Note: Descriptions are shown in the official language in which they were submitted.
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WO 96/40784 PCTIUS96/09980
METHOD OF SOLUBILIZING, PURIFYING, Al~D REFOLDING PROTEIN
Technical Field of the Invention
The invention relates to methods useful for refolding, solubilizing,
formulating
and purifying proteins. These methods are particularly useful for proteins
that have been
engineered by genetic recombination and produced in bacterial, yeast or other
cells in a
form that has a non-native tertiary structure.
Background of the Invention
To understand fully the entire process of gene expression, it is as important
to
understand the process for the folding of the peptide chain into a
biologically active
protein as it is important to understand the synthesis of the primary
sequence. The
biological activities of proteins depend not only on their amino acid
sequences but also
on the discrete conformations of the proteins concerned, and slight
disturbances to the
conformational integrity of a protein can destroy its activity. Tsou et crl. (
1988)
Biochemistry 27:1809-1812.
Under the proper conditions, the in vitro refolding of purified, denatured
proteins
to achieve the native secondary and tertiary structure is a spontaneous
process. To
avoid formation of stable, but undesired, structures, it is necessary to use
the tertiary
interactions (which are formed late in folding) with their high degree of
selectivity power
to select and further stabilize those early local structures that are on the
correct folding
pathway. Thus, the finite, but very low, stability of local structures could
be the kinetic
"proofreading" mechanism of protein folding. The activated state of folding
with the
highest energy is a distorted form of the native protein, and the slowest,
rate-limiting
step ofunfolding and refolding appears to be close to the native state in
tenors of ordered
structure. In addition, the refolding of many proteins is not completely
reversible in
vitro, and reactivation yields of less than 100% are frequently observed,
which holds true
in particular for experiments at high protein concentration, and competing
aggregation
of unfolded or partially refolded protein molecules may be the major reason
for a
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WO 96140784 PCT/US96/09980
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lowered reversibility, as described in Fischer and Schmid, (1990) Biochemistry
29:2205-
2212.
In the case of sufficiently large protein molecules, the nascent polypeptide
chain .
acquires its native three-dimensional structure by the modular assembly of
micro-
S domains. Variables including temperature, and cosolvents such as polyols,
urea, and
guanidinium chloride, have been tested to determine their role in stabilizing
and
destabilizing protein conformations. The action of cosolvents may be the
result of direct
binding or the alterations of the physical properties of water, as described
in Jaenicke et
al. (1991) Biochemistry 30 (13):3147-3161.
Experimental observations of how unfolded proteins refold to their native
three-
dimensional structures contrast with many popular theories of protein folding
mechanisms. Under conditions which allow for refolding, unfolded protein
molecules
rapidly equilibrate between different conformations prior to complete
refolding. The
rapid prefoIding equilibrium favors certain compact conformations that have
somewhat
1 S lower free energies than the other unfolded conformations. The rate-
limiting step occurs
late in the pathway and involves a high-energy, distorted form of the native
conformation. There appears to be a single transition through which
essentially all
molecules fold, as described in Creighton et al. (1988) Proc. Nat. Acad Sci.
USA
8S:S082-5086.
Various methods of refolding of purified; recombinantly produced proteins have
been used. For example, the protease encoded by the human immunodeficiency
virus
type I (HIV-1) can be produced in Escherichia coli, yielding inclusion bodies
harboring
the recombinant HIV-I protease as described by Hui et al. (1993) J. Prot.
Chem.12:
323-327. The purified HIV-I protease was refolded into an active enzyme by
diluting a
2S solution of the protein in SO% acetic acid with 2S volumes of buffer at pH
S.S. It was
found that a higher specific activity of protease was obtained if the purified
protein was
dissolved at approximately 2 mg/ml in SO% acetic acid fohowed by dilution with
2S
volumes of cold 0.1 M sodium acetate, pH S.S, containing S% ethylene glycol
and 10%
glycerol. Exclusion of glycerol and ethylene glycol led to gradual lass of
protein due to
precipitation. About 8S mg of correctly folded HIV-I protease per liter of E.
coli cell
culture was obtained by this method, and the enzyme had a high specific
activity.
CA 02451969 2003-12-19
_J_
Another example of refolding a recombinant protein is the isolation and
refolding of H-ras from inclusion bodies of E. coli as described by DeLoskey
et al.,
(1994) Arch. Biochem. and Biophys. 311:72-78. In this study, protein
concentration,
temperature, and the presence of 10% glycerol were varied during refolding.
The
yield of correctly folded H-ras was highest when the protein was refolded at
concentrations less than or equal 0.1 mg/ml and was independent of the
presence of
10% glycerol. The yield was slightly higher at 4° than at 25°C.
The refolding of Tissue Factor Pathway Inhibitor (also known variously as
Lipoprotein-Associated Coagulation Inhibitor (LACI), Extrinsic Pathway
Inhibitor
(EPI) and Tissue Factor Inhibitor (EFI) and hereinafter referred to as "TFPI")
produced in a bacterial expression system has been described by Gustafson et
al.,
(1994) P~~otein Expression and Purification 5: 233-241. In this study, high
level
expression of TFPI in recombinant E. coil resulted in the accumulation of TFPI
in
inclusion bodies. Active protein was produced by solubiization of the
inclusion
bodies in 8M urea, and purification of the fill-length molecule was achieved
by canon
exchange chromatography and renaturation in 6M urea. The refolded mixture was
then fractionated to yield a purified nonglycosylated TFPI possessing In vitro
biological activity as measure in the Prothombi~n clotting time assay
comparable to
TFPI purified from mammalian cells.
A non-glycosylated form of TFPI has also been produced and isolated from
Escherichia coli (E. coil) cells as disclosed in U.S. Patent No. 5,212,091.
The
invention described in U.S. Patent No. 5,212,091 subjected the inclusion
bodies
containing TFPI to sulfitolysis to form TFPI-S-sulfonate, purified TFPI-S-
sulfonate
by anion exchange chromatography, refolded TFPI-S-sulfonate by disulfide
exchange
using cysteine and purified active TFPI by cation exchange chromatography. The
form of TFPI described in U.S. Patent No. 5,212,091 has been shown to be
active in
the inhibition of bovine factor Xa and in the inhibition of human tissue
factor-induced
coagulation in plasma. In some assays, the E. coil- produced TFPI has been
shown to
be more active than native TFPI derived from SK hepatoma cells. However, TFPI
produced in E. coli cells is modified in ways that increase heterogeneity of
the
protein.
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A need exists in the art of refolding recombinantly produced proteins to
increase
the amount of correctly folded TFPI during the refolding process. A need also
exists for
increasing the solubility of TFPI. Presently the yields of recombinantly
produced TFPI
have been lower than desirable, and a need exists in the art of producing
correctly folded
TFPI. See for example Gustafuson et al. (1994) Protein expression and
Purification S:
233-241.
TFPI inhibits the coagulation cascade in at least two ways: preventing
formation
of factor VIIaJtissue factor complex and by binding to the active site of
factor Xa. The
. primary sequence of TFPI, deduced from cDNA sequence, indicates that the
protein
contains three Kunitz-type enzyme inhibitor domains. The first of these
domains is
required for the inhibition of the factor VIIa/tissue factor complex. The
second Kunitz-
type domain is needed for the inhibition of factor Xa. The fi~nction of the
third Kunitz-
type domain is unknown. TFPI has no known enzymatic activity and is thought to
inhibit its protease targets in a stoichiometric manner; namely, binding of
one TFPI
Kunitz-type domain to the active site of one protease molecule. The carboxy-
terminal
end of TFPI is believed to have a role in cell surface localization via
heparin binding and
by interaction with phospholipid. TFPI is also known as Lipoprotein Associated
Coagulation Inhibitor (LACI), Tissue Factor Inhibitor (TFI), and Extrinsic
Pathway
Inhibitor (EPI).
Mature TFPI is 276 amino acids in length with a negatively charged amino
terminal end and a positively charged carboxy-terminal end. TFPI contains 18
cysteine
residues and forms 9 disulphide bridges when correctly folded. The primary
sequence
also contains three Asn-X-Ser/Thr N-Iinked glycosylation consensus sites, the
asparagine residues located at positions 145, 195 and 256. The carbohydrate
component of mature TFPI is approximately 30% of the mass of the protein.
However,
data from proteolytic mapping and mass spectral data imply that the
carbohydrate
moieties are heterogeneous. TFPI is also found to be phosphorylated at the
serine
residue in position 2 of the protein to varying degrees. The phosphorylation
does not
appear to affect TFPI fixnction.
TFPI has been isolated from human plasma and from human. tissue culture cells
including HepG2, Chang liver and SK hepatorna cells, Recombinant TFPI has been
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WO 96/40784 PCT/US96/09980
expressed in mouse C 127 cells, baby hamster kidney cells, Chinese hamster
ovary cells
and human SK hepatoma cells. Recombinant TFPI from the mouse C127 cells has
been
shown in animal models to inhibit tissue-factor induced coagulation.
A non-glycosylated form of recombinant TFPI has been produced and isolated
fromFscherichia coli (E, coli) cells as disclosed in U.S. Pat. No. 5,212,091:
This form
of TFPI has been shown to be active in the inhibition of bovine factor Xa and
in the
inhibition of human tissue factor-induced coagulation en plasma. Methods have
also
been disclosed for purification of TFPI from yeast cell culture medium, such
as in
Petersen et al; ~.Biol.Chem. 18:13344-13351 (1993).
Recently, another protein with a high degree of structural identity to TFPI
has
been identified. Sprecher et al, Prod Nat. Acad. Sci., USA 91:3353-3357
(1994). The
predicted secondary structure of this protein, called TFPI-2, is virtually
identical to TFPI
with 3 Kunitz-type domains, 9 cysteine-cysteine linkages, an acidic amino
terminus and
a basic carboxy-terminal tail. The three Kunitz-type domains of TFPI-2 exhibit
43%,
35% and 53% primary sequence identity with TFPI Kunitz-type domains 1, 2, and
3,
respectively. Recombinant TFPI-2 strongly inhibits the amidolytic activity of
factor
VIIa/tissue factor. By contrast, TFPI-2 is a weak inhibitor of factor Xa
amidolytic
activity.
TFPI has been shown to prevent mortality in a lethal ~scherichia coli (E.
coli)
septic shock baboon model. Creasey et al, J. Clin. Invest. 91:2850-2860
(1993).
Administration of TFPI at 6 mglkg body weight shortly after infusion of a
lethal dose of
E coli resulted in survival in all five TFPI-treated animals with significant
improvement
in quality of life compared with a mean survival time for the five control
animals of 39.9
hours. The administration of TFPI also resulted in signif cant attenuation of
the
coagulation response, of various measures of cell injury and significant
reduction in
pathology normally observed in E. coli sepsis target organs, including
kidneys, adrenal
glands, and lungs.
Due to its clot-inhibiting properties, TFPI rnay also be used to prevent
thrombosis during nvcrovascular surgery: For example, U.S. 5,276,015 discloses
the
use of TFPI in a method for reducing thrombogenicity of microvascular
anastomoses
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wherein TFPI is administered at the site of the microvascular anastomoses
contemporaneously with microvascular reconstruction.
TFPI is a hydrophobic protein and as such, has very limited solubility in
aqueous solutions. This limited solubility has made the preparation of
pharmaceutically acceptable formulations of TFPI difficult to manufacture,
especially
for clinical indications which may benefit from administration of high doses
of TFPI.
Thus, a need exists in the art for pharmaceutically acceptable compositions
containing
concentrations of TFPI which can be administered to patients in acceptable
amounts.
Brief Description of the Drawings
Figure 1 is a coomassie stained SDS-PAGE analysis of TFPI peak fractions
from the Phenyl SepharoseTM HIC refolding procedure.
Figure 2 is a plot of the recovery of native TFPI from the HIC column.
Figure 3 is a plot of the recovery of native TFPI from a second HIC column.
Figure 4 is the amino acid sequence of TFPI.
Figure 5 shows the solubility of TFPI at different pH conditions. About 10
mg/mL TFPI in 2M urea was dialyzed against 20 mM acetate, phosphate, citrate,
glycine, L-glutamate and succinate in 150 mM NaC 1. The concentration of
remaining soluble TFPI after dialysis was measured by UV absorbance after
filtering
out the precipitates through 0.22 mm filter units.
Figure 6 shows the solubility of TFPI as a function of concentration of
citrate
in the presence of 10 mM Na phosphate at pH 7. TFPI solubiity increases with
increasing concentration of citrate.
Figime 7 shows the solubility of TFPI as a function of concentration of NaC 1.
TFPI solubiity increases with increasing salt concentration, indicating salt
promotes
solubility of TFPI.
Figure 8 shows effect of pH on the stability of TFPI prepared in 10 mM Na
phosphate, 150 mM NaCl and 0.005% (w/v) polysorbate-80. Stability samples
containing 150 mg/mL TFPI were incubated at 40°C for 20 days. Kinetic
rate
constant for the remaining soluble TFPI was analyzed by following decrease of
the
main peak on canon exchange chromatograms.
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WO 96140784 PCT/US96/09980
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Figure 9 shows the percentage of remaining soluble TFPI measured by ration
exchange HPLC (A) and remaining active TFPI by prothrombin time assay (B) as a
function of phosphate concentration. The formulation contains 150 mglmL TFPI
prepared in 150 mM NaCl and 0:005 % (w/v) polysorbate-80 at pH 7 with varying
concentrations of phosphate.
Figure 10 shows loss of soluble TFPI at 40°C measured by both cation-
exchange HPLC (triangle) and prothrombin time assay (circle) for 0.5 mg/mL
TFPI
formulated in 10 mM Na citrate, pH 6 and 150 mM NaCI.
Figure 11 shows loss of soluble TFPI at 40° C measured by both ,
cation-
exchange HPLC (open symbol) and prothrombin time assay (closed symbol) for 0.5
mg/mL TFPI formulated in 10 mM Na phosphate, pH 6 and either 150 mM NaCI
(triangle) or 500 mM NaCI (circle).
Figure 12 shows loss of soluble TFPI at 40°C measured by both cation-
exchange HPLC (open symbol) and prothrombin time assay (closed symbol) for 0.5
mg/mL TFPI formulated in 10 mM Na acetate and pH 5.5 containing 150 mM NaCI
(triangle) or 8 % (w/v) sucrose (square) or 4.5 % mannitol (circle).
Figure 13 shows two non-reducing SDS gels for TFPI formulation samples at
pH 4 to 9 stored at 40°C for O and 20 days.
Figure 14 shows the time course of a polyphosphate-facilitated rhTFPI refold
monitored using SDS PAGE.
Figure 15 shows the absorbance at 280 nm during the loading and elution of
the S-Sepharose HP column used to purify rhTFPI from a polyphosphate-
facilitated
refold.
Figure 16 shows SDS PAGE analysis of fractions collected during elution of
the S-Sepharose HP column used to purify rhTFPI from a polyphosphate-
facilitated
refold: Figure 17 shows the absorbance at 280 nm during the loading and
elution of the Q-Sepharose HP column used to purify rhTFPI from a S-Sepharose
pool prepared from a polyphosphate-facilitated refold.
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WO 96/40784 PCT/US96109980
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Figure 18 shows SDS PAGE analysis of fractions collected during elution of
the Q-Sepharose HP column used to purify rhTFPI from a S-Sepharose pool
prepared
from a polyphosphate-facilitated refold.
Figure 19 shows the time course of a polyethyleneimine-facilitated rhTFPI
refold monitored using SDS PAGE.
Figure 20 shows the absorbance at 280 nm during the loading and elution of
the S-Sephamse HP column used to purify rhTFPI from a
polyethyleneimine-facilitated refold.
Figure 21 shows SDS PAGE analysis of fractions collected during elution of
ZO the S-Sepharose HP column used to purify rhTFPI from a polyethyleneimine
facilitated refold.
Figure 22 shows the absorbance at 280 nm during the loading and elution of
the Q-Sepharose HP column used to purify rhTFPI from a S-Sepharose pool
prepared
from a polyethyleneimine-facilitated refold.
1S Figure 23 shows SDS PAGE analysis of fractions collected during elution of
the Q-Sephamse HP column used to purify rhTFPI from a S-Sepharose pool
prepared
from a polyethyleneimine-facilitated refold.
Figure 24 shows the ration exchange HPLC analysis of a 0.4 qb
polyphosphate-facilitated rhTFPI refold in the absence of urea.
20 Figure 25 shows results of canon exchange HPLC analysis of an evaluation of
different levels of cysteine on a rhTFPI refold in 0.4 ~ polyphosphate, SO mM
Tris in
the absence of urea.
Figure 26 shows the effect of polyphosphate chain length on the course of a
polyphosphate facilitated refold of rhTFPI inclusion bodies as monitored by
ration
25 exchange HPLC.
Figure 27 shows the effect of concentration of polyphosphate (Glass H) on the
refolding of rhTFPI from inclusion bodies as monitored by ration exchange
HPLC.
Figure 28 shows the ration exchange HPLC analysis of polyethyleneimine
- and polyphosphate-facilitated refolding of purified and reduced rhTFPI.
CA 02451969 2003-12-19
_C~_
Summary of the Invention
It is an object of an aspect of the present invention to describe a method of
refolding protein.
It is another object of an aspect of the invention to provide aqueous
formulations of TFPI.
It is another object of an aspect of the invention to provide methods for
modifying a protein's solubility using charged polymers.
It is still another object of an aspect of the present invention to describe a
method of refolding TFPI including the steps of adding charged polymers to a
solution of denatured TFPI prior to allowing the TFPI to refold.
Additionally, it is another object of an aspect of the invention to describe a
method of refolding TFPI including the step of immobilizing charged polymers
on a
column and passing a solution of denatured TFPI through the column and eluting
the
refolded TFPI after the refolding has occurred.
It has now been found that solubility of TFPI is strongly dependent on pH and,
surprisingly, that polyanions such as citrate, isocitrate, and sulfate have
profound
solubilizing effects on TFPI. This finding is surprising in light of the
hydrophobic
nature of TFPI and the hydrophilic character of these counterions. Thus,
citrate,
isocitrate, sulfate as well as other solubilizers described hereinbelow can be
used to
produce pharmaceutically acceptable compositions having TFPI concentrations
sufficient for administration to patients. It has also been shown that other
organic
molecules can act as secondary solubilizers. These secondary solubilizers
include
PEG, sucrose, mannitol, and sorbitol.
The invention relates to pharmaceutically acceptable compositions wherein
TFPI is present in a concentration of more than 0.2 mg/mL solubilizing agents.
The
solubilizing agents may be acetate ion, sodium chloride, citrate ion,
isocitrate ion,
glycine, glutamate, succinate ion, histidine, imidazole and sodium dodecyl
sulfate
(SDS) as well as charged polymers. In some compositions, TFPI may be present
in
concentrations of more than 1 mg/mL and more than 10 mg/mL. The composition
may also have one or more secondary solubilizers. The secondary solubilizer or
solubilizers may be polyethylene glycol (PEG), sucrose, mannitol, or sorbitol.
CA 02451969 2003-12-19
-~ a-
Finally, the composition may also contain sodium ,phosphate at a concentration
greater than 20 mM.
Although the solubility of TFPI is quite low between pH 5 and 10, it has been
found that L-arginine can increase the solubility by a factor of 100. The
solubility is
very dependent on the concentration of arginine, as 300 mM is about 30 times
more
effective than 200 mM. Urea also is quite effective in solubilization of TFPI.
Further, it has been found that aggregation of TFPI appears to be the major
degradation route at neutral and basic pH conditions and that fragmentation
occurs at
acidic pH conditions.
It has also been found that active TFPI monomers can be separated away from
TFPI oligomers that are produced during the process of folding recombinant
TFPI
produced E. coil. Some misfolded/modified monameric forms of TFPI are also
removed during this process. The separation employs hydrophobic interaction
chromatography. The oligomeric species of TFPI bind more tightly to a
hydrophobic
resin than does the active TFPI monomers. Resins such as Pharrnacia octyl
sepharose and Toyopearl butyl 650-M have been successful. The process is
carried
out in the presence of high salt, such as 1 M ammonium sulfate or 0.5 M sodium
citrate.
According to an aspect of the invention, there is provided a method of
removing bacterial contaminants from a recombinant polypeptide expressed in
bacteria, wherein the recombinant polypeptide is selected frorr~ the group
consisting of
(i) human TFPI having the amino acid sequence shown in Figure 4, (ii) ala-
human
TFPI having the amino acid sequence shown in Figure 4 and having one further
amino acid which is an amino-terminal alanine, and (iii) mutei:ns of (i) or
(ii) having
from 1 to 5 amino acid substitutions, comprising the steps o~
adding a cationic polymer to a solution comprising a soluble form of the
recombinant polypeptide, to precipitate bacterial contaminants; and
removing the precipitated bacterial contaminants.
According to a further aspect of the invention, there is provided a method of
purifying a polypeptide expressed in Escherichia coli, wherein the polypeptide
is
selected from the group consisting of (i) full length, properly folded human
TFPI
having the amino acid sequence shown in Figure 4; (ii) full length, properly
folded
ala-human TFPI having the amino acid sequence shown in Figure 4 and having one
CA 02451969 2003-12-19
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further amino acid which is an amino-terminal alanine; and (iii) a full
length,
properly folded mutein selected from the group consisting of muteins having
from 1
to 5 amino acid substitutions of (i) or (ii); the method comprising the steps
of:
applying a first preparation comprising the polypeptide to a hydrophobic
interaction chromatography resin to bind the polypeptide to the resin; and
washing the resin with an elution buffer having a lower salt concentration
than
the first preparation to elute from the resin a second preparation enriched in
the
polypeptide compared to the first preparation.
According to another aspect of the invention, there is provided a method of
purifying a polypeptide expressed in Escherichia coli, wherein the polypeptide
is
selected from the group consisting of (i) full length, properly folded human
TFPI
having the amino acid sequence shown in Figure 4; (ii) full length, properly
folded
ala-human TFPI having the amino acid sequence shown in Figure 4 and having one
further amino acid which is an amino-terminal alanine; and (iii) a full
length, properly
folded mutein selected from the group consisting of muteins having from 1 to 5
amino
acid substitutions of (i) or (ii); the method comprising the steps of:
applying a first preparation comprising the polypeptide to an anion exchange
chromatography resin to bind the polypeptide to the resin;
washing the resin with a wash buffer; and
washing the resin with an elution buffer comprising a polyionic compound to
elute from the resin a second preparation enriched in the polypeptide compared
to the
first preparation.
According to a further aspect of the invention, there is provided a method of
purifying a polypeptide expressed in Escherichia coli, wherein the polypeptide
is
selected from the group consisting of (i) full length, properly folded human
TFPI
having the amino acid sequence shown in Figure 4; (ii) full length, properly
folded
ala-human TFPI having the amino acid sequence shown in Figure 4 and having one
further amino acid which is an amino-terminal alanine; and (iii) a full
length, properly
folded mutein selected from the group consisting of mutezns having from 1 to 5
amino
acid substitutions of (i) or (ii); the method comprising the steps of
applying a first preparation comprising the polypeptide to an ion exchange
chromatography resin, wherein the first preparation further comprises urea and
a
charged polymer whose net charge is the same as th.e net charge of the resin,
to bind
the polypeptide to the resin;
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washing the resin with a wash buffer which is essentially urea-free and
comprises the charged polymer; and
washing the resin with an elution buffer which is essentially urea-free and
which comprises the charged polymer at a higher concentration than in the wash
buffer to elute from the resin a second preparation enriched in the
polypeptide
compared to the first preparation.
According to another aspect of the invention, there is provided a method of
purifying a polypeptide expressed in Escherichia coli, wherein the polypeptide
is
selected from the group consisting of (i) full length, properly folded human
TFPI
having the amino acid sequence shown in Figure 4; (ii) full length, properly
folded
ala-human TFPI having the amino acid sequence shown in Figure 4 and having one
further amino acid which is an amino-terminal alanine; and (iii) a full
length, properly
folded mutein selected from the group consisting of muteins having from 1 to 5
amino
acid substitutions of (i) or (ii); the method comprising the steps of:
applying a first preparation comprising the polypeptide to an ion exchange
chromatography resin, wherein the first preparation further comprises urea and
a
charged polymer whose net charge is the same as the net charge of the resin,
to bind
the polypeptide to the resin;
washing the resin with a wash buffer which comprises urea and the charged
polymer;
and
washing the resin with an elution buffer which comprises urea and which
comprises the charged polymer at a higher concentration than in the wash
buffer to
elute from the resin a second preparation enriched in the polypeptide compared
to the
first preparation.
According to a further aspect of the invention, there is provided a method tv
aid in purification of a polypepetide selected from the group consisting of
(i) full
length, properly folded human TFPI having the amino acid sequence shown in
Figure
4; (ii) full length, properly folded ala-human TFPI having the amino acid
sequence
shown in Figure 4 and having one fuuther amino acid which is an amino-terminal
alanine; and (iii) a full length, properly folded mutein selected from the
group
consisting of muteins having from 1 to 5 amino acid substitutions of (i) or
(ii); the
method comprising the step of
adding a second charged polyner to a composition comprising the polypeptide
and a first charged polymer, the second charged polymer bearing a net charge
which
CA 02451969 2003-12-19
is opposite to the net charge of the first charged polymer, wherein the amount
of the
second charged polyer added is sufficient to essentially neutralize the first
charged
polymer.
According to another aspect of the invention, there is provided a method of
purifying a polypeptide expressed in Escherichia coli, wherein the polypeptide
is
selected from the group consisting of (i) full length, properly folded human
TFPI
having the amino acid sequence shown in Figure 4; (ii) full length, properly
folded
ala-human TFPI having the amino acid sequence shown in Figure 4 and having one
further amino acid which is an amino-terminal alanine; and (iii) a full
length, properly
folded mutein selected from the group consisting of muteins having from 1 to 5
amino
acid substitutions of (i) or (ii); the method comprising the steps of:
(a) refolding the polypeptide which is present in a first preparation;
(b) purifying the refolded polypeptide from step (a) by canon exchange
chromatography;
(c) further purifying the polypeptide from step (b) by anion exchange
chromoatography to form a second preparation enriched in the polypeptide
compared
to the first preparation.
According to a further aspect of the invention, there is provided a method of
purifying a polypeptide expressed in Escherichia coli, wherein the polypeptide
is
selected from the group consisting of (i) full length, properly folded human
TFPI
having the amino acid sequence sho~m in Figure 4; (ii) full length, properly
folded
ala-human TFPI having the amino acid sequence shown in Figure 4 and having one
further amino acid which is an amino-terminal alanine; and (iii) a full
length, properly
folded mutein selected from the group consisting of muteins having from 1 to 5
amino
acid substitutions of (i) or (ii); the method comprising the steps of:
adding polyethylene glycol (PEG) and polyphosphate to a first preparation
comprising the polypeptide to form an aqueous two-phase system; and
collecting a PEG-rich phase to form a second preparation enriched in the
polypeptide compared to the first preparation.
CA 02451969 2003-12-19
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Detailed Description of the Invention
It is a discovery of the present inventors that polyionic polymers such as
polyethylene
imine and polyphosphate, can modify the ionic interactions within proteins.
The
masking of certain areas of high charge density within proteins using polyions
can
have numerous effects. Proteins whose solubility is reduced through the infra-
and/or
inter- molecular neutralization of oppositely charged areas can have their
solubility
improved by masking one of the charged regions with polycations or polyanions.
Barriers to conformational flexibility and specific attractive or repulsive
forces which
interfere with the refolding process can be modulated as well. Proteins which
require
strong denaturants such as urea or guanidine hydrochloride to solubilize and
maintain
solubiity during purification operations can be solubilized and processed
effectively
using polyions.
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Many proteins lacking a clear region of charge localization in the primary
sequence can still demonstrate areas of charge localization due to their
secondary
structure. Thus many proteins can have their solubility, refolding, and
purification
characteristics modified through interactioW vith charged polymeric templates.
The
nature of the modification will depend on the specific protein structure, the
chain
length, charge, and charge density of the ionic polymer.
We have characterized refolding of pure TFPI in a guanidine or a urea based
refolding buffer and the results indicate that refolding efficiencies and
lanetics can be
significantly improved by the addition of charged polymers including heparin,
dextran
sulfate, polyethyleneimine (PEI) and polyphosphates. These polymers increase
TFPI
solubility and enhance refolding through ionic interactions with either the N-
terminus ,
or the C-terminus. In addition to the polymer additives, refolding pure TFPI
requires
a cysteine/cys6ne redox buffer where the refolding reaction can be completed
within
48 hours. Refolding yields are a strong function of pH, redox concentration,
and
polymer additives; however refolding efficiencies as high as 60% can be
achieved for
pure TFPI under optimum refolding conditions.
It has been found by the inventors of this invention that addition of
glycosaminoglycans or sulfated polysaccharides such as, for example, heparin
and
dextran sulfate, to a solution containing a denatured protein prior to
refolding
increases the amount of correctly folded, active protein where the protein is
capable
of binding to the glycosaminoglycan or sulfated polysaccharide and is
subjected to
renaturing conditions.
Recombinant DNA technology has allowed the high level expression of many
proteins that could not normally be isolated from natural sources in any
appreciable
quantities. In E. coli and several other expression systems, the protein is
frequently
expressed in an inactive, denatured state where the primary amino acid
sequence is
correct, but the secondary and tertiary structure and any cysteine disulfide
bonds are
not present. The denatured protein present in an inclusion body may be in such
a
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conformation that charged residues of different parts of the amino acid
backbone that
are not normally in contact are able to interact and form strong ionic bonds
between
positively charged and negatively charged amino acid residues. The formation
of
these ionic bonds may limit the hydration that must occur to effect
dissolution of the
inclusion body. The protein in an inclusion body may also be complexed with
other
cellular components such as membrane components and nucleic acid which may
also
limit the access of solvent (water) to charged and normally hydrated residues.
Also
found in the unfolded state is that hydrophobic residues. which are normally
found
buried in the interior of a protein are more exposed to the polar aqueous
environment.
Such occurrences may work to prevent the dissolution of inclusion bodies in
solvents
other than strong chaotropic agents such as urea or guanidine or detergents
such as
SDS.
Charged polymers preferably in aqueous solution can interfere with and
disrupt the undesirable ionic interactions that occur within a polypeptide
chain as
found in an inclusion body or other environment. The charged polymers may help
disrupt the undesirable ionic interactions and facilitate solvation of ionic
and polar
residues, promoting dissolution without the need for strong chaotropes or
detergents.
The charge, charge density, and molecular weight (chain length) of the charged
polymer may vary depending on the specific protein. Suitable polymers include:
sulfated polysaccharides, heparins, dextran sulfates, agaropectins, carboxylic
acid
polysaccharides,alginic acids, carboxymethyl celluloses, polyinorganics,
polyphosphates, polyaminoacids, polyaspartates, polyglutamates,
polyhistidines;
polyorganics, polysaccharides,DEAE Dextrans, polyorganic amines,
polyethyleneinimes, polyethyleneinime celluloses, polyamines, polyamino acids,
polylysines, and polyarginines.
Proteins with pI greater than 7 may benefit more from interactions with
negatively charged polymers, as these proteins will have a positive charge at
pH 7.
Proteins with pIs below 7 may interact more strongly with positively charged
polymers at neutral pH. Changing the solution pH will modify the otal charge
and
charge distribution of any protein, and is another variable to be evaluated.
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Recombinant DNA technology has allowed the high level expression of many
proteins that could not normally be isolated from natural sources in any
appreciable
quantities. In E. cola and several other expression systems, the protein is
frequently
expressed in an inactive, denatured state where the primary amino acid
sequence is
correct, but the secondary and tertiary structure and any cysteine disulfide
bonds are
not present. The denatured protein must be refolded to the proper active
conformation, which often requires overcoming significant energy barriers
imposed
by ionic attraction and repulsion, restrictions on bond rotation; and other
types of
conformationally induced stresses. Specific ionic attraction between opposite
charges
and/or repulsion between like charges can severely limit the refolding
pathways
available to the denatured protein and reduce the efficiency of the refolding
process.
Some proteins have specific areas of charge whose interactions may limit
conformational flexibility or promote aggregation. Many proteins lacking a
clear
region of charge localization in the primary sequence can still demonstrate
areas of
charge localization due to their secondary structure found in refolding
intermediates,
misfolds and improperly folded protein. Proteins which are particularly
suitable
according to the present invention include but are not limited to TFPI, TFPI
muteins,
TFPI-2, tissue plasminogen activator, BST, PST. While it is believed that
these
methods will be suitable for use with proteins generically, those which are
most
suitable are those which are improperly folded, aggregated, oligomerized, or
inactive.
These will likely be proteins which have at least one highly charged domain,
and
possibly more, which can interact. In the case of TFPI, as well as other
proteins,
two oppositely charged domains interact with each other to prohibit proper
folding
and to cause oligomerization and aggregation. Proteins having many disulfide
bonds
will also be most likely to benefit from the present methods. Preferably the
protein
will have at least 2 and more preferably the protein will have at least 4 or 6
disulfide
bonds.
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Charged polymers can be used to modify the charge, charge density; and
reduce or eliminate sonically mediated limitations to conformation that rnay
arise in
the unfolded state. The juxtaposition of charged groups that are not normally
in
proximity may have result in dead-end refolding pathways from which the
refolding
process may never recover.
The introduction of extra positive or negative charges through the
complexation with charged polymers may allow refolding to proceed more
facilely
for several reasons: first different types of charge distributions may better
accommodate the refolding process; second, the addition of the charged polymer
may
enhance the solubility of the unfolded protein, reducing or eliminating the
need for
chaotropes which have a negative effect on protein conformation.
Because of the frequently unique structure associated with most proteins the
charged polymer that demonstrates preferred characteristics may vary.
Evaluation of
the isoelectric pH (pI) of the protein can serve as a starting point. At
neutral pH a
protein with a pI less than 7 will possess a net negative charge, and will
thus be more
likely to bind a positively charged polymer. The protein with a pI greater
than 7 will
possess a net positive charge at neutral pH , and will have a stronger
tendency to bind
a negatively charged polymer. However, it is well established that charges are
unevenly distributed around a protein, and significant charge localization can
occur.
The possibility of localized concentrations of charges reduce the ability to
predict
which type of charged polymer may be most effective for any application.
Theoretically, for any protein with a specific distribution of interacting
charges and
conformational requirements, there exists a charged polymer of appropriate
compositions in terms of molecular weight, charge, and charge distribution
which
would maximize refolding efficiency. Other variables, such as pH and solvent
ionic
strength; would also be evaluated. Initial screening would involve
polyethyleneimine,
DEAF dextran, dextran sulfate, and polyphosphate at several different
concentrations
and molecular weights. Work with rhTFPI has demonstrated the significant
impact
that polyphosphate chain length and concentration can have on the course of
the TFPI
refolding reaction. Relatively short chain length (n=5) produces high levels
of
CA 02451969 2003-12-19
WO 96140784 PCT/US96/09980
-15-
aggregate. The optimal polyphosphate chain length for refolding rhTFPI was
approximately 25 repeating units. Longer chain length polyphosphates (n= 75)
also
produced more aggregate and less properly folded monomer.
S
Proteins consist of chains of amino acids, the exact composition and sequence
of which constitutes one of the primary structure determinants of the protein.
The
secondary determinant of protein structure is the result of the conformational
guidance that the individual amino acid bonds have on protein conformation:
Thirdly,
the specific amino acid sequence directs the formation of tertiary structures
such as
~i-sheets and a- helices. The three dimensional nature of protein conformation
often
brings into proximity amino acid residues that are not normally close to each
other
based on the direct sequence of the polypeptide chain. The functional form of
a
protein is generally a modestly stable conformation held together by a
combination of
cysteine disulfide bonds, ionic bonds, and hydrophobic and Van der Waals
interactions.
In general, protein solubility can be related to the number of charged and to
a
lesser extent polar amino acids that make up the protein. These charged and
polar
groups become solvated with water molecules in the aqueous solution and this
interaction keeps the polypeptide chain in solution. Polypeptides with
insufficient
numbers of positively or negatively charged amino acids can have limited
aqueous
solubility. In some cases, the positive and negatively charged groups present
in a
protein can interact with each other, displacing the water of solvation and
leading to
reduced aqueous solubility. Many proteins lacking a clear region of charge
localization in the primary sequence can still demonstrate areas of charge
localization
due to their secondary structure. Thus many proteins can have their solubility
modified through interaction with charged polymeric templates. The nature of
the
modification will depend on the specific protein structure, the chain length,
charge,
and charge density of the ionic polymer.
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Complexation with charged polymers with relatively high charge density
represents one approach to increasing the charge density of any protein. A
protein
with a small number of positively charged residues (lysine or arginine) can be
complexed with a negatively charged polymer such as polyphosphate. Some of the
negatively charged groups of the polymer will interact with the positively
charged
groups present in the protein. The remaining charged groups on the polymer
will be
free to interact with the solvent, in most cases water, and effectively
increase the
charge density and solvation of the protein. Alternatively, a positively
charged
polymer such as polyethyleneimine can be used to complex with the negatively
charged residues of the protein. In some cases both types of charged polymers
may
work equally effectively, in other cases, one charge type may be more
effective than
others. The effectiveness of any particular charged polymer, will depend on
protein
amino acid composition, protein amino acid distribution, protein conformation,
charged polymer charge density, charged polymer chain length, solution pH, and
other variables. However, it is likely that for any given protein, a
complementary
charged polymer that will bind to the protein and essentially increase the
charge
density of protein can be found that will improve the solubility
characteristics of that
protein in aqueous medium.
DEfinixinns
The term "processing" as used herein refers to the steps involved in the
purification and preparation of pharmaceutically-acceptable amounts of
proteins.
Processing may include one or more steps such solubilization, refolding,
chromatographic separation, precipitation, and formulation.
The term °charged polymer" and "charged polymeric template" refer
to any
compound composed of a backbone of repeating structural units linked in linear
or
non linear fashion, some of which repeating units contain positively or
negatively
charged chemical groups. The repeating structural units may be polysaccharide,
hydrocarbon, organic, or inorganic in nature. The repeating 'units may range
from n
= 2 to n=several million
CA 02451969 2003-12-19
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The term "positively charged polymer" as used herein refers to polymers
containing chemical groups which carry, can carry, or can be modified to carry
a
positive charge such as ammonium, alkyl ammonium, dialkylammonium, trialkyl
ammonium, and quaternary ammonium.
The term "negatively charged polymer" as used herein refers to polymers
containing chemical groups .which carry, can carry, or can be modified to
carry a
negative charge such as derivatives of phosphoric and other phosphorous
containing
acids, sulfuric and other sulfur containing acids, nitrate and other nitrogen
containing
acids, formic and other carboxylic acids
The term "polyethyleneimine" as used herein refers to polymers consisting of
repeating units of ethylene imine (H3N+-(CH2-CH2-NH2+)x-CH2-CH2-NH3+).
The molecular weight can vary from 5,000 to greater than 50,000.
The term "polyphosphate" as used herein refers to polymers consisting of
repeating units of orthophosphate linked in a phospho anhydride linkage. The
number
of repeating units can range from 2 (pyrophosphate) to several thousand.
Polyphosphate is frequently referred to as sodium hexametaphosphate (SHMP).
Other common names include Grahams salt, CalgonTA'', phosphate glass, sodium
tetrametaphosphate, and Glass HTM.
The term "refold" as used herein refers to the renaturation of protein.
Typically, the goal of refolding is to produce a protein having a higher level
of
activity than the protein would have if produced without a refolding step. A
folded
protein molecule is most stable in the conformation that has the least free
energy.
Most water soluble proteins fold so that most of the hydrophobic amino acids
are in
the interior part of the molecule, away from the water. The weak bonds that
hold a
protein together can be disrupted by a number of treatments that cause a
polypeptide
to unfold, i.e. denature. A folded protein is the product of several types of
interactions between the amino acids themselves and their environment,
including
ionic bonds, Van der Waals interactions, hydrogen bonds, disulfide bonds and
covalent bonds.
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The term "denature° as used herein refers to the treatment of a
protein or
polypeptide in which results in the disruption of the ionic and covalent bonds
and the
Van der Waals interactions which exist in the molecule in its native or
renatured
state. Denaturation of a protein can be accomplished, for example, by
treatment with
8 M urea, reducing agents such as mercaptoethanol, heat, pH, temperature and
other
chemicals. Reagents such as 8 M urea disrupt both the hydrogen and hydrophobic
bonds, and if mercaptoethanol is also added, the disulfide bridges (S-S) which
are
formed between cysteines are reduced to two -S-H groups. Refolding of proteins
which contain disulfide linkages in their native or refolded state may also
involve the
oxidation of the -S-H groups present on cysteine residues for the protein to
reform the
disulfide bonds.
The term "glycosaminoglycan" as used herein refers to polysaccharides
containing alternating residues of uronic acid and hexosamine and usually
contain
sulfate. The binding of a protein in a refolding reaction as described herein
to a
glycosaminoglycan is through ionic interactions.
The term "dextran sulfate" as used herein refers a polyanionic derivative of
dextran, ranging in molecular weight from 8,000 to 500,000 daltons. Dextrans
are
polymers of glucose in which glucose residues are joined by a1,6 linkages.
The term "heparin" as used herein refers to 2 glucoaminoglycans or
heparinoids which are based on a repeating disaccharide (-4DGIcA(p)(~1,
4GlcNAca1-
)n but are subject to extensive modification after assembly. Heparin is stored
with
histamine in mast cell granules and is thus found in most connective tissues.
In general
heparins have shorter chains than heparin.
The term "HIC" as used herein refers to hydrophobic interaction chromatography
which employs a hydrophobic interaction between the column and the molecule of
interest to separate the sulfated polysaccharides and other contaminants from
the
refolded product.A.
Negatively charged polymers include sulfated polysaccharides, such as
heparins,
dextran sulfates, and agaropectins, as well as carboxylic acid polysaccharides
such
asalginic acids and carboxymethyl celluloses. Polyinorganics such as
polyphosphates are
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-19-
also included. Polyamino acids such as polyasparatate, polyglutamate, and
polyhistidine
can also be used.
Positively charged polymers include polysaccharides such as DEAE dextran; '
polyorgnic amines, such as polyethyleneimines, polyethyleneimine celluloses,
and
polyamines, as well as the polyamino acids, polylysine and polyarginine.
Combinations
of polymers may be used, of either charge polarity. In addition, amphoteric co-
polymers
rnay also be used.
As used herein, "TFPI" refers to mature Tissue Factor Pathway Inhibitor. As
noted above, TFPI is also known in the art as Lipoprotein Associated
Coagulation
Inhibitor (LACI), Extrinsic Pathway Inhibitor (EPI) and Tissue Factor
Inhibitor (TFI).
Muteins of TFPI which retain the biological activity of TFPI are encompassed
in this
definition. Further, TFPI which has been slightly modifaed for production in
bacterial
cells is encompassed in the definition as well. For example, a TFPI analog has
an alanine
residue at the amino-terminal end of the TFPI polypeptide has been produced in
Escherichia coli. See U.S. 5,212,091.
As used herein, "pharmaceutically acceptable composition" refers to a
composition that does not negate or reduce the biological activity of
formulated TFPI,
and that does not have any adverse biological erects when formulated TFPI is
administered to a patient.
As used herein, "patient" encompasses human and veterinary patients.
As used herein, the term "solubilizer" refers to salts, ions, carbohydrates,
amino
acids and other organic molecules which, when present in solution, increase
the
solubility of TFPI above 0.2 mg/mL. Solubilizers may also raise the
concentrations of
TFPI above 1 mg/mL and above 10 mglmL. It should be noted that solubilizers
may act
as stabilizing agents. Stabilizing agents preserve the unit activity of TFPI
in storage and
may act by preventing formation of aggregates, or by preventing degradation of
the
TFPI molecule (e.g. by acid catalyzed reactions).
As used herein, the term "secondary solubilizersy' refers to organic salts,
ions,
carbohydrates, amino acids and other organic molecules which, when present in
solution
with a solubilizer, further increase the solubility of TFPI. Secondary
solubilizers may
CA 02451969 2003-12-19
-20-
have other effects as well. For example, secondary stabilizers may be useful
in
adjusting tonicity (e.g. to isotonicity).
The amino acid sequence of TFPI is disclosed in U.S. Patent No. 5,106,833 and
Figure 4. Muteins of TFPI and TFPI-2 are disclosed in U.S. Patent No.
6,103,500. As
described in U.S. Patent No.6,103,500, muteins of TFPI and TFPI-2, with single
or
multiple point mutations, and chimeric molecules of TFPI and TFPI-2 can be
prepared.
For instance, the lysine residue in the P 1 site of the first Kunitz-type
domain of TFPI
may be replaced with arginine. Muteins, containing one to five amino acid
substitutions, may be prepared by appropriate mutagenesis of the sequence of
the
recombinant cloning vehicle encoding TFPI or TFPI-2. Techniques for
mutagenesis
include, without limitation, site specific mutagenesis. Site specific
mutagenesis can be
carried out using any number of procedures known in the art. These techniques
are
described by Smith (1985) Annual Review of Genetics, 19:423, and modifications
of
some of the techniques are described in METHODS IN ENZYMOLOGY, 154, part E,
(eds.) Wu and Grossman (1987), chapters 17, 18, 19, and 20. A preferred
procedure
when using site specific mutagenesis is a modification of the Gapped Duplex
site
directed mutagenesis method. The general procedure is described by Kramer, et
al., in
chapter 17 of the Methods in Enzymology, above. Another technique for
generating
point mutations in a nucleic acid sequence is overlapping PCR. The procedure
for
using overlapping PCR to generate point mutations is described by Higuchi in
Chapter
22 of PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, (eds.)
Innis, Gelfand, Sninsky and White (Academic Press, 1990).
Alternatively, hybrid proteins containing the first Kunitz-type domain of TFPI-
2
and the second and third Kunitz-type domains of TFPI could be produced. One
skilled
in the art of DNA cloning in possession of the DNA encoding TFPI and TFPI-2
would
be able to prepare suitable DNA molecules for production of such a chimeric
protein
using known cloning procedures. Alternatively, synthetic DNA molecules
encoding
part or all of each Kunitz-type domain and peptide sequences linking the
Kunitz-type
domains can be prepared. As a further alternative, the overlapping PCR
technique may
CA 02451969 2003-12-19
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-21 -
also be used to prepare DNA encoding chimeric molecules containing TFPI and
TFPI-2
sequences.
TFPI can be prepared in yeast expression systems as described in U. S. Serial
No.
08!286,530 which is herein incorporated by reference. Methods have also been
disclosed for purification of TFPI from yeast cell culture medium, such as in
Petersen et
al, JLBiol.Chem. 18:13344-13351 (1993). In these cases, recombinant TFPI is
secreted
from the yeast cell. TFPI recovered in such protocols is also frequently
heterogeneous
due to'N-terminal modification, proteolytic degradation, and variable.
glycosylation.
Therefore, a need exists in the art to produce mature TFPI that is authentic
(i.e. having
the correct N-terminal amino acid sequence, full-length and homogeneous.
TFPI can be produced in E coli as described in U.S. Patent No. 5,212,091 which
discloses a method of producing TFPI by expression of a non-glycosylated form
of TFPI
in an ~ coli host.
In one aspect of the invention recombinantly produced proteins which have the
ability to bind polymers of sulfated polysaccharides such as, for example,
heparin or
dextran sulfate are refolded. The invention provides a method that facilitates
refolding
of a denatured recombinantly produced protein product using polymers of
sulfated
polysaccharides which act as a templates for the refolding protein. Without
being
limited to any particular theory, the inventors believe that the interactions
between the
refolding protein and the polymeric template may minimize aggregation of the
refolding
intermediates and provide an environment for the protein to refold to its
native
conformation. The polymer acting as a template may bind a domain or region of
protein
to stabilize the intermediate and allow further folding to occur without
aggregation. The
protein aggregates, if formed, are generally less active than non-aggegated
refolded
protein, and generally result in a reduced overall yield of active refolded
protein. The
NaCI concentration of the refolding conditions is considered important and is
selected to
achieve the maximum efficiency of refolding by maximizing the interaction
between the
template polymer and the refolding protein. For example, it has been found by
the
inventors that approximately 0.2 M concentration of NaCI or lower promotes
binding of
the C-terminal and/or the third Kunitz domain of TFPI to heparin or other
sulfated
polysaccharide polymer. The binding of polymer to the interanediate is
presumed to
CA 02451969 2003-12-19
-22-
facilitate the solubility of the intermediate and provide an environment for
the rest of the
protein to refold by reducing aggregation of the refolding intermediates.
General Methods
TFPI may be prepared by recombinant methods as disclosed in U.S. 5,212,091.
Briefly, TFPI is expressed in Escher-ichia coil cells and the inclusion bodies
containing
TFPI are isolated from the rest of the cellular material. The inclusion bodies
are
subjected to sulfitolysis, purified using ion exchange chromatography,
refolded by
disulfide interchange reaction and the refolded, active TFPI purified by
cation exchange
chromatography. TFPI may also be produced in yeast as disclosed in U.S. Patent
No.
6,103,500.
TFPI activity may be tested by the prothrombin time assay (PTT assays).
Bioactivity of TFPI was measured by the prothrombin clotting time using a
model RA4
Coag-A-MateTM from Organon Teknika Corporation (Oklahoma City, OK). TFPI
samples were first diluted to 9 to 24 ug/mL with a TBSA buffer (50 mM Tris,
100 mM
NaCl, 1 mg/mL BSA, pH 7.a). Then 10 uL of Varify 1TM (pooled normal plasma
from
Organon Teknika Corp.) was mixed with 90 uL of diluted TFPI samples in a
sample tray
and warmed to 37 C in the instrument. Finally SimplastinTM Excel
(Thromboplastin
from Organon Teknika Corp.) was added to start the clotting. The time delay in
clotting
due to anticoagulant activity of TFPI was measured and converted into TFPI
concentration in the measured samples by comparison to a TFPI standard curve.
The amount of soluble TFPI may also be quantified by measuring the area of the
main peak on a cation exchange chromatogram. HPLC analysis of TFPI samples was
performed using a Waters 626 LC system (Waters Corporation, Milford, MA)
equipped
with a Water 717 plus heater/cooler autosampler . Data acquisition was
processed by a
Turbochrom system from Perkin-Elmer.
The canon exchange (IEX) method used a Pharmacia Mono S HR 5/5 glass
column. The column was equilibrated in 80% buffer A (20 mM sodium acetate
trihydrate:acetonitrile solution (70:3 0 v/v) at pH 5.4) and 20% buffer B (20
mM sodium
acetate trihydrate - 1.0 M ammonium chloride:acetonitrile solution (70:30 v/v)
at pH
5.4). After a sample was injected, a gradient was applied to elute the TFPI at
a flow rate
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- 23 -
of 0.7 mIJmin from 20% buil'er B to 85% buffer B in 21 minutes. Eluting TFPI
species
were detected by absorbance at 214 nm. The main peak (monomer TFPI) was found
to
elute at about 18 minutes. Loss of soluble TFPI was quantified by integrating
remaining ,
peak area of the main peak.
All reagents are U.S.P. or A.C.S. grade. Suppliers include J.T. Baker and
Sigma
Co. (St. Louis, MO).
The present invention will now be illustrated by reference to the following
examples, which set forth certain embodiments. However, it should be noted
that these
embodiments are illustrative and are not to be construed as restricting the
invention in
any way.
EXAMPLES
Example 1 - Refolding Denatured TFPI
The following example describes the making of stock solutions, the HIC column
preparation, the initial recovery and purification of TFPI prior to refolding,
the refolding
of TFPI, and the recovery of active TFPI.
The TFPI stock was prepared from refractile bodies resulting from the
expression of recombinant TFPI in bacteria. The refractile bodies were
solubilized at 10
mglml in 8 M urea, 50 mM Tris pH 8.5 containing 10 mM DTT, and this solution
was
clarified by centrifugation at 10,000 x g for 10 minutes.
The column preparation for the initial purification of the solubilized TFPI
was
prepared with S-Sepharose beads mixed in 7.5 M urea, l0 mlVl Tris and 10 mM
sodium
phosphate (pH 6:5) containing 5 mM DTT and 1 mM EDTA. The solubilized TFPI at
a
concentration of 5 mg/ml was then run over the S-Sepharose column and eluted
with a
sodium chloride gradient of 0 to 1 M. The purified TFPI had an absorbency at
wavelength 280 nm of 3.2 (which is equivalent to 4.1 mg/ml using an extinction
coe~cient of 0.78).
The dextran sulfate stock consisted of dextran sulfate of molecular weight
8000
daltons available from Sigma, item number D-4911, made up at 50 mg/ml (6.25 ~
in
50 mM Tris (pH 8.8) in 0.1 M sodium chloride, and stored at -20 degrees
centigrade
between uses.
CA 02451969 2003-12-19
-24-
The heparin stock, if heparin was used to conduct the refolding, was of
molecular weight 6000 to 30,000 daltons, (with an average molecular weight of
18,000 daltons) prepared as a sodium salt available from Sigma Co. (St. Louis,
MO),
item number H-3393, made up at 60 mg/ml (3.33 mM) in 50 mM Tris (pH8.8) in 0.1
sodium chloride, and stored at -20°centigrade between uses.
To the S-Sepharose purified TFPI either dextran sulfate stock solution or
heparin stock solution can be added. Dextran or heparin was added to TFPI
under
denaturing conditions in 6 to 8 M urea. With 4°C reagents, the
denaturing solution
containing TFPI was diluted to 3 M urea, 50 mM Tris (pH 8.8), 0.2 M sodium
chloride, and 0.5 mg/ml TFPI, and to a final dextran sulfate concentration of
0.6
mg/ml (75 MM) or a final heparin concentration of 1.5 mg/ml (83 pM), depending
on
which was used to facilitate the refolding. Cystine was added to the refolding
solution to a final concentration equal to the final DTT concentration. The
refolding
solution was incubated at 4°C with gentle agitation for from 4 to 6
days, preferably 5
days.
As an illustration of this procedure the following is a detail of a protocol
for
refolding a 5 ml solution of TFPI in dextran sulfate or heparin.
To 610 p l of TFPI stack either 60 ~1 of dextran sulfate with 65 ~1 of 50 mM
Tris (pH 8.8) in 0.1 M NaCl, or 125 p l of heparin stock solution with 50 mM
Tris
(pH 8.8) or 0.1 M NaCI was added. The refolding solution was mixed and allowed
to
incubate 10 minutes on ice. Next, 4.2 ml of refolding buffer containing 2.5 M
urea,
50 mM Tris (pH 8.8) and 165 mM sodium chloride was added to the refolding
solution and mixed. Finally, 61 ~ 1 of 50 mM Cystine made up in 120 mM sodium
hydroxide was added and the total solution was incubated at 4°C with
gentle agitation
for 4 days. The free sulfhydryl content was checked with Ellman's reagent
(also
called DTNB). Idoacetamide was added, to 20 mM, made up at 1 M in 100% ethanol
for storage at -20°C.
The hydrophobic interaction column (HIC) was prepared from Butyl-650M
Tosohaas ToyopearlTM resin particle size 40-90, part # 014702. The butyl resin
was
washed in 3 M urea, 1 M ammonium sulfate, 50 mM Tris, 10 mM sodium phosphate,
pH 6.5 and resuspended at a 50% slurry.
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The refolding samples, stored at -20~C remained in the standard refolding
buffer
containing 3 M urea, 50 mM Tris, pH 8.8, 1-4 mM redox, 0.5 mglml TFPI, and 0.2-
0.6
M NaC1 depending on condition. Samples refolded with dextran or heparin had
0.2 M
salt, and samples without dextran or heparin had 0.6 M lvlaCl.
The following steps were performed at room temperature to effect the further
purification of the refolded TFPI. To 300 u1 of refolded sample, an equal
volume of 2
M ammonium sulfate, 3 M urea, 50 mM Tris, and 10 mM sodium phosphate (pH 6.5)
was added. Next, 100 pI of washed Butyl-650M beads was added to the diluted
refolded sample. The solution with the beads was incubated with gentle rocking
or
mixing for 30 minutes at room temperature. The mix was then spun in an
ependorf
centrifuge for 5 seconds, and put in a rack and allowed to sit for one minute
for the
beads to settle flat in the tube. The supernatant was aspirated carefully, so
as not to
disturb the beads.
To wash the TFPI-bound beads, 1 ml of wash buffer composed of 1 M ammonium
sulfate, 3 M urea, SO mM Tris, 10 mM sodium phosphate (pH 6.5) was added to
the
beads to remove the remaining dextran sulfate or heparin. The washed mixture
was re-
spun in an ependorf centrifuge for 5 seconds, and allowed to sit for one
minute for the
beads to settle as before. The supernatant was removed, and the beads then
washed
with the wash buffer a final time, and spun and allowed to sit as before.
After the final
wash and settling, the supernatant was removed with a flame-pulled-tip Pasteur
pipette
very carefully.
To elute the refolded TFPI, 300 pi of elution buffer composed of 3 M urea, 0.1
M
ammonium sulfate, 50 mM Tris and 10 mM sodium phosphate (pH 6.5) was added to
the slurry of beads and rocked for more than 10 minutes. The beads were
pelleted by
spinning in an ependorf centrifuge, and the supernatant containing refolded
TFPI was
recovered. To avoid contamination of the beads with the product, some of the
supernatant was left behind.
CA 02451969 2003-12-19
WO 96140784 PCT/US96/09980
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Example 2 - HIC of Dextran Sulfate Refold
The sample of TFPI was renatured at a concentration of 0.5 mg/ml TFPI, 0.6
mg/mi
Dextran sulfate, 3.0 M Urea, 200 mM NaCI and 50 mM Tris (pH 5. S). The HIC
column
was prepared from TosoHaas Butyl beads for HIC, 4.6 mmD/100mmL, in a I.66 ml
slurry. The flow rate was set for 1.0 ml/min. Before loading the HIC column,
the
sample was diluted 2:3 with 3.0 M Urea and 3.0 M NH,S04 at a final pH of 5.68;
2 ml
of sample was loaded. The gradient start was 33 mM MES/33 mM HEPES/33 mM
sodium acetate, 1.0 M NH4S04, and 3.0 M Urea, pH 6.0; the gradient end was 33
mM
MESI33 mM HEPES/33 mM sodium acetate, 3.0 M Urea at pH 6Ø The gradient
volume was 5.0 CV. From this column; the recovery of native TFPI was 68%. The
results of this run are shown in Figure 2.
A second HIC column was also run. The sample of denatured TFPI was diluted 2:3
with 3.0 M Urea, 1.5 M NH4504 and two ml were loaded. The gradient start was
33
mM MESl33 mM HEPESi33 mM sodium acetate, 0.5 M NH4S0', and 3.0 M Urea, pH
6.0; the gradient end was 33 mM MES/33 mM HEPES/33 mM sodium acetate, 3.0M
Urea at pH 6Ø The gradient volume was 5.0 CV. The recovery of native TFPI
from
this second column was 74%. The results of this run are shown in Figure 3.
The samples were analyzed by non-reducing SDS-PAGE as illustrated in Figure
Z. Correctly refolded, active TFPI species (major band) are seen on the gel.
Example 3
About 10 mg/mL TFPI in 2M urea was dialyzed against one of the following: 20
mM acetate, 20 mM phosphate, 20 mM citrate, 20 mNi glycine, 20 mM L-glutamate
or
20 mM succinate in I50 mM NaCI as described above. 6-IO mg/mL TFPI bulk stock
was loaded into SpaclPor 7 dialysis tubings (MW cutoff 3,500). Dialysis was
carried
out either at 4iC or ambient temperature. Three changes of buffer at a protein
solution to buffer ratio: 1 to 50-100, were made during course of dialysis
over 12 to
24 hr time period. After dialysis, TFPI solution was filtered by Costar 0.22
micron
filter units to separate precipitated TFPI from soluble TFPI. The solubility
of TFPI
was then measured by UVIVis absorbance assuming an absorptivity 0.68 (mglmL)'~
cm'' at 278 nm. The solutions were prepared at various pH levels by titration
with
HCl or NaOH.
CA 02451969 2003-12-19
r WO 96140784 PCTIUS96109980
-27-
After completion of dialysis, the precipitates were filtered through 0.22 ~cm
filter units. The concentration of remaining soluble TFPI after dialysis was
measured
by UV absorbance. Figure 1 shows the results of these experiments. Solubility
of
TFPI increased greatly in solutions containing 20 mM acetate, 20 mM phosphate,
20
mM L-glutamate and 20 mM succinate at pH levels below 7 and particularly at or
below pH 4.5. Solubility of TFPI was also substantially increased in solutions
containing 20 mM glycine above pH 10. Figure 2 shows the solubility of TFPI as
a
function of concxntration of citrate ion in the presence of 10 mM Na phosphate
at pH
7. TFPI solubility increascs with increasing concentration of citrate. Figure
3 shows
the solubility of TFPI as a function of concentration of NaCI at pH 7Ø TFPI
solubility increases with increasing salt concentratian, indicating salt .
promotes
solubility of TFPI.
The solubility of TFPI was studied using a number of different solubilizers
and secondary solubilizers. Table 1 shows solubility of TFPI in varying buffer
solutions measured by UV absorbance after dialyzing 6 to 10 mg/mL TFPI into
these
buffer solutions.
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Table 1
Salt effect Solubilit
Content H c m /ml
uv
lomM NaP04. 7 0.21
~
lOmM NaP04, i50mM NaCI 7 0.72
2omM NaP04, i50mM NaCI 7 0.85
20mM NaP04, 0.5M NaCI 7 6.71
20mM NaP04, 1M NaCI 7 8.24
pH effect
Content H c m Iml
uv
20mM NaOAc, 150mM NaCI 3 10.27
20mM NaOAc, 150mM NaGI 3.510.25
20mM NaOAc, 150mM NaCI 4 7.54
20mM NaOAc, 150mM NaCI 4.51 .75
20mM NaOAc, 150mM NaCI S 1.15
20mM NaOAc, 150mM NaCI 5.50.85
20mM NaP04, 150mM NaCI S.50.89
_ 6 0.78
2omM NaP04, 150mM NaCI
20mM NaP04, 150mM NaCI 6.50.79
20mM NaP04, t50mM NaCI 7 O.gS
20mM NaP04, 150mM NaCI 7.5~ 0.82
2omM NaP04, 150mM NaCI 8 0.86
I
20mM NaCitrate, 150mM NaCI 4 2.7 7
20mM NaCitrate, 150mM NaCI 4.51.19
20mM NaCitrate, 150mM NaCI . 5 t .1
20mM NaCitrate, 150mM NaCI 5.51.84
20mM NaCitrate, 150mM NaCI 6 2.09
20mM NaCitrate, 150mM NaC) 6.52.12
20mM NaCitrate, 150mM NaG) 7 1.92
20mM GI cine, 150mM NaCI 9 0.32
20mM Glycine, 150mM NaCI 10 0.9
20mM GI cine, 150mM NaC! 1 13.94
1
20mM'L-Glutamate, 150rnM NaCI 4 9.07
20mM L-Glutamate, 150mM NaCI 5 7 .21
20mM Succinate, 150mM NaCI 4 8.62
20mM Succinate, 150mM NaCI 5 1.21
20mM Succinate, 150mM NaCI
6 1.07
Citrate
Content H c m Iml
uv
lOmM NaP04, 20mM NaCitrate 7 1 .1 6
CA 02451969 2003-12-19
WO 96!40784
PCTIUS96109980
Table 1 (cont.)
lOmM NaP04, 50mM NaCitrate ~ . 5.81
7
lOmM NaP04, 100mM NaCitrate ~ 7 12.7
lOmM NaP04, 200mM NaCitrate 7 15.9
lOmM NaP04, 300mM NaCitrate 7 8.36
~
M 2+, Ca2+ and of hos hate
Content H c m /ml
uv
lOmM NaP04, 150mM NaCI, 1mM M CI2 7 0.66
IOmM NaP04, 150mM NaCI, lOmM M CI2 7 1.02
1 OmM NaP04, 150mM NaCI, 0.1 mM CaCl2 7 0.67
1 OmM NaP04, 150mM NaCI, 1 mM CaCl2 7 0.71
lOmM NaP04, 150mM NaCI, iOmM tri hos hate7 3.64
lOmM NaP04, 5% PEG-400 7 0.07
lOrnM NaP04, lOmM EDTA ~ 7_ 0.36
lOmM NaP04, 100mM Na2S04 7 5.08
lOmM NaP04, 100mM L-as artic acid 7 0.4
lOmM NaP04, 100mM Succinic acid 7 2.33
lOmM NaP04, t 00mA4 Tartaric acid 7 2.56
20mM NaP04, 100mM Malefic acid 7 _0_.11
20mM NaP04, 100mM Malic acid 7 _1.87
lOmM NaP04, 100mM L- lutamic acid 7 0
IOmM NaP04, 750mM NaCI 7 0.25
_ 7 10.83
lOmM NaP04, 100mM isocitrate
NaOAc, NaP04 and NaCI
Content H c m !ml
uv
lOmM NaOAc, 150mM NaCI , 4.5 1.76
lOmM NaOAc 4.5 4.89
lOmM NaOAc 5.5 4.95
lOmM NaOAc _ w 5.1
6.5
lOmM NaOAc 7 5.87
1 OmM NaP04, i 50mM NaCI 4.5 0.14
lOmM NaP04 4. 4.97
lOmM NaP04 5.5 0.79
lOmM NaP04 6.5 0.091
t OmM NaP04 7 0.94
50mM NaOAc 5 5.24
SmM NaOAc 5.5 _4.59
lOmM NaOAc 5.5 5.05
20mM NaOAc 5.5 5.04
___ 5.5 5.71
50mM NaOAc
100mM NaOAc b.5 ~ 1 .4
200mM NaOAc
5.5 i .32
CA 02451969 2003-12-19
W~196140'784 PCTIUS96I09980
-30-
Table 1 (cost.)
5mM NaOAc. 5mM NaCI ~ ~ ~ 5.5 ! 4.85'
SmM NaOAc. lOmM NaCI I 5.5 5.04
5mM NaOAc. 50mM NaCI I 5.5 ' 0.56
5mM NaOAc, 100mM NaCI 5.5 0.43
5mM NaOAc, 200mM NaCI ( 5.5 ~ 0.8
5mM NaOAc 4.5 7.27
__ 4.5 ! 6.5
lOmM NaOAc
20mM NaOAc 4.5 ~ 8,32
50mM NaOAc ~ 4.5 I 9.17
5mM NaOAc ! 5.5 8.98
lOmM NaOAc ' S.5 B,Og
20mM NaOAc ! 5.5 8.
99
50mM NaOAc ~ _
5.5 ~ 2.92
5mM NaOAc, 150mM NaCI 4.5' 2.6
~
_ _ _ _ 4.5 2.59
lomM NaOAc, 150mM NaCI '
20mM NaOAc, 150mM NaCI 4.5 2.55
50mM NaOAc. 150mM NaCt j 4.5 ; 2.1
5mM NaOAc, 150mM NaCI 5.5 ~ 0.65
lOmM NaOAc, 150mM NaC! j 5.5 0.69
20mM NaOAc, 7 50mM NaCI _ 5.5 0.71
50mM NaOAc, 150mM NaCI 5.5 0.91
H dro hobtc chain ten th l
Content
! H i c m ml
uv
1 OmM NaP04, 50mM Formic acid
7 0.12
7 OmM NaP04, 50mM Acetic acid
7 ( 0.16
iOmM NaP04, SOmM Pro anoic acid
7 0.76
lOmM NaP04, 50mM Butanoic acid
' i
lOmM NaP04, 50mM Pentanoic acid 7
0.14
tOmM NaP04, 50mM Hexanoic acid 7 0,' 1
Others '
Content ~ pH t c m
/ml uv
20mM NaOAc. 3% Mannitol. 2% Sucrose, 5% 4 19.9
PEG-400 !
20mM Na Citrate, 3% Mannitot. 2% Sucrose,6.5 0.72
5% PEG-400
20mM Na Citrate, 150mM NaCl, S% PEG-400 6.5 2.18
20mM NaOAc, 150mM NaCI, S% PEG-400 4 t 9.8
20mM Na Citrate, 130mM NaCI, 1% GI cine, 8.5 1.48
0.25% Tween-80.
5% PEG-400
20mM Na Citrate, 130mM NaCI, 1 % G( tine,6.5 t .32
0.25% Tween-80
! Sotubilit
Content ~ pH ' c (mglml),uv
SmM NaAcetate - _ - ! 5.5 i 8.9
5mM NaAcetate, 8~ Sucrose j 5.5 t t
5mM NaAcetate; O.Ot~ Poivsorbate-80 ! 5.5 ~ 7
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Table 1 (cost.)
5mM NaAcetate, 8% Suaose, 0.01% Polysor_bate-80 5.5 12
_
lOmM NaAcetate _ 5.5 7.6
~
lOmM NaAcetate, 8% Sucrose 5.5 1 0
lomM NaAcetate, 8% Sucrose, 0.01% 5.5 12.1
Pol sorbate-80
5mM NaAcetate, 5% Sorbitol 5.5 7.8
5mM NaAcetate, 4.5% Mannitol 5.5 9.2
5mM Histidine 6 5.5
5mM Histidine 6.5 1
5mM NaCitraie 5.5 ' 0.1
SmM NaCitrate 6 0.1
5mM NaCitrate 6.5 0.1
5mM NaSuccinate - 5.5 0.6
5mM NaSuccinate 6 0.3
5mM NaSuccinate 6:5 0.2
lOmM Imidazole 6.5 2.5, 10.8
lOmM Imidazole 7 0.8
lOmM Imidazole, 8% Sucrose 6.5 12.2
5mM NaAcetate 6 8.2
lOmM Imidazole, 5mM NaAcetate 6.5 12.8
tOmM NaCitrate 6 0_2
100mM NaCitrate 6 8.1
100mM NaCitrate 7 9.3
t OmM Naphosphate, 260mM Na2S04 6 9.1
IOmM NaPhosphate, lOOmM NaCitrate 8 8.8
t OmM NaCitrate, 1 % (_- lutamic acid 6 4.6
lOmM NaCitrate, 2% L-I sine 6 1.1
t OmM NaCitrate, 0.5% l-as artic acid 6 0.4
lOmM NaCitrate, 0.1 % Phos hate lass 7 5.9
lOmM Tris, 100mM NaCitrate 8 8.5
10mM NaCitrate, 1 M GI cine ' 6 0
3
lOmM NaCitrate, 300mM GI cine 6 .
0.3
tOmM NaCitrate, 280mM GI cerol 6 0.3
lOmM NaCitrate, 0.5M NH4 2504 6 _
~ 8.3
lOmM NaCitrate, 120mM NH4 2S04 6 8.8
iOmM NaCitrate, 260mM Na2S04 6 9.4
lOmM NaP04, 0.19' Pho hatelass 7 15.8
lOmM NaCitrate, 0.1 /a SOS 6 1 1.2
lOmM NaCitrate, 0.02% SOS 6 7.8
lOmM NaAcetate, 8% PEG-400 5.5 13_7
lOmM NaAcetate,.1~50mM NaCI, 8% PEG-400 5.5 0.6
tOmM NaAcetate, 8% PEG-400 6 16_2
~
t OmM NaCitrate, 8% PEG-400 - 6 0.2
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The stability of TFPI stored at various pH conditions was tested. TFPI was
prepared by dialysis as above in 10 mM Na phosphate, 1S0 mM NaCI and 0.005
(wlv) polysorbate-80. Stability samples containing 150 mglmL TFPI were
incubated
S at 40iC for 20 days. Kinetic rate constant for the remaining soluble TFPI
was
analyzed by following decrease of the main peak on cation exchange
chromatograms.
As can be seen in Figure 5, the decay rate constant increases at pH above 6.0,
indicates more aggregation at higher pH conditions.
TFPI was also formulated at a concentration of 1S0 mg/mL.in 1S0 mM NaCI
and O.OOS 9b (w!v) polysorbate-80 at pH 7 with varying concentrations of
phosphate.
Figure SA shows the percentage of remaining soluble TFPi measured by the canon
exchange HPLC. Increasing concentrations of phosphate ion in solution resulted
in
higher levels of soluble TFPI remaining after incubation at 40°C.
Higher levels of
phosphate ion also resulted in higher levels of active TFPI as assayed by the
1 S prothrombin time assay. These results are shown in Figure SB.
Stability of TFPI at a concentration of O.S mg/mL and formulated in 10 mM
Na citrate, pH 6 and 1S0 mM NaCl was also tested at 40°C over a 40 day
period,
As seen in Figure 6, ration-exchange HPLC (triangle) shows the presence of
soluble
TFPI at levels greater than 60°~ initial, even after the 40 day
incubation. In like
24 manner, the prothrombin time assay (circle) shows the presence of active
TFPI at
levels greater than 609 initial, even after the 40 day incubation.
Figure 7 shows loss of soluble TFPI at 40°C measured by both cation-
exchange HPLC (open symbol) and prothrombin time assay (closed symbol) for 0.5
mg/mL TFPI formulated in 10 mM Na phosphate, pH 6 and either 150 mM NaCl
25 (triangle) or S00 mM NaCl (circle).
Figure 8 shows loss of soluble TFPI at 40°C measured by both cation-
exchange HPLC (open symbol) and prothrombin time assay (closed symbol) for 0.5
mg/mL TFPI formulated in 10 mM Na acetate and pH S.S containing 1S0 mM NaCI
(triangle) or 8l (wlv) sucrose (square) or 4.S ~b (w/v) mannitol (circle).
CA 02451969 2003-12-19
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Figure 9 shows two non-reducing SDS gels for TFPI formulation samples in
mM NaP04, 150 mM NaCI, and 0.005 to polysorbate-80 at pH 4 to pH 9 stored at
40°C for 0 days (lower) and 20 days (upper). No loss on TFPI is seen at
0 days.
However, at 20 days cleavage fragments of TFPI may be seen at the lower pH
range
5 (i.e. pH 4 and pH 5). Without being bound to a particular theory, it is
believed that
these fragments may result from an acid catalyzed reaction.
Finally, Table 2 shows the half Life of remaining soluble TFPI at
40°C for
various formulations. 0.5 mglmL TFPI was formulated in these formulation
' conditions and incubated at 40°C. Samples were withdrawn at
predetermined dme
10 intervals and loss of soluble and active TFPI were examined by the TEX-HPLC
and
the PT assay. half life for remaining soluble TFPI was then calculated by
performing a single exponential fitting to the IEX-HPLC and PT assay results.
Elution of TFPI in displacement mode from chromatography resins using
polyionic compounds.
API is first bound to a resin in a low salt buffer. Next a buffer containing
the polyionic compound used to elute TFPI in displacement mode, is pumped
through
the column. This compaund binds stronger to the resin than TFPI and displaces
TFPi. For a positively charged resin (anion exchanger) a negatively charged
compound is used and for a negatively charged resin (ration exchanger) a
positively
charged compound is used.
Partially purified TFPI was used as starting material. TFPI, in 6 M urea, 20
mM Tris, pH 8.0 was loaded onto a column packed with an anion exchange resin,
Q
Sepharose HP, to 20 mg/mL resin. After loading, .the column was washed with 6
M
urea, ~ 20 mM Tris, pH 9Ø TFPI was eluted and 10 mg/ml of Glass H
(polyphosphate) in 6 M urea, 10 mM Tris, pH 9Ø
Elution of TFPI from chromatography resin in aqueous buffer using polyionic
compounds.
CA 02451969 2003-12-19
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Table 2
t1!2 d a at 40C
0.5 m ml TFPI formulated in: (EX-HPi.C PT assa
mM Na Acetate, 150 mM NaCI, 10.8 17.2
pH 5.5
10 mM Na Citrate, 150 mM NaCI, 12.2 24.4
H 5.5 ~
10 mM Na Acetate, 8% w/v Sucrose,43.2 42.2
H 5:5
10 mM Na Acetate, 4.5% Mannitol, 47.7 46.8
H 5.5
10 mM Na Succinate, 150 mM NaCI, 7.8 11.0
pH 6.0
10 mM Na Citrate, 150 mM NaCI, 13.0 18.8
H 6.0
10 mM Na Phos hate, 150 mM NaCI, 7:8 1 1.2
H 6.0
10 mM Na Phosphate, 500 mM NaCI, 52.2 68.9
pH 6.0
10 mM Na Citrate, 150 mM NaCI, 10.0 14.8
H 6:5
CA 02451969 2003-12-19
-35-
For a positively charged resin, a positively charged compound is used and for
a negatively charged resin, a negatively charged compound is ~xsed.
TFPI, in 3.5. M urea, 1 mg~'ml polyphosphate, 50 mM Tris, pH 5.9 was loaded
onto a canon exchange resin, SP Sepharose HP. ~?.fter loading the column was
washed with a non-urea containing buffer, 10 mghnl polyphosphate, 10 mM sodium
phosphate, pH 5Ø TFPI was eluted in the same buffer at pH 7.5, without urea.
Example 7
Selective elution of TFPI from ion exchange resins using polyionic
compounds.
Because of the charged ends of TFPI, oppositely charged polyionic
compounds can bind to these ends. When the polyionic compound has a higher
strength of binding to TFPI than does the resin, the TFPI may be selectively
eluted
from the chromatography resin.
TFPI, in 3.5. M urea, 1 mg/m1 polyphosphate, 50 rnM Tris, pH 5.9 was loaded
onto a canon exchange resin, SP Sepharose HP. After loading, the column is
washed
with 6 M urea, lmg/ml polyphosphate, 10 mM sodium phosphate, pH 5.9. TFPI was
eluted in a 25 column volume gradient up to 20 mg/ml of polyphosphate. TFPI
starts
to elute at about 2-3 mg/ml of polyphosphate.
Example 8
Neutralization of pol.yionic compounds prior to chromatographic separation of
TFPI. TFPI can interact with charged polymers. This interaction may prevent
binding and purification to chromatographic resins. By neutralizing the
charged
polymer with an oppositely charged polymer, TFPI may bind to the resin.
In a buffer containing polyphosphate (Glass H), TFPI does not bind to Express
Ion ST'S (Whatman) and no purification is achieved. By mixing PEI into the
column
load, TFPI now binds to the resin and TFPI can be purified.
CA 02451969 2003-12-19
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Refolding and purification of recombinant human TFPI (rhT'FPI) using
Polyphosphate (Glass H) Facilitated Refolding Process.
Inclusion bodies containing about 40 g of rhTFPI were thawed by removing
the containers from the -20°C freezer and incubating them in a cold
room at 4-10°C
for approximately 196 hours. The thawed inclusion bathes were then dispersed
with
a high shear mixer to reduce the clumping that occurs during freezing. The
thawed
inclusion bodies were added to 80 L of 3 M urea, 50 mM Tris-Cl, pH 10.5 buffer
containing 2 g/L Glass H contained in a 100 L polyethylene tank equipped with
an
overhead stirrer. The contents were mixed for approximately 15 minutes, and
then
the absorbance of the solution is measured at 280 nm. If the absorbance is
greater
than the mixture was diluted with sufficient dissolution buffer to obtain an
absorbance
at 280 nm of 1.0-1.1. The solution was incubated with gentle agitation for 15-
30
minutes, and then sufficient cysteine was added to give a cysteine
concentration of
0.1 mM. The solid L-cysteine was dissolved in approximately 50 ml of purified
water and added to the refold mixture. The pH was checked and adjusted to pH
10.2
if necessary. The refold mixture was incubated with gentle agitation for 96-
120
hours.
After approximately 96 h, the refolding process was terminated by adjusting
the pH of the refold mixture to pH 5.9 using glacial acetic acid. Stirring was
continued for 90 minutes and the pH checked. More acid was added, if necessary
to
adjust the pH to 5.9 +!- 0.1. A two-step filtration process was used to remove
the
particulates that formed during previous steps and prepare the acidified
refold mixture
for SP-Sepharose HP chromatography. First the acidified refold mixture is
passed
through A Cuno 60LP depth filter (filter housing model 8ZP1P) using a
peristaltic
_ pump ('~ -'/a inch inner diameter silicon tubing).
The filter system was washed with 8-10 L of deionized 6 M urea before use.
The filtrate was collected in a 100 L polyethylene tank. Back pressure was
maintained at a constant 20 PSI. Initial flow rate for a new filter was
approximately
5-6 L per minute. Filters were replaced when the flow rate dropped below 1 L
per
CA 02451969 2003-12-19
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_37_
minute in order to maintain the back pressure at 20 PSI. T'he second stage of
the
filtration used a 0.45 micron filter cartridge (Sartorius Sartobran pH or
equivalent)
with a peristaltic pumping system. After filtration, the pH was checked, and
adjusted '
to pH 5.9 if necessary.
The acidified, filtered refold was loaded onto the equilibrated SP Sepharose
HP column at a flow rate of approximately 80.0 mllmin. Flow rate was adjusted
to
accommodate overnight loading of the acidified filtered refold mixture. The
column
was equilibrated in 6 M urea, 20 mM sodium phosphate buffer pH 5.9 prior to
loading. After loading, the column is washed with 2 CF of G M urea, 0.3 M
NaCI,
20 mM sodium phosphate buffer, pH 5.9 prior to the gradient elution step. The
column flow rate was increased to 190-200 ml/min for the wash step and all
subsequent steps (linear velocity =- 47 cmlhr). The product was eluted from
the
column using a linear salt gradient from 0.3 to 0.5 M NaCI in 6 M urea, 20 mM
sodium phosphate buffer, pH 5.9. The gradient was formed by delivering 6 M
urea,
0.5 M NaCI, 20 mM sodium phosphate buffer into 6 M urea, 0.3 M NaCI, 20 mM
sodium phosphate buffer, Limit buffer was pumped with a Masterflex pump (model
7553-20) with a Mastertlex head (model 7015.21) at a flow rate of
approximately 100
ml/min. with vigorous mixing using a Paratrol A. mixer from Parametrics (model
250210). The total volume of the gradient was 71.0 liters or 13.0 CV. The pH
of
the gradient buffers was 5.92 (+/- .02). Fractions are evaluated qualitatively
using
r SDS PAGE and pooled based on the content of the correctly folded SC-59735
relative
to other misfolds and impurities. After pooling the process stream is referred
to as
the S pool.
The pH of the S pool was next adjusted to pH 8.0 with 2.5 N NaOH. The S
pool was concentrated 2-3 fold to approximately 2 L using an Amicon DC-lOL
ultrafiltration unit containing an Amicon YM10 spiral cartridge (10,000 M.W.
cut-off
membrane). After concentration, the concentrated S pool was diafiltered
against 7
volumes of 6 M urea, 20 mM Tris-HCI buffer, pH 8Ø The diafiltration was
considered complete when the conductivity of the retentate was below 2 mS. The
diafiltered concentrate was drained from the ultrafiltration unit and the unit
was
CA 02451969 2003-12-19
WO 96!40784 PCT/US96109980
-38-
washed with approximately 1 L of diafiltration buffer. The was is combined
with the
concentrate to form the Q-load.
An Amican column (7.0 cm diameter) was packed with approximately 700 mI
of Q-Sepharose high performance medium (Pharmacia Q-Sepharose HP). The
column was packed with 209 ethanol at 20 psi. The bed height after packing was
approximately 18 cm. The column was equilibrated with 5 CF of 6 M urea, 0.02 M
TrisIHCl buffer; pH $. The target for protein laading is 8-10 mg proteinlml Q
Sepharose resin. The Q load was applied to the column at a flow rate 30-35
mUmin
(50 cm/hr). After loading, the column was washed with appraximately 5 CV of 6
M
urea, 20 mM Tris/HCl buffer, pH 8.0, or until the absorbance at 280 um
returned to
baseline. The product was eluted using a sodium chloride gradient from 0-0.15
M
NaCl in b M urea, 20 mM Tris/HCl buffer, pH 8.0 over 25 column volumes. The
first seven column volumes were collected as a single fraction, followed by 30
fractions of 0.25 column volume each.
Fractions are routinely analyzed by reducing and non-reducing SDS-PAGE
and size exclusion chromatography. Fractions are pooled based on aggregate
content
( < 5 °~ by SEC HPLC Method MSL 13929) and qualitative evaluation by
SDS PAGE
to assess purity. The fractions are stored frozen at -20°C until
pooled.
Acceptable Q Sepharose fractions were pooled, and the pH of the pool was
adjusted to 7.2 using 2 M HCI. The pool was then concentrated approximately 5
fold
in an Amicon DC-1 ultrafiltration system containing a S1Y1 Amicon YM-10
cartridge (10,000 MWCO spiral cartridge membrane). The concentrated Q Pool was
then diafiltered against seven column volumes of 2 M urea, 0.15 M NaCI, 20 mM
sodium phosphate buffer, pH 7.2. Following ultrafiltration, the solution was
drained
from the ultrafiltratian system. Approximately 100 mI of 2 M urea, 0.15 M
NaCI,
20 mM sodium phosphate buffer, pH 7.2 was circulated through the
ultrafiltration
system for approximately ~ min. The rinse solution was combined with the
original
concentrate and the solution was filtered through .a 0.45 micron vacuum filter
unit
(Nalgene).
CA 02451969 2003-12-19
WO 96/40784 PCT/US96/09980
-39-
Refolding and Purification of rhTFPI using Polyethyleneimine (PEI)
Facilitated Refolding Process.
Inclusion bodies containing about 40 g of rhTFPI were thawed by removing
the containers from the -20°C freezer and incubating them in a cold
room at 4-10°C
for approximately 96 hours. The thawed inclusion bodies were then dispersed
with a
high shear mixer to reduce the clumping that occurs during freezing. The
inclusion
body slurry was vigorously blended for approximately 1 minute using a polytron
homogenizer (Brinkman model PT45180) or until the inclusion bodies were then
added to 40 L of 6 M urea 100 mM TrisIHCI buffer pH 9.8 containing 300 mM
NaCl and 0.4 gIL PEI contained in a Z00 L polyethylene tank equipped with an
overhead stirrer. The mixture was vigorously stirred for 20-30 min. The pH was
monitored and adjusted to pH 9.8 as necessary. The absorbance of the dissolved
inclusion body mixture was measured at 280 nm, and if the absorbance was
greater
than 2.1, the sample was diluted with 10 liters of the dissolution buffer
described
above to obtain an A280 value of 2.0-2.1. Gentle agitation was continued for
another
15-30 minutes. Next, the dissolved inclusion body solution was diluted with an
equal
volume of 1.0 M urea, 300 mM NaCl solution. Finally, L-cysteine was added to
give a final concentration of 0.25 mM. The solid L-cysteine was dissolved in
50 ml
of WFI and added as a solution to the diluted refold. The pH was checked and
adjusted, if necessary. The refold continued with gentle mixing for 96-I20
hours
with periodic checks of the pH, and adjustment to pH 9.8, if necessary. The
progress
of the refold was monitored by Mon-S cation exchange and prothrombin time
assays.
After approximately 96 h, the refolding process was terminated by adjusting
the pH of the refold to pH 5.9 using glacial acetic acid. Sdrririg was
continued for
90 minutes and the pH checked. More acid was added, if necessary to adjust the
pH
to 5.9 /- 0.1.
A two-step filtration process was used to remove the particulates that formed
during previous steps and prepare the acidified refold for SP-Sepharose HP
chromatograph. First, the acidified refold is passed through a Cuno 60LP depth
filter
CA 02451969 2003-12-19
-40-
(filter housing model 8ZP1P) using a peristaltic pump (1/4 - 3/8 inch inner
diameter
silicon tubing).
The filter system was washed with 8-10 L of deionized 6 M urea before use.
The filtrate was collected in a 100 L polyethylene tank. Back pressure was
maintained at a constant 20 PSI. Initial flow rate for a new filter was
approximately
5-6 L per minute. Filters were replaced when the flow rate dropped below 1 L
per
minute in order to maintain the back pressure at 20 PSI. The second stage of
the
filtration used a 0.45 micron filter cartridge (Sartorius Sartobran pH or
equivalent)
with a peristaltic pumping system. After filtration, the pH was checked, and
adjusted
to pH 5.9, if necessary.
The acidified, filtered refold was loaded onto the equilibrated SP Sepharose
HP column at a flow rate of approximately 80.0 ml/mm. Flow rate was adjusted
to
accommodate overnight loading of the acidified filtered refold. The column
~~as then
washed with 5.5 column volumes of G M urea, 0.3 M NaCl, 20 mM sodium phosphate
buffer, pH 5.9. The column flow rate was increased to 190-200 ml/mm for the
wash
step and all subsequent steps (linear velocity = ~ 47 cm/hr). The product was
eluted
from the column using a linear salt gradient from 0.3 to 0.5 M NaC 1 in 6 M
urea, 20
mM sodium phosphate buffer, pH 5.9. The gradient vvas formed by delivering 6 M
urea, 0.5 M NaCI, 20 mM sodium phosphate buffer into 6 M urea, 0.3 M NaCl, 20
mM sodium phosphate buffer into G M urea, 0.3 M NaCI, 20 mM sodium phosphate
buffer. Limit buffer was pumped with a Masterflex'''M pump (model 7553-20)
with a
MasterflexTM head (model 7015.21 ) at a flow rate of approximately 100 mi/mm
with
vigorous mixing using a ParatrolT~ A mixer from Parametrics (model 250210).
The
total volume of the gradient was 71.0 liters or 13.0 CV. The pH of the
gradient
buffers was 5.92 (+/-.02).
Fraction collection was started when the column inlet conductivity reached
28.0 - 28.5 mS/cm as measured by the in-line Radiometer conductivity meter.
Forty
500 ml fractions (0.1 CV) were collected. A Pharnnacia Frac-300 fraction
collector
was used with numbered, 500 ml polypropylene bottles. When the fi-action
collection
was stopped, the remainder of the gradient was collected as a pool.
CA 02451969 2003-12-19
WO 96140784 PCT/US96/09980
_41
Column fractions were assayed by A280, size exclusion HPLC, and in
addition; for informational purposes, SDS PAGE, reverse phase HPLC, and PT
assays. Fractions were pooled if they met the pooling criteria of containing
20 % of '
less aggregate as determined by the in process SEC HPLC: Pooled SP Sepharose
fractions are referred to as the S Pool.
The pH of the S-pool was next adjusted to pH 8.0 with 2.5 N NaOH. The S
Pool was concentrated 2-3 fold to approximately 2 L using an Amicon DC-lOL
ultrafiltration unit containing an Amicon YM10 spiral cartridge (10,000 N.W.
cut-off
membrane). After concentration, the concentrated S Pool was diafiltered
against 7
volumes of 6 M urea, 20 mM Tris-HCl buffer, pH 8Ø The diafiltration was
considered complete when the conductivity of the retentate was below 2 mS. The
diafiltered concentrate was drained from the ultrafiltration unit and the unit
was
washed with approximately 1 L of diafiltradon buffer. The was is combined with
the
concentrate to form the Q-load.
An Amicon column (7.0o cm diameter) was packed with approximately 700
ml of Q-Sepharose high performance medium (Pharmacia Q-Sepharose HP). The
column was packed in 20 % ethanol at 20 psi. The bed height after packing was
approximately 18 c.m The column was equilibrated with 5 C'V of 6 M urea, 0.02
M
TrisIHCl buffer, pH $. The target for protein loading is 8-10 mg protein/ml Q
Sepharose resin. The Q load was applied to the column at a flow rate 30-35
mUmin
(50 crn/hr). After loading, the column was washed with approximately 5 CV of 6
M
urea, 20 mM Tris/HCl buffer, pH 8.0, or until the absorbance at 280 nm
returned to
baseline. The product was eluted using a sodium chloride gradient from 0-0.15
M
NaCI in 6 M urea, 20 mM Tris/HCl buffer, pH 8.0 over 25 column volumes. The
first seven column volumes were collected as a single fraction, followed by 30
fractions of 0.25 column volume cach.
Fractions are routinely analyzed by reducing and non-reducing SDS-PAGE
and size exclusion chromatography. Fractions are polled based on aggregate
content
(5 % by SEC HPLC) and qualitative evaluation by SDS PAGE to assess purity. The
fractions are stored frozen at -20°C until pooled.
CA 02451969 2003-12-19
WO 96/40784 PCT/US96/09980
-42-
The Q-Sepharose fractions to be pooled were thawed by incubation at 2-
8°C,
pooled, and the pH of the pool was adjusted to 7.2 using 2 MHCI. The pool was
then concentrated approximately 5 fold in an Amicon DC-1 ultrafiltration
system
containing a SIYl Amicon YM-10 cartridge (10,000 MWCO spiral cartridge
membrane). The concentrated Q Pool was then diafiltered against seven column
volumes of 2 M urea, O. IS M NaCI, 20 mM sodium phosphate buffer, pH 7.2.
Following ultrafiltxation; the solution was drained from the ultrafiltration
system.
Approximately 100 ml of 2 M urea, 0.15 M NaCI, 20 mM sodium phosphate buffer,
pH 7.2 was cizculated through the ultrafiltration system for approximately 5
min.
The rinse solution was combined with the original concentrate and filtered
through a
0.45 micron vacuum filter unit (Nalgene).
Fxampl~e~1
Solubilization, refolding, and purification of rhTFPI from inclusion bodies
using polyphosphate in the absence of chaotropes such as urea (GDS 5327089,92)
About 2 g of rhTFPI (43 ml inclusion body slurry containing 46 mgl ml
rhTFPT) was dissolved with mixing in 4 L of 50 mM Tris buffer, pH 10.5
containing
4 gll polyphosphate (Glass H, FMC Corporation) 2-8°C. Sufficient
cysteine and
cystine was added to make the solutions 0.1 mM and 0.05 mM respectively. The
pH
was maintained at pH 10.5 with 1 N NaOH. The refold solution was incubated at
2-
8 ° C with gentle mixing for ?2-96 h.
The refold was next adjusted to pH 6 using glacial acetic acid and then
filtered
thmugh a 0.2 micron filter. An aliquot of the filtered refold was applied to a
200 ml
column of SP-Sepharose HP (Pharmacia) previously equilibrated in 0.4 % Glass
H,
20 mM sodium phosphate pH 6 buffer after loading, the column was washed with 4
column volumes of 0.4 % Glass H, 20 mM sodium phosphate pH6 buffer. The
column was eluted using a linear pH gradient from 0.4 % Glass H, 20 mM sodium
phosphate buffer pH 6 to 0.4 % Glass H, 50 mM Tris pH 8 buffer. Fractions were
collected and analyzed by SDS PAGE. Relatively pure rhTFPI could be refolded
and
purified in this manner.
CA 02451969 2003-12-19
WO 96/40784 PCT/US96/09980
43 -
Improved solubility of rhTFPI in water by formation of a complex between
TFPI and polyphosphate (GDS 532?046-4?) '
About 10 g of purified rhTFPI in about 1 liter of 2 M urea, 125 mM sodium
chloride, 20 mM sodium phosphate pH 7.4 buffer was thawed by incubation at 2-
8°C
for 18-36 h. Sufficient dry urea was added to make the solution 6 M in urea.
The
solution was then filtered through a 0.2 micron filter. Five g of
polyphosphate glass
(Glass H, FMC) was dissolved in 50 ml of b M urea, adjusted to pH ? with 1 N
NaOH, and added to the protein solution. The solution was then concentrated by
ultrafiltration using 1 square foot of membrane (Amicon S 1 Y3) to about 400
ml (-25
mg/ml) and diafiltered against 10 volumes (about 4 liters) of purified water
to remove
residual urea. After diafiltration, the solution was concentrated to about 250
ml and
removed from the ultrafiltration unit. The ultrafiltradon unit was washed with
about
150 ml of purified water and the was added to the protein concentrate. The
final
protein concentrate contained almost 10 g of protein in 400 ml of water (about
24
mg/ml.protein). The normal solubility of rhTFPI in water is less than 0.5
mg/ml.
Use of cationic polymers for removal of E. coli contaminants from TFPI cell
lysates and refracdle bodies.
The use of cationic polymers to precipitate and remove E coli contaminants
from crude TFPI intermediates (lysates, refractile bodies) can significantly
improve
subsequertce process operations (refolding, chromatography etc.) A random
screening of cationic polymers identified candidates which selectively
precipitate
bacterial contaminants while TFPI remains in solution. Specifically, Betz
polymer
b24 precipitated substantial amounts of bacterial contaminants, while leaving
TFPI in
solution in an aqueous environment.
Solubilized TFPI refractile bodies (in 3.5 M guanidine hydrochloride, 2 M
sodium chloride, 50 mM TRIS, 50 mM dithiothreitol, pH?.l) was the starting
material used for a polymer screening experiment. This material was diluted 10
fold
into a 0.5 % solution of various polymers. The precipitates from this
experiment
CA 02451969 2003-12-19
WO 96140784 PCT/US96I09980
-44-
were analyzed by SDS-PAGE for the presence of TFPI. Betz polymer 624
precipitated substantial amounts of contaminants, no TFPI, and resulted in a
clear
aqueous solution.
The use of aqueous two phase extraction with a polyethylene glycol (PEG),
polyphosphate, urea system offers processing advantages for TFPI purification.
Typical aqueous two phase systems consist of two polymer systems (e.g., PEG
and
dextran) or a polymer and salt (e.g., PEG and sulfate). The system described
here
has advantages in that the polyphosphate chain length can be optimized for the
separation, is inexpensive and is specific in removing problematic
contaminants from
TFPI refractile bodies known to interfere with refolding and chromatography
(native
polyphosphate and associated divalent metals).
TFPI refractile bodies were solubilized in 7 M urea, I0 mM CAPS; 1
monothioglycerol pHlO. Polyphosphate and PEG of different chain lengths were
added to form two phases. hTpon phases separation, the TFPI partitioned into
the
PEG rich upper phase, leaving the polyphosphates and associated contaminants
in the
lower phase. Separation is effected by both PEG and polyphosphate chain length
and
can be optimized by varying both.
Charged polymer facilitated refolding of recombinant tissue plasminogen
activator (t-PA) from E. coli inclusion bodies
Five grams (wet weight) of inclusion bodies containing about 2 grams of
recombinant tissue plasminogen activator are added to about 1 liter of 0.5 %
Glass H,
50 mM Tris buffer pH 10.8 containing 1 mM reduced glutathione (GSH) and 0.2
mM glutthione disulfide (GSSG). The mixture is thoroughly blended using a
polytron (Brinkman) homogenizes for 2-3 minutes to thoroughly disperse the
inclusion bodies. The mixture is incubated with mixing using an overhead
stirrer for
15 minutes while the pH is maintained at 10.5-10.9 using I N NaOH. The mixture
is
then gently mixed for 48-72 hours at 2-8oC.
Fxamy_t~ 1 C,
CA 02451969 2003-12-19
-45-
Charged polymer facilitated refolding of hovine somatotropin from ~. coli
inclusion bodies
Ten grams (wet weight) of inclusion bodies containing 5 grains of bovine
somatotropin are added to about 1 liter of 1% Glass H, 50 mM Tris buffer pH
10.5.
The mixture is thoroghly blended using a polytron (Brinkman) homogenizer for 2-
3
minutes to thoroughly disperse the inclusion bodies. The mixture is incubated
with
mixing using an overhead stirrer for 15 minutes while the pH is maintained at
10.4
10.6 using 1 N NaOH. Solid cysteine (121 mg) is added to make the reaction 1
mM
cysteine, and the refolding reaction is mixed for 48-72 hours
The present invention has been described with reference to specific
embodiments. However, this application is intended to cover those changes and
substitutions which may be made by those skilled in t:he art without departing
from
the spirit and the scope of the appended claims.
