changes in pH by the action of its acid-base
conjugate components.
[0043] The "bacteria" for purposes herein include eubacteria and
archaebacteria. In certain embodiments of the invention,
eubacteria, including gram-positive and gram-negative bacteria, are
used in the methods and processes described herein. In one
embodiment of the invention, gram-negative bacteria are used, e.g.,
Enterobacteriaceae. Examples of bacteria belonging to
Enterobacteriaceae include Escherichia, Enterobacter, Erwinia,
Klebsiella, Proteus, Salmonella, Serratia, and Shigella. Other
types of suitable bacteria include Azotobacter, Pseudomonas,
Rhizobia, Vitreoscilla, and Paracoccus. In one embodiment of the
invention, E. coli is used. Suitable E. coli hosts include E. coli
W3110 (ATCC 27,325), E. coli 294 (ATCC 31,446), E. coli B, and E.
coli X1776 (ATCC 31,537). These examples are illustrative rather
than limiting, and W3110 is one example. Mutant cells of any of the
above-mentioned bacteria may also be employed. It is, of course,
necessary to select the appropriate bacteria taking into
consideration replicability of the replicon in the cells of a
bacterium. For example, E. coli, Serratia, or Salmonella species
can be suitably used as the host when well-known plasmids such as
pBR322, pBR325, pACYC177, or pKN410 are used to supply the
replicon. See further below regarding examples of suitable
bacterial host cells.
[0044] As used herein, the expressions "cell," "cell line,"
"strain," and "cell culture" are used interchangeably and all such
designations include progeny. Thus, the words "transformants" and
"transformed cells" include the primary subject cell and cultures
derived therefrom without regard for the number of transfers. It is
also understood that all progeny may not be precisely identical in
DNA content, due to deliberate or inadvertent mutations. Mutant
progeny that have the same function or biological activity as
screened for in the originally transformed cell are included. Where
distinct designations are intended, it will be clear from the
context.
[0045] As used herein, "polypeptide" refers generally to peptides
and proteins from any cell source having more than about ten amino
acids. "Heterologous" polypeptides are those polypeptides foreign
to the host cell being utilized, such as a human protein produced
by E. coli. While the heterologous polypeptide may be prokaryotic
or eukaryotic, preferably it is eukaryotic, more preferably
mammalian, and most preferably human. In certain embodiments of the
invention, it is a recombinantly produced, or recombinant
polypeptide.
Heparin Binding Proteins
Isolating Heparin Binding Protein
[0046] Insoluble, misfolded heparin binding protein (HBP) is
isolated from prokaryotic host cells expressing the protein by any
of a number of art standard techniques. For example, the insoluble
HBP is isolated in a suitable isolation buffer by exposing the
cells to a buffer of suitable ionic strength to solubilize most
host proteins, but in which the subject protein is substantially
insoluble, or disrupting the cells so as to release the inclusion
bodies or the protein form the periplasmic or intracellular space
and make them available for recovery by, for example,
centrifugation. This technique is well known and is described in,
for example, U.S. Pat. No. 4,511,503. Kleid et al., disclose
purification of refractile bodies by homogenization followed by
Centrifugation (Kleid et al., (1984) in Developments in Industrial
Microbiology, (Society for Industrial Microbiology, Arlington, Va.)
25:217-235). See also, e.g., Fischer et al., (1993) Biotechnology
and Bioengineering 41:3-13.
[0047] U.S. Pat. No. 5,410,026 describes a typical method for
recovering protein from inclusion bodies and is summarized as
follows. The prokaryotic cells are suspended in a suitable buffer.
Typically the buffer consists of a buffering agent suitable for
buffering at between pH 5 to 9, or about 6 to 8 and a salt. Any
suitable salt, including NaCl, is useful to maintain a sufficient
ionic strength in the buffered solution. Typically an ionic
strength of about 0.01 to 2 M, or 0.1 to 0.2 M is employed. The
cells, while suspended in this buffer, are disrupted or lysed using
techniques commonly employed such as, for example, mechanical
methods, e.g., Homogenizer (Manton-Gaulin press, Microfluidizer, or
Niro-Soavi), a French press, a bead mill, or a sonic oscillator, or
by chemical or enzymatic methods.
[0048] Examples of chemical or enzymatic methods of cell disruption
include spheroplasting, which entails the use of lysozyme to lyse
the bacterial wall (H. Neu et al., (1964) Biochem. Biophys. Res.
Comm., 17:215), and osmotic shock, which involves treatment of
viable cells with a solution of high tonicity and with a cold-water
wash of low tonicity to release the polypeptides (H. Neu et al.,
1965 J. Biol. Chem., 240(9):3685-3692). Sonication is generally
used for disruption of bacteria contained in analytical scale
volumes of fermentation broth. At larger scales high pressure
homogenization is typically used.
[0049] After the cells are disrupted, the suspension is typically
centrifuged at low speed, generally around 500 to 15,000.times.g,
e.g., in one embodiment of the invention about 12,000.times.g is
used, in a standard centrifuge for a time sufficient to pellet
substantially all of the insoluble protein. Such times can be
simply determined and depend on the volume being centrifuged as
well as the centrifuge design. Typically about 10 minutes to 0.5
hours is sufficient to pellet the insoluble protein. In one
embodiment the suspension is centrifuged at 12,000.times.g for 10
minutes.
[0050] The resulting pellet contains substantially all of the
insoluble protein fraction. If the cell disruption process is not
complete, the pellet may also contain intact cells or broken cell
fragments. Completeness of cell disruption can be assayed by
resuspending the pellet in a small amount of the same buffer
solution and examining the suspension with a phase contrast
microscope. The presence of broken cell fragments or whole cells
indicates that further sonication or other means of disruption is
necessary to remove the fragments or cells and the associated
non-refractile polypeptides. After such further disruption, if
required, the suspension is again centrifuged and the pellet
recovered, resuspended, and reexamined. The process is repeated
until visual examination reveals the absence of broken cell
fragments in the pelleted material or until further treatment fails
to reduce the size of the resulting pellet.
[0051] The above process can be employed whether the insoluble
protein is intracellular or in the periplasmic space. In one
embodiment of the invention, the conditions given herein for
isolating heparin binding protein are directed to inclusion bodies
precipitated in the periplasmic space or intracellular space and
relate particularly to VEGF. However, the processes and procedures
are thought to be applicable to heparin binding proteins in general
with minor modifications as noted throughout the following text. In
certain embodiments of the invention, the processes and procedures
are applicable to manufacturing or industrial scale production,
refolding, and purification of the HBP.
Refolding Heparin Binding Proteins
[0052] The isolated insoluble, misfolded heparin binding protein is
incubated in a first buffered solution containing an amount of a
chaotropic agent and a reducing agent sufficient to substantially
solubilize the heparin binding protein. This incubation takes place
under conditions of concentration, incubation time, and incubation
temperature that will allow solubilization of some or substantially
all the heparin binding protein, and for unfolding to occur.
[0053] Measurement of the degree of solubilization in the buffered
solution can be simply determined and is suitably carried out, for
example, by turbidity determination, by analyzing fractionation
between the supernatant and pellet after centrifugation, on reduced
SDS-PAGE gels, by protein assay (e.g., the Bradford reagent protein
assay (e.g., Pierce, Bio-Rad etc.)), or by HPLC.
[0054] The first buffered solution comprises a buffering agent
suitable for maintaining the pH range of the buffer at least about
7.0, with the typical range being 7.5-10.5. In one embodiment, the
pH for VEGF is pH 8.0. Examples of suitable buffers that will
provide a pH within this latter range include TRIS--HCl
(Tris[hydroxymethyl]aminomethane),
HEPPS(N-[2-Hydroxyethyl]piperazine-N'-[3-propane-sulfonic acid]),
HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid])),
CAPS 0 (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP
(2-Amino-2-methyl-1-propanol), CAPS
(3-[Cyclohexylamino]-1-propanesulfonic acid), CHES
(2-[N-Cyclohexylamino]ethanesulfonic acid), glycine, and sodium
acetate. In one embodiment of the invention, the buffer herein is
HEPPS at about pH 8.0. In a further embodiment, the buffers, e.g.,
such as HEPPS, are sulfated.
[0055] Chaotropic agents suitable for practicing this invention
include, e.g., urea and salts of guanidine or thiocyanate, e.g.,
urea, guanidine hydrochloride, sodium thiocyanate, etc. The amount
of chaotropic agent necessary to be present in the buffer is an
amount sufficient to unfold the HBP in solution. In certain
embodiments of the invention, a chaotrope is present at about
between about 4 and 10 molar. In one embodiment of the invention,
the chaotropic agent is urea at about 5-8 M, or at about 7 M. In
another example, the chaotropic agent is guanidine hydrochloride at
about 6-8 M.
[0056] Examples of suitable reducing agents include, but are not
limited to, dithiothreitol (DTT), dithioerythritol (DTE),
3-mercaptoethonol (BME), cysteine, DTE, etc. The amount of reducing
agent to be present in the buffer will depend mainly on the type of
reducing agent and chaotropic agent, the type and pH of the buffer
employed, the amount of oxygen entrained in or introduced to the
solution, and the concentration of the protein in the buffer. For
example, with 0.5-1.5 mg/ml protein in a buffered solution at pH
7.0-10.0 containing 4-8 M urea, and reducing agent is, e.g., DTT
with a concentration at about 1-15 mM, or BME with a concentration
at about 0.2-2 mM, or cysteine with a concentration at about 2-10
mM. In one embodiment, the reducing agent is DTT at about 0.5 to
about 4 mM, or 2-4 mM. FIG. 9 illustrates the effect of urea and
DTT on the extraction of VEGF. Peak 3 VEGF refers to properly
folded biologically active VEGF. In one embodiment, the reducing
agent is DTT at about 10 mM. A single reducing agent or a
combination of reducing agents can be used in a buffer herein.
[0057] The concentration of the protein in the buffered solution
must be such that the protein will be substantially solubilized as
determined by optical density. The exact amount to employ will
depend on, e.g., the concentrations and types of other ingredients
in the buffered solution, particularly the protein concentration,
reducing agent, and the pH of the buffer. In one embodiment of the
invention, the concentration of heparin binding protein is in the
range of 0.5-5.5 mg per ml, or 1.5-5.0 mg/ml. The solubilization is
typically carried out at about 0-45.degree. C., or about
20-40.degree. C., or about 23-37.degree. C., or about 25-37.degree.
C., or about 25.degree. C. for at least about one to 24 hours. In
one embodiment, the solubilization is carried out for at least
about two hours at room temperature. Typically, the temperature is
not apparently affected by salt, reducing agent and chaotropic
agent levels.
[0058] After the polypeptide is solubilized, it is placed or
diluted into a second buffered solution containing the chaotropic
agent and a sulfated polyanionic agent as described above however
at a concentration of chaotropic agent which allows for refolding
of the heparin binding protein.
[0059] The conditions of this second incubation of the soluble,
misfolded protein will generally be such that some or substantial
or complete refolding of the protein will take place. The exact
conditions will depend on, for example, the pH of the buffer and
the types and concentrations of sulfated polyanionic agents and of
chaotropic and reducing agents, if any, present. The incubation
temperature is generally about 0-40.degree. C., or 10-40.degree. C.
and the incubation will generally be carried out for at least about
1 hour to effect refolding. In certain embodiments, the reaction is
carried out, e.g., at about 15-37.degree. C., or at 20-30.degree.
C., for at least about 6 hours, for at least about 10 hours, or
between about 10 and 48 hours, or between about 15 and 20 hours, or
between 6 and 20 hours, or between 12 and 24 hours.
[0060] The degree of refolding is suitably determined by
radio-immuno assay (RIA) titer of the HPB or by high performance
liquid chromatography (HPLC) analysis using e.g., a POROS HE2/M
column (PerSeptive BioResearch Products) or other appropriate
heparin affinity column. Increasing RIA titer or correctly folded
HBP peak size directly correlates with increasing amounts of
correctly folded, biologically active HPB present in the buffer.
The incubation is carried out to maximize the ratio of correctly
folded HPB to misfolded HPB recovered, as determined by RIA or
HPLC.
[0061] In one embodiment, the quality and quantity of
properly-folded VEGF is assessed using a heparin-binding assay.
Samples containing the diluted heparin binding protein are loaded
on a e.g., POROS HE2/M column (4.6.times.100 mm, PerSeptive
BioResearch Products, Cambridge, Mass.) or other suitable heparin
affinity column. For example, the heparin affinity column is
equilibrated in 10 mM sodium phosphate, pH 7 containing 0.15 M
sodium chloride. At a flow rate of 1 ml/min or 2 ml/min, the column
is eluted using a linear gradient from 0.15-2 M sodium chloride in,
10 mM sodium phosphate, pH 7 over 10 minutes. The eluant is
monitored at 280 nm. In one embodiment, the protein is recovered in
a single peak corresponding to the biologically active properly
refolded HBP. In one embodiment of the invention, an assay for
determining properly refolded HBP is RPHPLC. Disulfide linkages can
optionally be confirmed by peptide map. Circular dichroism can also
be used in for determining 2 & 3D structure/folding.
[0062] The buffer for the second buffered solution can be any of
those listed above for the first buffered solution, e.g., HEPPS pH.
8.0, e.g., at a concentration of about 50 mM for refolding VEGF.
The polypeptide may be diluted with the refolding buffer, e.g., at
least five fold, or at least about ten fold, about 20 fold, or
about 40 fold. Alternatively, the polypeptide may be dialyzed
against the refolding buffer.
[0063] The second buffered solution contains a chaotropic agent at
a concentration such that refolding of the HPB occurs. Generally a
chaotrope is present at about between about 0.5 and 2 molar. In one
embodiment of the invention, the chaotropic agent herein is urea at
about 0.5-2 M, 0.5-2 M, or at about 1 M. In one embodiment, the
chaotropic agent is urea at about 1.3 M concentration. In another
embodiment of the invention, the chaotropic agent is guanidine
hydrochloride at about 1 M. FIG. 10 illustrates the effect of urea
and reducing agent DTT on the refolding of VEGF. Peak 3 VEGF refers
to properly folded biologically active VEGF.
[0064] As noted, the solution optionally also contains a reducing
agent. The reducing agent is suitably selected from those described
above for the solubilizing step in the concentration range of about
0.5 to about 10 mM for cysteine, 0.1-1.0 mM for DTT, and/or less
than about 0.2 mM for BME. In one embodiment of the invention, the
reducing agent is DTT at about 0.5-2 mM. In one embodiment of the
invention, the reducing agent is DTT at about 0.5 mM. Examples of
suitable reducing agents include, but are not limited to, e.g.,
dithiothreitol (DTT), f3-mercaptoethonol (BME), cysteine, DTE, etc.
Whereas DTT and BME can be used in connection with the procedures
provided herein for heparin binding proteins in general, a
combination of cysteine at about 0.1 to about 10 mM and about 0.1
to about 1.0 mM DTT as described herein is an example for the
recovery of VEGF.
[0065] The refolding step includes a sulfated polyanionic agent at
a concentration sufficient to achieve complete refolding of the
solubilized protein. Examples of suitable polyanionic agents are
described herein above, e.g., a sulfate derivative of a
polysaccharide as noted above with sulfated polyanionic agents such
as heparin sulfate, dextran sulfate, heparin sulfate, and
chrondroitin sulfate as well as sulfated heparin fragments. For
heparin sulfates used in the context of the invention, the
molecular weight are generally between about 3,000 and 10,000
daltons, or between about 3,000 and 6,000 dalton.
[0066] In one embodiment of the invention, dextran sulfate is
employed in the context of the invention. The molecular weight of
the sulfated polyanionic or other agent such as dextran sulfate
employed in the invention depends upon the size of the particular
heparin binding protein being recovered. Generally, dextran sulfate
between about 3,000 and 10,000 daltons is employed. In one
embodiment of the invention, dextran sulfate between about 5,000
daltons and 10,000 daltons is used, e.g., for the recovery of VEGF.
In another embodiment, a dextran sulfate between about 5,000 and
8,000 daltons is used for recovery of the HBP. FIG. 3A-3D shows the
recovery of VEGF with various concentrations of and molecular
weights of dextran sulfate (FIGS. 3A-C) and heparin (FIG. 3D) as
analyzed by heparin affinity chromatography. Peak 3 corresponds to
properly folded VEGF.
[0067] The concentration of the polyanionic compound employed
depends upon the protein being recovered and its concentration and
conditions such as temperature and pH of the refolding buffer.
Typical concentrations are between about 50 and 500 mM for sodium
sulfate, between about 10 and 200 .mu.g/ml for low molecular weight
heparins such as 6,000 dalton heparin (Sigma Chemical Co.), between
about 10 and 200 .mu.g/ml for high molecular weight heparins such
as porcine heparin I-A (Sigma Chemical Co.) and between about 10
and 400 .mu.g/ml, or between about 10 and 200 .mu.g/ml for dextran
sulfates.
[0068] The refolding buffer can optionally contain additional
agents such as any of a variety of non-ionic detergents such as
TRITON.TM. X-100, NONIDET.TM. P-40, the TWEEN.TM. series and the
BRIJ.TM. series. The non-ionic detergent is present at about
between 0.01% and 1.0%. In one example, the concentrations for non
ionic detergent are between about 0.025% and 0.05%, or about
0.05%.
[0069] Optionally, positively charged amino acids, e.g., arginine
(e.g., L-arginine/HCl), lysine, etc., can be present in the
refolding buffer. In certain embodiments of the invention, the
concentration of arginine is e.g., about 0-1000 mM, or about 25 to
750 mM, or about 50-500 mM, or about 50-250 mM, or about 100 mM
final concentration, etc. In certain embodiments of the invention,
the protein is in a buffer solution at pH 7.0-9.0 containing, 0.5-3
M urea, 0-30 mg/L dextran sulfate, 0-0.2% Triton X-100, 2-15 mM
cysteine, 0.1-1 mM DTT and 0-750 mM arginine, final concentration.
In one embodiment, 50 mM HEPPS is used. In one embodiment, the
final concentration of the refolding buffer solution is 1 M urea,
50 mM HEPPS, 15 mg/L dextran sulfate, 0.05% Triton X-100, 7.5 mM
cysteine, 100 mM arginine, pH 8.0. In one embodiment, the final
concentration of the refolding buffer solution is 1.3 M urea, 50 mM
HEPPS, 15 mg/L dextran sulfate, 0.05% Triton X-100, 7.5 mM
cysteine, 0.5 mM DTT, 100 mM arginine, pH 8.0.
Recovery and Purification of Heparin Binding Proteins
[0070] Although recovery and purification of the heparin binding
protein from the culture media can employ various methods and known
procedures for the separation of such proteins such as, for
example, salt and solvent fractionation, adsorption with colloidal
materials, gel filtration, ion exchange chromatography, affinity
chromatography, immunoaffinity chromatography, electrophoresis and
high performance liquid chromatography (HPLC), an example of a four
step chromatographic procedure is described which comprises
contacting said refolded heparin binding protein with a
hydroxyapatite chromatographic support; a first hydrophobic
interaction chromatographic support, a cationic chromatographic
support and a second hydrophobic interaction chromatographic
support and selectively eluting the heparin binding protein from
each support. Alternatively, another chromatographic procedure is
described which comprises contacting said refolded heparin binding
protein with a cation exchange support; a hydrophobic interaction
chromatographic support, and an ion exchange chromatographic
support and selectively eluting the heparin binding protein from
each support. It is contemplated that the steps of either procedure
can be performed in any order. In one embodiment of the invention,
the steps are performed sequentially.
[0071] A suitable first step in the further recovery and
purification of the heparin binding protein characteristically
provides for the concentration of the heparin binding protein and a
reduction in sample volume. For example, the second incubation step
described above, may result in a large increase in the volume of
the recovered heparin binding protein and concommitant dilution of
the protein in the refolding buffer. Suitable first chromatographic
supports provide a reduction in volume of recovered heparin binding
protein and may advantageously provide some purification of the
protein from unwanted contaminating proteins. Suitable first
chromatographic steps include chromatographic supports which can be
eluted and loaded directly onto a first hydrophobic interaction
chromatographic support. For example, chromatographic supports from
which the heparin binding protein can be eluted in a high salt
concentration suitable for loading a hydrophobic interaction
chromatographic support are used.
[0072] Exemplary first chromatographic supports include, but are
not limited to, hydroxyapatite chromatographic supports, e.g., CHT
ceramic type I and type II (formally known as MacroPrep ceramic),
Bio-Gel HT, Bio-Gel HTP, Biorad, Hercules, Calif., etc.; metal
chelating chromatographic supports consisting of an inert resin of
immobilized metal ions such as copper, nickel, etc.; as well as
non-derivatized silica gels. In one embodiment of the invention,
the first chromatographic supports for the purification and
recovery of VEGF are hydroxyapatite chromatographic supports. In
another embodiment of the invention, the first chromatographic
supports for the purification and recovery of VEGF are cation
exchange supports, e.g., described below in more detail.
[0073] Elution from the first chromatographic support is
accomplished according to art standard practices. Suitable elution
conditions and buffers will facilitate the loading of the eluted
HPB directly onto the first hydrophobic interaction chromatographic
support as described below.
[0074] Hydrophobic interaction chromatography is well known in the
art and is predicated on the interaction of hydrophobic portions of
the molecule interacting with hydrophobic ligands attached to
"chromatographic supports." A hydrophobic ligand coupled to a
matrix is variously referred to as an HIC chromatographic support,
HIC gel, or HIC column and the like. It is further appreciated that
the strength of the interaction between the protein and the HIC
column is not only a function of the proportion of non-polar to
polar surfaces on the protein but of the distribution of the
non-polar surfaces as well.
[0075] A number of matrices may be employed in the preparation of
HIC columns. The most extensively used is agarose, although silica
and organic polymer resins may be used. Useful hydrophobic ligands
include but are not limited to alkyl groups having from about 2 to
about 1.0 carbon atoms, such as butyl, propyl, or octyl, or aryl
groups such as phenyl. Conventional HIC supports for gels and
columns may be obtained commercially from suppliers such as GE
Healthcare, Uppsala, Sweden under the product names
butyl-SEPHAROSE.TM., phenyl-SEPHAROSE.TM. CL-4B, octyl
SEPHAROSE.TM. FF and phenyl SEPHAROSE.TM. FF and Tosoh Corporation,
Tokyo, Japan under the product names TOYOPEARL.TM. butyl 650M
(Fractogel TSK Butyl-650) or TSK-GEL phenyl 5PW. In one embodiment,
the purification and recovery of VEGF is a first HIC
chromatographic support that is butyl-agarose and a second
hydrophobic chromatographic support that is a phenyl agarose. In
another embodiment, the first HIC chromatographic support is phenyl
agarose.
[0076] Ligand density is an important parameter in that it
influences not only the strength of the interaction of the protein
but the capacity of the column as well. The ligand density of the
commercially available phenyl or octyl phenyl gels is on the order
of 5-40 .mu.mol/ml gel bed. Gel capacity is a function of the
particular protein in question as well as pH, temperature and salt
concentration but generally can be expected to fall in the range of
3-20 mg/ml gel.
[0077] The choice of particular gel can be determined by the
skilled artisan. In general the strength of the interaction of the
protein and the HIC ligand increases with the chain length of the
alkyl ligands but ligands having from about 4 to about 8 carbon
atoms are suitable for most separations. A phenyl group has about
the same hydrophobicity as a pentyl group, although the selectivity
can be different owing to the possibility of pi-pi interaction with
aromatic groups of the protein.
[0078] Adsorption of the protein to a HIC column is favored by high
salt concentration, but the actual concentration can vary over a
wide range depending of the nature of the protein and the
particular HIC ligand chosen. In general salt concentration between
about 1 and 4 M are useful.
[0079] Elution from an HIC support, whether stepwise or in the form
of a gradient, can be accomplished in a variety of ways such as a)
by changing the salt concentration, b) by changing the polarity of
the solvent or c) by adding detergents. By decreasing salt
concentrations adsorbed proteins are eluted in order of increasing
hydrophobicity. Changes in polarity may be effected by additions of
solvents such as ethylene glycol or isopropanol thereby decreasing
the strength of the hydrophobic interactions. Detergents function
as displacers of proteins and have been used primarily in
connection with the purification of membrane proteins.
[0080] Various anionic constituents may be attached to matrices in
order to form cationic supports for chromatography. Anionic
constituents include carboxymethyl, sulfethyl groups, sulfopropyl
groups, phosphate and sulfonate (S). Cellulosic ion exchange resins
such as SE52 SE53, SE92, CM32, CM52, CM92, P11, DE23, DE32, DE52,
EXPRESS ION.TM. S and EXPRESS ION.TM. C are available from Whatman
LTD, Maidstone Kent U.K. SEPHADEX.TM. and SEPHAROSE.TM. based and
cross linked ion exchangers are also known under the product names
CM SEPHADEX.TM. C-25, CM SEPHADEX.TM. C-50 and SP SEPHADEX.TM. C-25
SP SEPHADEX.TM. C-50 and SP-SEPHAROSE.TM. High Performance,
SP-SEPHAROSE.TM. Fast Flow, SP-SEPHAROSE XL, CM-SEPHAROSE.TM. Fast
Flow, and CM-SEPHAROSE.TM., CL-6B, all available from GE
Healthcare. Examples of ion exchangers for the practice of the
invention include but are not limited to, e.g., ion exchangers
under the product names MACROPREP.TM. such as for example
MACROPREP.TM. S support, MACROPREP.TM. High S support and
MACROPREP.TM. CM support from BioRad, Hercules, Calif.
[0081] Elution from cationic chromatographic supports is generally
accomplished by increasing salt concentrations. Because the elution
from ionic columns involves addition of salt and because, as
mentioned, HIC is enhanced in salt concentration the introduction
of HIC step following the ionic step or other salt step is
optionally used. In one embodiment of the invention, a cationic
exchange chromatographic step precede the HIC step.
[0082] Examples of methods for purifying VEGF is described herein
below, e.g., see Example V and VI. After refolding, insoluble
material in the pool is removed by depth filtration. The clarified
pool is then loaded on to a ceramic hydroxyapatite (Bio Rad,
Hercules, Calif.) equilibrated in 5-mM HEPPS/0.05% TRITON.TM.
X100/pH 8. The non-binding protein is removed by washing with
equilibration buffer and the VEGF eluted using an isocratic step of
50 mM HEPPS/0.05% TRITON.TM. X100/0.15 M sodium phosphate/pH 8. The
pool of VEGF is loaded onto a column of Butyl SEPHAROSE.TM. Fast
Flow (GE Healthcare, Uppsala, Sweden) equilibrated in 50 mM
HEPPS/0.05% TRITON.TM. X100/0.15 M sodium phosphate/pH 8. The
column is washed with equilibration buffer and the VEGF collected
in the column effluent. The Butyl SEPHAROSE.TM. pool is loaded onto
a column of Macro Prep High S (BioRad, Hercules, Calif.) that is
equilibrated in 50 mM HEPES/pH 8. After washing the effluent
absorbance at 280 nm to baseline, the column is washed with two
column volumes of 50 mM HEPES/0.25 M sodium chloride/pH 8. The VEGF
is eluted using a linear, 8-column-volume gradient from 0.25-0.75 M
sodium chloride in 50 mM HEPES/pH 8. Fractions are collected and
those which contained properly-folded VEGF, as determined by a
heparin-binding assay, are pooled.
[0083] The Macro Prep High S pool is conditioned with an equal
volume of 50 mM HEPES/0.8 M sodium citrate/pH 7.5. The conditioned
pool is then loaded on to a column of Phenyl 5PW TSK (Tosoh
Bioscience LLC, Montgomeryville, Pa.) that is equilibrated with 50
mM HEPES/0.4 M sodium citrate/pH 7.5. After washing non-binding
protein through the column with equilibration buffer, the VEGF is
eluted from the column using a 10-column-volume gradient from 0.4-0
M sodium citrate in 50 mM HEPES, pH 7.5. Fractions are assayed by
SDS-polyacrylamide gel electrophoresis and those containing VEGF of
sufficient purity pooled.
Expressing Heparin Binding Protein in Host Cells
[0084] In brief, expression vectors capable of autonomous
replication and protein expression relative to the host prokaryotic
cell genome are introduced into the host cell. Construction of
appropriate expression vectors is well known in the art including
the nucleotide sequences of the heparin binding proteins described
herein. See, e.g., Sambrook et al., Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor,
N.Y.) (2001); Ausubel et al., Short Protocols in Molecular Biology,
Current Protocols John Wiley and Sons (New Jersey) (2002); and,
Baneyx, (1999) Current Opinion in Biotechnology, 10:411-421.
Appropriate prokaryotic cell, including bacteria, expression
vectors are available commercially through, for example, the
American Type Culture Collection (ATCC), Rockville, Md. Methods for
the large scale growth of prokaryotic cells, and especially
bacterial cell culture are well known in the art and these methods
can be used in the context of the invention.
[0085] For example, prokaryotic host cells are transfected with
expression or cloning vectors encoding the heparin binding protein
of interest and cultured in conventional nutrient media modified as
appropriate for inducing promoters, selecting transformants, or
amplifying the genes encoding the desired sequences. The nucleic
acid encoding the polypeptide of interest is suitably RNA, cDNA, or
genomic DNA from any source, provided it encodes the polypeptide(s)
of interest. Methods are well known for selecting the appropriate
nucleic acid for expression of heterologous polypeptides (including
variants thereof) in microbial hosts. Nucleic acid molecules
encoding the polypeptide are prepared by a variety of methods known
in the art. For example, a DNA encoding VEGF is isolated and
sequenced, e.g., by using oligonucleotide probes that are capable
of binding specifically to the gene encoding VEGF.
[0086] The heterologous nucleic acid (e.g., cDNA or genomic DNA) is
suitably inserted into a replicable vector for expression in the
microorganism under the control of a suitable promoter. Many
vectors are available for this purpose, and selection of the
appropriate vector will depend mainly on the size of the nucleic
acid to be inserted into the vector and the particular host cell to
be transformed with the vector. Each vector contains various
components depending on the particular host cell with which it is
compatible. Depending on the particular type of host, the vector
components generally include, but are not limited to, one or more
of the following: a signal sequence, an origin of replication, one
or more marker genes, a promoter, and a transcription termination
sequence.
[0087] In general, plasmid vectors containing replicon and control
sequences that are derived from species compatible with the host
cell are used in connection with microbial hosts. The vector
ordinarily carries a replication site, as well as marking sequences
that are capable of providing phenotypic selection in transformed
cells. For example, E. coli is typically transformed using pBR322,
a plasmid derived from an E. coli species (see, e.g., Bolivar et
al., (1977) Gene, 2: 95). pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR322 plasmid, or other
bacterial plasmid or phage, also generally contains, or is modified
to contain, promoters that can be used by the host for expression
of the selectable marker genes.
[0088] (i) Signal Sequence
[0089] Polypeptides of the invention may be produced recombinantly
not only directly, but also as a fusion polypeptide with a
heterologous polypeptide, which is typically a signal sequence or
other polypeptide having a specific cleavage site at the N-terminus
of the mature protein or polypeptide. The heterologous signal
sequence selected typically is one that is recognized and processed
(i.e., cleaved by a signal peptidase) by the host cell. For
prokaryotic host cells that do not recognize and process the native
polypeptide signal sequence, the signal sequence is substituted by
a prokaryotic signal sequence selected, for example, from the group
of the alkaline is phosphatase, penicillinase, 1 pp, or heat-stable
enterotoxin II leaders.
[0090] (ii) Origin of Replication Component
[0091] Expression vectors contain a nucleic acid sequence that
enables the vector to replicate in one or more selected host cells.
Such sequences are well known for a variety of microbes. The origin
of replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria such as E. coli.
[0092] (iii) Selection Gene Component
[0093] Expression vectors generally contain a selection gene, also
termed a selectable marker. This gene encodes a protein necessary
for the survival or growth of transformed host cells grown in a
selective culture medium. Host cells not transformed with the
vector containing the selection gene will not survive in the
culture medium. This selectable marker is separate from the genetic
markers as utilized and defined by this invention. Typical
selection genes encode proteins that (a) confer resistance to
antibiotics or other toxins, e.g., ampicillin, neomycin,
methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies other than those caused by the presence of the genetic
marker(s), or (c) supply critical nutrients not available from
complex media, e.g., the gene encoding D-alanine racemase for
Bacilli.
[0094] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. In this case, those cells that are
successfully transformed with the nucleic acid of interest produce
a polypeptide conferring drug resistance and thus survive the
selection regimen. Examples of such dominant selection use the
drugs neomycin (Southern et al., (1982) J. Molec. Appl. Genet., 1:
327), mycophenolic acid (Mulligan et al., (1980) Science 209: 1422)
or hygromycin (Sugden et al., (1985) Mol. Cell. Biol., 5: 410-413).
The three examples given above employ bacterial genes under
eukaryotic control to convey resistance to the appropriate drug
G418 or neomycin (geneticin), xgpt (mycophenolic acid), or
hygromycin, respectively.
[0095] (iv) Promoter Component
[0096] The expression vector for producing the heparin binding
protein of interest contains a suitable promoter that is recognized
by the host organism and is operably linked to the nucleic acid
encoding the polypeptide of interest. Promoters suitable for use
with prokaryotic hosts is include the beta-lactamase and lactose
promoter systems (Chang et al., (1978) Nature, 275: 615; Goeddel et
al., (1979) Nature, 281: 544), the arabinose promoter system
(Guzman et al., (1992) J. Bacteriol., 174: 7716-7728), alkaline
phosphatase, a tryptophan (trp) promoter system (Goeddel, (1980)
Nucleic Acids Res., 8: 4057 and EP 36,776) and hybrid promoters
such as the tac promoter (deBoer et al., (1983) Proc. Natl. Acad.
Sci. USA, 80: 21-25). However, other known bacterial promoters are
suitable. Their nucleotide sequences have been published, thereby
enabling a skilled worker operably to ligate them to DNA encoding
the polypeptide of interest (Siebenlist et al, (1980) Cell, 20:
269) using linkers or adaptors to supply any required restriction
sites. See also, e.g., Sambrook et al., supra; and Ausubel et al.,
supra.
[0097] Promoters for use in bacterial systems also generally
contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding the polypeptide of interest. The promoter can be removed
from the bacterial source DNA by restriction enzyme digestion and
inserted into the vector containing the desired DNA.
[0098] (v) Construction and Analysis of Vectors
[0099] Construction of suitable vectors containing one or more of
the above-listed components employs standard ligation techniques.
Isolated plasmids or DNA fragments are cleaved, tailored, and
re-ligated in the form desired to generate the plasmids
required.
[0100] For analysis to confirm correct sequences in plasmids
constructed, the ligation mixtures are used to transform E. coli
K12 strain 294 (ATCC 31,446) or other strains, and successful
transformants are selected by ampicillin or tetracycline resistance
where appropriate. Plasmids from the transformants are prepared,
analyzed by restriction endonuclease digestion, and/or sequenced by
the method of Sanger et al., (1977) Proc. Natl. Acad. Sci. USA, 74:
5463-5467 or Messing et al., (1981) Nucleic Acids Res., 9: 309, or
by the method of Maxam et al., (1980) Methods in Enzymology, 65:
499. See also, e.g., Sambrook et al., supra; and Ausubel et al.,
supra.
[0101] The nucleic acid encoding the heparin binding protein of
interest is inserted into the host cells. Typically, this is
accomplished by transforming the host cells with the
above-described expression vectors and culturing in conventional
nutrient media modified as appropriate for is inducing the various
promoters.
Culturing the Host Cells
[0102] Suitable prokayotic cells for use to express the heparin
binding proteins of interest are well known in the art. Host cells
that express the recombinant protein abundantly in the form of
inclusion bodies or in the perplasmic or intracellular space are
typically used. Suitable prokaryotes include bacteria, e.g.,
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, E. coli, Bacilli such as B. subtilis, Pseudomonas species
such as P. aeruginosa, Salmonella typhimurium, or Serratia
marcescens. One example of an E. coli host is E. coli 294 (ATCC
31,446). Other strains such as E. coli B, E. coli X1776 (ATCC
31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These
examples are illustrative rather than limiting. Strain W3110 is a
typical host because it is a common host strain for recombinant DNA
product fermentations. In one aspect of the invention, the host
cell should secrete minimal amounts of proteolytic enzymes. For
example, strain W3110 may be modified to effect a genetic mutation
in the genes encoding proteins, with examples of such hosts
including E. coli W3110 strains 1A2, 27A7, 27B4, and 27C7 described
in U.S. Pat. No. 5,410,026 issued Apr. 25, 1995. For example, a
strain for the production of VEGF is E. coli stain W3110 having the
genotype tonA.DELTA. ptr3 phoA.DELTA.E15 .DELTA.(argF-lac)169
degP41 ilvg designated 49B3. In another example, a strain for the
production of VEGF is the E. coli strain (62A7) having the genotype
.DELTA.fhuA (.DELTA.tonA) ptr3, laI.sup.q, lacL8, .DELTA.ompT
.DELTA.(nmpC-fepE) .DELTA.degP ilvG.sup.+. See also, e.g., table
spanning pages 23-24 of WO2004/092393.
[0103] Prokaryotic cells used to produce the heparin binding
protein of interest are grown in media known in the art and
suitable for culture of the selected host cells, including the
media generally described by Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring
Harbor, N.Y.) (2001). Media that are suitable for bacteria include,
but are not limited to, AP5 medium, nutrient broth, Luria-Bertani
(LB) broth, Neidhardt's minimal medium, and C.R.A.P. minimal or
complete medium, plus necessary nutrient supplements. In certain
embodiments, the media also contains a selection agent, chosen
based on the construction of the expression vector, to selectively
permit growth of prokaryotic cells containing the expression
vector. For example, ampicillin is added to media for growth of
cells expressing ampicillin resistant gene. Any necessary
supplements besides carbon, nitrogen, and inorganic phosphate
sources may also be included at appropriate concentrations
introduced alone or as a mixture with another supplement or medium
such as a complex nitrogen source. Optionally the culture medium
may contain one or more reducing agents selected from the group
consisting of glutathione, cysteine, cystamine, thioglycollate,
dithioerythritol, and dithiothreitol.
[0104] Examples of suitable media are given in U.S. Pat. Nos.
5,304,472 and 5,342,763. C.R.A.P. phosphate-limiting media consists
of 3.57 g (NH.sub.4).sub.2(SO.sub.4), 0.71 g Na citrate-2H.sub.2O,
1.07 g KCl, 5.36 g Yeast Extract (certified), 5.36 g
HycaseSF.TM.-Sheffield, adjusted pH with KOH to 7.3, qs volume
adjusted to to 872 ml with deionized H.sub.2O and autoclaved;
cooled to 55.degree. C. and supplemented with 110 ml 1 M MOPS pH
7.3, 11 ml 50% glucose, 7 ml 1M MgSO.sub.4). Carbenicillin may then
be added to the induction culture at a concentration of 50
.mu.g/ml.
[0105] The prokaryotic host cells are cultured at suitable
temperatures. For E. coli growth, for example, the temperature
ranges from, e.g., about 20.degree. C. to about 39.degree. C., or
from about 25.degree. C. to about 37.degree. C., or at about
30.degree. C.
[0106] Where the alkaline phosphatase promoter is employed, E. coli
cells used to produce the polypeptide of interest of this invention
are cultured in suitable media in which the alkaline phosphatase
promoter can be partially or completely induced as described
generally, e.g., in Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring
Harbor, N.Y.) (2001). The culturing need never take place in the
absence of inorganic phosphate or at phosphate starvation levels.
At first, the medium contains inorganic phosphate in an amount
above the level of induction of protein synthesis and sufficient
for the growth of the bacterium. As the cells grow and utilize
phosphate, they decrease the level of phosphate in the medium,
thereby causing induction of synthesis of the polypeptide.
[0107] If the promoter is an inducible promoter, for induction to
occur, typically the cells are cultured until a certain optical
density is achieved, e.g., a A.sub.550 of about 200 using a high
cell density process, at which point induction is initiated (e.g.,
by addition of an inducer, by depletion of a medium component,
etc.), to induce expression of the gene encoding the polypeptide of
interest.
Any necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art,
introduced alone or as a mixture with another supplement or medium
such as a complex nitrogen source. The pH of the medium may be any
pH from about 5-9, depending mainly on the host organism. For E.
coli, the pH is, e.g., from about 6.8 to about 7.4, or about
7.0.
Formulations of Heparin Binding Proteins
[0108] The polypeptide recovered, e.g., using the methods described
herein, may be formulated in a pharmaceutically acceptable carrier
and is used for various diagnostic, therapeutic, or other uses
known for such molecules. For example, VEGF described herein can be
used in immunoassays, such as enzyme immunoassays. Therapeutic uses
for the heparin binding proteins obtained using the methods
described herein are also contemplated. For example, a growth
factor or hormone, e.g., VEGF, can be used to enhance growth as
desired. For example, VEGF can be used to promote wound healing of,
e.g., an acute wound (e.g., burn, surgical wound, normal wound,
etc.) or a chronic wound (e.g., diabetic ulcer, pressure ulcer, a
decubitus ulcer, a venous ulcer, etc.), to promote hair growth, to
promote tissue growth and repair (e.g., bone, liver, etc.),
etc.
[0109] Therapeutic formulations of heparin binding proteins are
prepared for storage by mixing a molecule, e.g., a polypeptide,
having the desired degree of purity with optional pharmaceutically
acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences 18th edition, Gennaro, A. Ed. (1995)), in
the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0110] In certain embodiments, the formulations to be used for in
vivo administration are sterile. This is readily accomplished by
filtration through sterile filtration membranes. HBP can be stored
in lyophilized form or as an aqueous solution or gel form. The pH
of the HBP preparations can be, e.g., from about 5 to 8, although
higher or lower pH values may also be appropriate in certain
instances. It will be understood that use of certain of the
excipients, carriers, or stabilizers can result in the formation of
salts of the HBP.
[0111] Typically for wound healing, HBP is formulated for
site-specific delivery. When applied topically, the HBP is suitably
combined with other ingredients, such as carriers and/or adjuvants.
There are no limitations on the nature of such other ingredients,
except that they must be pharmaceutically acceptable and
efficacious for their intended administration, and cannot
significantly degrade the activity of the active ingredients of the
composition. Examples of suitable vehicles include ointments,
creams, gels, sprays, or suspensions, with or without purified
collagen. The compositions also may be impregnated into sterile
dressings, transdermal patches, plasters, and bandages, optionally
in liquid or semi-liquid form.
[0112] For obtaining a gel formulation, the HBP formulated in a
liquid composition may be mixed with an effective amount of a
water-soluble polysaccharide or synthetic polymer such as
polyethylene glycol to form a gel of the proper viscosity to be
applied topically. The polysaccharide that may be used includes,
for example, cellulose derivatives such as etherified cellulose
derivatives, including alkyl celluloses, hydroxyalkyl celluloses,
and alkylhydroxyalkyl celluloses, for example, methylcellulose,
hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl
methylcellulose, and hydroxypropyl cellulose; starch and
fractionated starch; agar; alginic acid and alginates; gum arabic;
pullullan; agarose; carrageenan; dextrans; dextrins; fructans;
inulin; mannans; xylans; arabinans; chitosans; glycogens; glucans;
and synthetic biopolymers; as well as gums such as xanthan gum;
guar gum; locust bean gum; gum arabic; tragacanth gum; and karaya
gum; and derivatives and mixtures thereof. In certain embodiments
of the invention, the gelling agent herein is one that is, e.g.,
inert to biological systems, nontoxic, simple to prepare, and/or
not too runny or viscous, and will not destabilize the HBP held
within it.
[0113] In certain embodiments, the polysaccharide is an etherified
cellulose derivative, in another embodiment one that is well
defined, purified, and listed in USP, e.g., methylcellulose and the
hydroxyalkyl cellulose derivatives, such as hydroxypropyl
cellulose, hydroxyethyl cellulose, and hydroxypropyl
methylcellulose. In one embodiment, methylcellulose is the
polysaccharide. If methylcellulose is employed in the gel, e.g., it
typically comprises about 2-5%, or about 3%, or about 4% or about
5%, of the gel, and the HBP is present in an amount of about
300-1000 mg per ml of gel.
[0114] The polyethylene glycol useful for gelling is typically a
mixture of low and high molecular weight polyethylene glycols to
obtain the proper viscosity. For example, a mixture of a
polyethylene glycol of molecular weight 400-600 with one of
molecular weight 1500 would be effective for this purpose when
mixed in the proper ratio to obtain a paste.
[0115] The term "water soluble" as applied to the polysaccharides
and polyethylene glycols is meant to include colloidal solutions
and dispersions. In general, the solubility of the cellulose
derivatives is determined by the degree of substitution of ether
groups, and the stabilizing derivatives useful herein should have a
sufficient quantity of such ether groups per anhydroglucose unit in
the cellulose chain to render the derivatives water soluble. A
degree of ether substitution of at least 0.35 ether groups per
anhydroglucose unit is generally sufficient. Additionally, the
cellulose derivatives may be in the form of alkali metal salts, for
example, the Li, Na, K, or Cs salts.
[0116] The active ingredients may also be entrapped in
microcapsules, or sustained-release preparations. See, e.g.,
Remington's Pharmaceutical Sciences 18th edition, Gennaro, A. Ed.
(1995). See also Johnson et al., Nat. Med., 2:795-799 (1996);
Yasuda, Biomed. Ther., 27:1221-1223 (1993); Hora et al.,
Bio/Technology, 8:755-758 (1990); Cleland, "Design and Production
of Single Immunization Vaccines Using Polylactide Polyglycolide
Microsphere Systems," in Vaccine Design The Subunit and Adjuvant
Approach, Powell and Newman, eds, (Plenum Press: New York, 1995),
pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; U.S. Pat. No.
5,654,010; DE 3,218,121; Epstein et al., (1985) Proc. Natl. Acad.
Sci. USA, 82: 3688-3692; Hwang et al., (1980) Proc. Natl. Acad.
Sci. USA, 77: 40304034; EP 52,322; EP 36,676; EP 88,046; EP
143,949; EP 142,641; Japanese patent application 83-118008; U.S.
Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.
[0117] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Recombinant human VEGF expressed in Escherichia coli
[0118] Recombinant human VEGF was expressed in Escherichia coli.
During synthesis, the protein was secreted into the periplasmic
space and accumulated as refractile bodies. Studies were therefore
conducted to achieve extraction and refolding of the protein. These
studies revealed at least 3 species of VEGF (FIG. 1) were isolated
using standard recovery techniques without the addition of a
polyanionic agent. Studies with native VEGF showed that heparin
addition increased resistance to chaotrope- and thiol-induced
denaturation (FIG. 2). In addition, heparin significantly increased
the amount of properly refolded VEGF in small scale refolding
experiments. To adapt this result to a large-scale process,
conditions were discovered which allowed for refolding VEGF in the
presence of dextran sulfate, a molecule structurally analagous to
heparin. Addition of dextran sulfate improved yields of properly
folded biologically active VEGF 3-5-fold relative to controls.
Methods
[0119] Plasmid for VEGF.sub.165 expression--The plasmid pVEGF171
was designed for the expression of human VEGF.sub.165 (see, e.g.,
Leung et al., (1989) Science, 246:1306-1309) in the E. coli
periplasm. Transcription of the VEGF coding sequence was placed
under tight control of the alkaline phosphatase (AP) promoter (see,
e.g., Kikuchi et al., (1981) Nucleic Acids Research, 9:5671-8),
while sequences required for translation initiation were provided
by the trp Shine-Dalgarno region (see, e.g., Yanofsky et al.,
(1981) Nucleic Acids Research, 9:6647-68). The VEGF coding sequence
was fused downstream of the bacterial heat-stable enterotoxin II
(STII) signal sequence (see, e.g., Lee et al., (1983) Infect.
Immun. 42:264-8; and, Picken et al., (1983) Infect. Immun.
42:269-75) for subsequent secretion into the E. coli periplasm.
Codon modifications in the STII signal sequence provided for an
adjusted translation level, which resulted in an optimal level of
VEGF accumulation in the periplasm (see, e.g., Simmons and Yansura,
(1996) Nature Biotechnoloy, 14:629-34). The lambda to
transcriptional terminator (see, e.g., Scholtissek and Grosse,
(1987) Nucleic Acids Research 15:3185) was located downstream of
the VEGF translational termination codon. The replication origin,
and both ampicillin and tetracycline resistance genes, were
provided by the plasmid pBR322. See, e.g., Bolivar et al., (1977)
Gene 2:95-113.
[0120] Cell Homogenization and Refractile body
preparation--Harvested Escherichia coli cells were frozen and
stored at -70 C..degree.. Cells were harvested by BTUX (centrifuge,
Alfa laval) centrifugation and freezing using BEPEX (freezer at
large scale). Cells were suspended in 5 volumes of 50 mM HEPES/150
mM NaCl/5 mM EDTA pH 7.5 (5 L/kg pellet) and homogenized in a model
15 M laboratory homogenizer Gaulin 15M (small scale) or M3 (large
scale) (Gaulin Corporation, Everett, Mass.). The cell suspension
was then diluted with an equal volume of the same buffer and
refractile bodies were harvested by centrifugation in a BTPX 205
(Alfa Laval Separation AB (Tumba, Sweden) continuous feed
centrifuge. Intermediate scale centrifuge used SA1. Alternatively,
cells can be homogenized and the pellet can be harvested directly
without freezing in BEPEX and rehydrating.
Example II
Extracting and Refolding of Recombinant Human VEGF Expressed in
Escherichia coli-I
Methods
[0121] Extraction and Refolding--The refractile pellet was
suspended in extraction buffer containing 7 M Urea/50 mM HEPPS/pH 8
(final concentration) at 5 L of buffer/kg pellet. Solid
dithiothreitol was then added at 3.7 g/kg pellet for a final
concentration of 4 mM. See, e.g., FIG. 9 for the effect of urea and
DTT on extraction of VEGF. The suspension was thoroughly mixed for
1-2 h at 20.degree. C. The pH may be adjusted with 50% sodium
hydroxide (w/w) to pH 8.0. Refolding was initiated by addition of
19 volumes of refolding buffer per volume of extraction buffer. The
refolding buffer contained 50 mM HEPPS/1 M-2M Urea/2-5 mM
cysteine/0.05%-0.2% TRITON.TM. X100/pH 8, final concentration. See,
e.g., FIG. 10 for the effect of urea and DTT concentration present
during refolding. Dextran sulfate, heparin or sodium sulfate was
added as indicated. Refold incubation was conducted at room
temperature for 4-24 hours. Optionally, the incubation can be
conducted at room temperature for up to about 48 hours. The folding
was monitored by SDS-PAGE and/or Heparin HPLC. The product was
clarified by depth filtration with a Cuno 90SP filter followed by
0.45 .mu.m filtration.
[0122] Heparin-binding HPLC Assay--The quality and quantity of
properly refolded VEGF was determined using a column containing
immobilized heparin. The column POROS HE2/M (4.6.times.100 mm,
HE2/M by PerSeptive BioResearch Products, Cambridge, Mass.) was
equilibrated in 10 mM sodium phosphate, pH 7 containing 0.15 M
sodium chloride. At a flow rate of 1 mL/min or 2 ml/min, the
columns were eluted using a linear gradient from 0.15 M to 2 M
sodium chloride in equilibration buffer over 10 min. In some
assays, elution was done in 16 min The eluant was monitored at 280
nm. Typically, the majority of protein was eluted in the void
volume and 3 classes of VEGF could be identified. The highest
affinity, latest-eluting species was identified as correctly folded
VEGF and was subsequently identified as "Peak 3 VEGF".
Results
[0123] Heparin protects VEGF against cysteine-mediated
denaturation--Addition of 10 mM cysteine to native VEGF resulted in
a large decrease in the properly-folded molecule (FIG. 2). This
denaturation was prevented by the addition of 2 different forms of
heparin at concentrations as low as 20 mM.
TABLE-US-00001 TABLE Ia The Effect of Heparin and Dextran sulfate
on VEGF Refolding Concentration (.mu.g/ml) % Increase or Addition 0
10 55 100 200 400 Fold Increase None 5.3* -- -- Low (3 kd) 12.2
14.2 14.8 14.1 179% 2.8 MW heparin High MW 15.3 16.6 13.9 15.3 213%
3.1 (6 kD) heparin Dextran 15.9 15.4 13.6 7.4 8.3 191% 2.9 sulfate
(10 Kd) Values in the table are the amount of Peak 3 VEGF formed
(in mg) per g of refractile pellet. Concentration of each addition
is as indicated. *Average control (5.6 + 5.0 = 5.3)
TABLE-US-00002 TABLE Ib The Effect of Sodium Sulfate on VEGF
Refolding Concentration (.mu.g/ml) % Increase or Addition 0 50 98
195 293 455 Fold Increase None 5.3 -- -- sodium sulfate 6.9 9.1
10.4 10.9 10.4 106% 2.1 Values in the table are the amount of Peak
3 VEGF formed (in mg) per g of refractile pellet. Concentration of
sodium sulfate is as indicated
TABLE-US-00003 TABLE II The Effect of Heparins and Dextran sulfates
on VEGF Refolding Concentration (.mu.g/ml) % Increase or Addition 0
2.5 12.5 50 100 Fold Increase None 2.2 -- -- dextran sulfate 10.1
13.7 13.4 11.2 523% 6.2 (5 Kd) dextran sulfate 9.9 17.2 14.0 12.9
682% 7.8 (8 Kd) dextran sulfate 13.8 19.2 14.6 10.1 773% 8.7 (10
Kd) Low MW (3 Kd) 10.4 16.9 14.7 668% 7.7 Heparin High MW (6 Kd)
14.1 18.8 20.2 818% 9.2 Heparin Values in the table are the amount
of Peak 3 VEGF formed (in mg) per g of refractile pellet.
Summary
[0124] Heparin and dextran sulfate increase refolding yields--Due
to the protective properties against denaturation described above,
the effect of several different forms of sulfated polymers on
refolding VEGF was investigated. As seen in TABLE Ia (and in FIG.
5), both low and high molecular weight forms of heparin increased
the yield of refolded VEGF approximately 3-fold. As seen in TABLE
Ib (and in FIG. 6), sodium sulfate increased the yield of refolded
VEGF by approximately 2-fold. The 10 Kd form of dextran sulfate was
also effective at increasing refold yields; however, the higher
concentration range investigated lead to substrate inhibition.
Further investigation demonstrated that 5 Kd, 8 Kd and 10 Kd forms
of dextran sulfate all significantly increased the yield of VEGF on
refolding (TABLE II). See FIG. 7. See also FIG. 8.
Example III
Effect of Different Buffers and TRITON.TM. X-100 on the Recovery of
VEGF
Results
TABLE-US-00004 [0125] Buffer VEGF (mg/g pellet) HEPES, pH 8 13.3
HEPES, pH 8 with TRITON .TM. 14.3 HEPPS, pH 8 16.6 TrisHCl, pH 8
12.8 HEPES, pH 7.2 9.1 HEPPS, pH 7.2 10.7 HEPES, pH 8 10.3 HEPPS,
pH 8 12.8 HEPES, pH 8 + TRITON .TM. X-100 12.4 HEPPS, pH 8 + TRITON
.TM. X-100 13.9
Summary
[0126] The combined data of Example I, II and III demonstrate a
significant (2-5 fold) improvement in yield by including either
heparin sulfates or dextran sulfates when refolding VEGF, a
heparin-binding growth factor as well as the conditions of
recovery. This method has been implemented successfully at
industrial scale. It is expected that that this method is
applicable in the refolding of other basic growth factors and other
proteins that bind heparin.
Example IV
Extracting and Refolding of Recombinant Human VEGF Expressed in
Escherichia coli-II
Methods
[0127] Extraction and Refolding--The refractile pellet was
suspended in extraction buffer where the final concentration was 7
M Urea, 2-30 mM DTT (e.g., 10 mM DTT), 50 mM HEPPS/pH 7-9 (e.g., pH
8) at 5 L of buffer/kg pellet. The suspension was thoroughly mixed
for 1-2 h at room temperature. Refolding was initiated by addition
of 19 volumes of refolding buffer per volume of extraction buffer.
The refolding buffer contained as the final concentration 1 M or
1.3 M urea, 2-15 mM cysteine (e.g., 7.5 mM cysteine), 0.5 mM DTT,
0-0.75 M arginine (e.g., 100 mM arginine), 15 mg/L dextran sulfate,
50 mM HEPPS, 0.05% TRITON.TM. X100/pH 8. See, e.g., FIG. 12 for the
effect on refolding in the presence of charged amino acids, where
the addition of histidine produced the same effect as without amino
acid additives. Refold incubation was conducted at room temperature
for 12-24 hours. Optionally, the incubation can be conducted at
room temperature for up to about 48 hours. Optionally, air or
oxygen can be added during the refolding process (0.3-1 cc/min/L).
The folding was monitored by SDS-PAGE and/or Heparin HPLC. The
product was clarified by depth filtration with a Cuno 90SP filter
followed by 0.45 .mu.m filtration.
[0128] The overall dilution of the extraction and refolding steps
was 1:100. Increasing the overall dilution of the extraction and
refolding steps, e.g., to 1:100 to 1:200, increased the total
amount of active VEGF although the concentration is lower. See FIG.
13.
[0129] The efficiency of refolding can be determined by determining
the amount of dimer/monomer, where monomers can be determined by a
C18 reverse-phase HPLC column and dimer formation can be determined
by heparin column chromatography or SP-5PW cation exchange
chromatography assay.
Example V
Large-Scale Refolding
[0130] In order to test the scalability of the optimized refolding
conditions, studies were conducted to examine the kinetics of
refolding at small (0.1 L), intermediate (1 L) and pilot plant (250
L to 400 L) scale. As shown in FIG. 4, the kinetics of refolding at
large scale were indistinguishable from the smaller scales and the
final titer of refolded VEGF was slightly increased. These data
demonstrate the scalablility of refolding with dextran sulfate. The
product was further clarified by depth filtration with a Cuno 90SP
filter followed by 0.45 .mu.m filtration.
Example VI
Purification I of rhVEGF after Refolding
[0131] MacroPrep Ceramic Hydroxyapatite Chromatography--After
refolding, insoluble material in the pool was removed by depth
filtration. The clarified pool was then loaded on to a ceramic
hydroxyapatite column (35 D.times.31 H=30 L) (Bio Rad, Hercules,
Calif.) equilibrated in 50 mM HEPPS/0.05% TRITON.TM. X100/pH 8. The
non-binding protein was removed by washing with equilibration
buffer and the VEGF eluted using an isocratic step of 50 mM
HEPPS/0.05% TRITON.TM. X100/0.15 M sodium phosphate/pH 8. The flow
rate was 120 cm/hr. Pooling fractions were determined by Heparin
HPLC analysis of fractions.
[0132] Butyl SEPHAROSE.TM. Fast Flow Chromatography--The pool of
VEGF was loaded onto a column of Butyl SEPHAROSE.TM. Fast Flow (35
D.times.26 H=25 L) (GE Healthcare, Uppsala, Sweden) equilibrated in
50 mM HEPPS/0.05% TRITON.TM. X100/0.15 M sodium phosphate/pH 8. The
flow rate was 100 cm/hr. The column was washed with equilibration
buffer and the VEGF collected in the column effluent. Fractions
were collected and protein containing fractions were pooled, by
measuring A280 nm.
[0133] Macro Prep High S Chromatography--The Butyl SEPHAROSE.TM.
pool was loaded onto a column of Macro Prep High S (30 D.times.39
H=27 L) (BioRad, Hercules, Calif.) that was equilibrated in 50 mM
HEPES/pH 8. After washing the effluent absorbance at 280 nm to
baseline, the column was washed with two column volumes of 50 mM
HEPES/0.25 M sodium chloride/pH 8. The VEGF was eluted using a
linear, 8-column-volume gradient from 0.25-0.75 M sodium chloride
in 50 mM HEPES/pH 8. The flow rate was 75-200 cm/hr. Fractions were
collected and those which contained properly-folded VEGF, as
determined by a heparin-binding assay, e.g., Heparin HPLC, were
pooled.
[0134] Phenyl 5PW TSK Chromatography--The Macro Prep High S pool
was conditioned with an equal volume of 50 mM HEPES/0.8 M sodium
citrate/pH 7.5. The conditioned pool was then loaded on to a column
of Phenyl 5PW TSK (18 D.times.43 H=11 L) (Tosohaas,
Montgomeryville, Pa.) that was equilibrated with 50 mM HEPES/0.4 M
sodium citrate/pH 7.5. After washing non-binding protein through
the column with equilibration buffer, the VEGF was eluted from the
column using a 10-column-volume gradient from 0.4-0 M sodium
citrate in 50 mM HEPES, pH 7.5. Fractions were assayed by
SDS-polyacrylamide gel electrophoresis and those containing VEGF of
sufficient purity were pooled.
[0135] Ultrafiltration/Diafiltration--The pooled VEGF was
ultrafiltered on a 5 kD regenerated cellulose membrane (G30619);
Unit Pellicon; Feed Rate 17.1 L/min. The membrane was conditioned
with polysorbate 20. The pooled VEGF was ultrafiltered at a
concentration of 6 g/L (UF1). The sample was diafiltrated with 7-14
DV (Diavolume) with 5 mM Sodium Succinate/275 mM Trehalose/pH 5.0.
The final formulation was 5 mM Sodium Succinate/275 mM
Trehalose/0.01% polysorbate 20/pH5.0, at a concentration of 5
mg/ml.
Example VII
Purification II of rhVEGF after Refolding
[0136] Cation Exchange Liquid Chromatography--After refolding,
insoluble material in the pool can be removed by depth filtration.
The refold pool is conditioned to pH 5.0-7.5 and about 2 to 6.5
mS/cm. In one embodiment, the pool is conditioned to pH 6.5 and 5
mS/cm. The refold pool can be then loaded on to a sulfopropyl
extreme load column (SPXL) and eluted using a gradient of
increasing salt concentration. Pooling fractions can be determined
by Heparin HPLC analysis of fractions.
[0137] Hydrophobic Interaction Column (HIC):--The SPXL elution pool
of VEGF can be conditioned to 50 mS/cm for loading onto a Phenyl
TSK chromatography column (Tosohaas, Montgomeryville, Pa.).
Fractions are collected and protein containing fractions are
pooled.
[0138] IEX or mixed mode:--The Phenyl TSK pool can be loaded onto a
column of ion exchange chromatography (IEX) or mixed-mode
chromatography. Fractions are collected and those which contained
properly-folded VEGF, as determined by assays described herein are
pooled.
[0139] Ultrafiltration/Diafiltration--The pooled VEGF can be
ultrafiltered on a 5 kD regenerated cellulose membrane (G30619);
Unit Pellicon; Feed Rate 17.1 L/min. For example, the membrane is
conditioned with polysorbate 20. The pooled VEGF is ultrafiltered
at a concentration of 6 g/L (UF1). The sample is diafiltrated with
7-14 DV (Diavolume) with 5 mM Sodium Succinate/275 mM Trehalose/pH
5.0.
[0140] In methods and processes described herein, final purity
and/or activity can be assessed by peptide mapping, disulfide
mapping, SDS-PAGE (both reduced and non-reduced), circular
dichroism, limulus amobocyte lysate (LAL), heparin chromatography,
heparin HPLC (e.g., Heparin HPLC can be used to determine VEGF
dimer concentration), reverse phase (rp) HPLC chromatography (e.g.,
rpHPLC can be used to determine VEGF monomer concentration),
heparin binding, receptor binding (for example for VEGF e.g., KDR
receptor binding-Bioanalytic R&D, and/or Flt1 receptor
binding), SEC Analysis, cell assays, HUVEC potency assays, ELISAs
with VEGF antibodies, mass spec analysis, etc.
[0141] It is understood that the deposits, examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and scope of the appended
claims. All publications, citations, patents, and patent
applications cited herein are hereby incorporated by reference in
their entirety for all purposes.
Sequence CWU 1
1
11165PRTHomo sapiens 1Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His
His Glu Val Val1 5 10 15Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys
His Pro Ile Glu 20 25 30Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp
Glu Ile Glu Tyr 35 40 45Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg
Cys Gly Gly Cys 50 55 60Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr
Glu Glu Ser Asn65 70 75Ile Thr Met Gln Ile Met Arg Ile Lys Pro His
Gln Gly Gln His 80 85 90Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys
Cys Glu Cys Arg 95 100 105Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn
Pro Cys Gly Pro Cys 110 115 120Ser Glu Arg Arg Lys His Leu Phe Val
Gln Asp Pro Gln Thr Cys 125 130 135Lys Cys Ser Cys Lys Asn Thr Asp
Ser Arg Cys Lys Ala Arg Gln140 145 150Leu Glu Leu Asn Glu Arg Thr
Cys Arg Cys Asp Lys Pro Arg Arg 155 160 165
* * * * *