Note: Descriptions are shown in the official language in which they were submitted.
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w~ 96140784 PCTI~JS96~099$0
U y
Technical Field of the Invention
The invention relates to methods useful for refolding, solubilizing,
formulating
and purifying proteins. °I~hese methods are particularly useful for
proteins that have been
engineered by genetic recombination and produced i;n bacterial, yeast or other
cells in a
form that has a non-native tertiary structure.
Back r~oun~ of the Invention
To understand fully the entire process of gene e~cpressyon, 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 ~I. (
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 firr:~te, but very Low, stability of Local structures could
be the kinetic
"proofreading" mechanism of protein folding. The activated state of foldung
with the
highest energy is a distorted form of the native protein, and the slowest,
rate-Limiting
step of unfolding and refolding appears to be close to the native state in
terans of ordered
structure. In addition, the refolding of many proteins is not completely
reversible in
a'i1r~, and reactivation s2elds of Less than 100% are ~c~equent>ly observed,
which holds tnre
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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_2~
lowered reversibility, as described in Fischer and Schmid, (i990)
Biochertaistrjr 29:2205-
2212.
In the case of su~ciently large protein snolecuies, the nascent polypeptide
chain
acquires its native three-dimensional structure by the modular assembly of
micro-
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 eosolvents may be the
result of direct
binding or the alterations of the physical properties of water, as described
in Jaenicke e~
al. (1981) ~iochemlstry 30 (13):3I~7-3161.
Experimental observations of how unfolded proteins refold to their native
three-
dimensional structures contrast with rrrany popular theories of protein
folding
mechanisms. Under conditions which allow for refolding, unfolded protein
molecules
rapidly equilibrate between different conformations prior lo complete
refolding. The
rapid prefolding equilibrium favors certain compact conformations that have
samewhat
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 transiti~n through which
essentially all
molecules fold, as described in ~reighton et al. (198) Pn°oc. .fiat.
~4cac1 d~rci. f7,SA
85:5082-5086.
~larious methods of refolding of purified, recombinantly produced proteins
have
been used. For example, the protease encoded by the human immunodeficiency
vines
type I (HIV-I) can be produced in Fscherichia cola, yielding inclusion bodies
harboring
the recombinant HIV I protease as described by hlui et al. (1993) ,L 1'r~t.
Cherar.l2:
323-327. The purified ~il~l-I protease was refolded into an active enzyme by
diluting a
solution of the protein in 50% acetic acid with 25 volumes of buffer at phI
5.5. It was
found that a higher specific activity of protease was obtained if the purified
protein was
dissolved at approximately 2 mg/ml in 50°/~ acetic acid followed by
dilution with 25
volumes of cold 0.11VI sodium acetate, phi 5.5, containing 5% ethylene glycol
and 10%
glycerol. Exclusion of glycerol and ethylene glycol Led to gradual loss of
protein due to
precipitation. About 85 mg of correctly folded hiI'~JaI protease per Liter of
~:. codi cell
culture was obtained by this method, and the enzyme had a high specific
activity.
CA 02450800 2003-12-19
Another example of refolding a recombinant protein is the isolation and
refolding of H-ras from inclusion bodies of E. roll as described by DeLoskey
et al.,
(1994) arch. Biochem. an,d 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 mgiml 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) Protein Expression and Puri~catioh 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 ration
exchange chromatograpl~ay 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 Prothombin clotting time assay
comparable to
TFPI purified from mammalian cells.
A non-glycosylated form of TFPI has also been produced and isolated from
Escherichia roll (E. coil) cells as disclosed in U.S. Patent No. 5,212,t)91.
The
invention described in (.T.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 ration 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. roll 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 TF.PI during the refolding process. A need also
exists for
increasing the solubility of I. Presently the yields of recombinantly produced
TFPI
have been lower than desirable, and a need exists in the art of
pp°oducing correctly folded
TFPI. See for example Gustafuson et al. ( 1994) Prvtei'r ressior~ and
Purification S:
233-241.
TFPI inhibits the coagulation cascade in at least tyro ways: preventing
formation
of factor VIIaltissue factor complex and by binding to the active site of
factor Via. The
- primary sequence ~f TFP'I, deduced from cD?~TA sequence, indicates that the
protein
IO contains three Kunitz-type enzyme inhibitor domains. °The first of
these domains is
required for the inhibition of the factor ~IIIaltissue factor complex. The
second Kunitz-
type domain is needed for the inhibition of factor Xa. ~'he function of the
third Kunitz-
type domain is unknowra. TFPI has no lonourn enzymatic activity and is thought
to
inhibit its protease targets in a stoichiometric manner; na~raely, binding of
one T~'PI
Kunitz-type domain to the active site of one protease molecule. °The
carboxy-~ternunal
end of TFI.'I is believed tc have a role in cell surface localization via
heparin binding and
by interaction with phospholipid. fiFFI is also known as Lipoprotein
A.~soclated
Coagulation Inhibitor (LACI), Tissue Factor Inhibitor (TFI), and Extrinsic
Pathway
Inhibitor {EPI).
ll~ature TFPI is 2~6 amino acids in length with a negatively charged amino
terminal end and a positively charged carboxy-terminal end. TAI contains 1S
cysteine
residues and forms 9 disulphide baidges when correctly falded. T'he primary
sequence
also contains three Asn-X-Ser/Thr I~-linked glycosylation consensus sites, the
asparagine residues located at positions 145, 155 and 256. The carbohydrate
component of mature TF'PI is approximately 30°l° of the mass of
the protein. however,
data from proteoiytic mapping and mass spectral data imply that the
carbohydrate
moieties are heterogeneous. , I is also found to be phosphorylated at the
serine
residue in position 2 of the protein to varying degrees. °Fhe
phosphorylation does not
appear to affect TFPI function.
TFPI has 'been isolated frcsm human plasma and from human tissue culture cells
including F~iepG2, Char~g liver and SK hepatoma cells. F~ecombinant TFIaI has
been
CA 02450800 2003-12-19
~h'~ 96140784 PCTI~JS96I09980
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expressed in mouse C127 cells, baby hagnster kidney cells, Chinese hamster
ovary cells
and human SK hepatoma cells. Recombinant TFPI from the mouse 0127 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
fromErcherrchia coli (E. ø:o11) cells as disclosed in ~T.S. Pat. l~To.
5,2~12,09I. This form
of TFPI has been shown to be active in the inhibitioa~ of bovine factor %a and
in the
inhibition of human tissue factor-induced coagulation in plasma. Methods have
also
been disclosed for purification of TFPI from yeast cell culture medium, such
as in
Petersen et al, J.BioLChem. 18:13344-13351 (1993).
Recently, another protein with a high degree of stru~~tural identity to 3.~PI
has
been identified. Sprecher et al, Prop. lVat. Acid. Sci., IJSA 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 Iinkagzs, an acidic amino
terminus and
a basic carboxy-terminal tail. 'The three Kunitx-type domains of fiFPI-2
exhibit 43%,
I~ 35% and 53% primary sequence identity with TFPI Kunitz-type domains I, 2,
and 3,
respectively. Recombinant TFPI-2 strongly inhibits the anudolytic activity of
factor
'Tila/tissue factor. ~y contrast, TFPI-2 is a weak inhibitor of factor Xa
amidolytic
activity
TFPI has been shown to prevent mortality in a Iethal ~cherichi~a colt (~:
colt)
septic shock baboon model. Creasey et al, J. Clin. Invest. 91:2850-28b0
(1993).
Administration of TFPI at 6 mg/kg body weight shortly after infusion of a
Iethal dose of
~ colt resulted in survival in alI 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
2S coagulation response, of various measures of cell injury and significant
reduction in
pathology normally observed in E colt sepsis target organs, including kidneys,
adrenal
glands, and lungs.
Due to its clot-inhibiting properties, TFPi rnay also be used to prevent
thrombosis during microvascular surgery. For example, I).S. 5,27b,O1~
discloses the
use of TFPI in a method for reducing thrombogenicity o!° microvascular
anastomoses
CA 02450800 2003-12-19
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wherein TFPI is administered at the site of the microvascular anastomoses
contemporaneously with microvascular reconstrczctior~.
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 wruch may benefit from administration of high doses
of TFPI.
Thus, a need exists in the art for pharmaceutically ac<;eptable compositions
containing
concentrations of TFPI which can be administered to patients in acceptable
amounts.
Brief Descri tion of the Drawings
Figure 1 is a coomassie stained SDS-PAGE analysis of TFPI peak fractions
from the Phenyl SepharoseTM HIC refolding procedure.
1 S 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 TFPT at different pH conditions. About 10
mg/mL TFPi in 2M urea was dialyzed against 20 mM acetate, phosphate, citrate,
glycine, L-glutamate grad succinate in 150 mM NaCl. The concentration of
remaining soluble TFPI after dialysis was measured by TTtT 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 i'. TFI'I solubiity increases with
increasing concentration of citrate.
Figure 7 shows the solubility of TFPI as a function of concentration of NaCl.
TFPI solubiity increases with increasing salt concenta-ation, 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 arid 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 cation exchange chromatograms.
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w~ 96e40784 PI~1S96e~998~
_7_
Figure 9 shows the percentage of remaining soluble 'fFPI measured by cation
exchange HPLC (A) and remaining active 'TFPI by prothrombin time assay (B) as
a
function of phosphate concentration. Z'he foraraulation contains 1S0 mglmL
TFPI
prepared in 1S0 mIVI NaC1 and 0.005 % (wlv) polysorbate-80 at pH '~ with
'varying
concentrations of phosphate.
Figure 10 shows loss of soluble fiFPI at 4(1 ° C rr~easured by both
canon-
exchange HPLC (triangle) and prothrombin time assay (circle) for O.S
mg/mL'IFPI
formulated in, l0 mlvl Na citrate, pH 6 and 1S0 mM NaCl.
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/rnL TFPI formulated in 10 m~I Na phosphate, pH 6 and either 150 m:M NaCI
(triangle) or 500 mHi NaCl (circle).
Figure 12 shows loss of soluble '1'FPI at 40°C measured by both
cation-
exchange HPLC (open symbol) and prothrombin time assay (closed symbol;f for
0.5
mg/mL T'FPI formulated in 10 mN! l~Ta acetate and pH S.S containing 150 mM
NaCI
(triangle). or 8 ~ (w/v) sucrose (square) or 4.5 ~ mannitoi (circle).
Figure i3 shoves two non-reducing SIBS gels f~r 'I'FPI formulation samples at
pH 4 to 9 stored at 40°C for 0 and 20 days.
Figure 14 shows the time course of a polyphosphate-facilitated rh'1'FPI refold
monitored using SDS Pa~GE.
Figure 15 shows the absorbance at 280 em during the loading and elution of
the S-Sepharose HP column used to purify rhT'FPI from a polyphosphate-
facilitated
refold.
Figure 16 shows SDS PAGE analysis of fractions collected during elution of
2S the S-Sepharose HP column used to purify rhTFPI from a polyphosphate-
facilitated
refold. Figure 17 shows the absorbance at 280 em during the loading and
elution of the ~-Sepharose HP column used to purify rhTFPI from a S-Sepharose
pool prepared from a polyphosphate-facilitated refold.
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W~ 9614~~8~1 P~'I'/CJS96If199t30
Figure 18 shows SDS PAGE analysis of fractions collected during elution of
the Q-Sepharose HP column used to purify rhTFPI fro:~n 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-Sepharose coluann used to purify rhTFPI from a
polyethyleneimine-facilitated refold,
Figure 21 shows SDS PAGE analysis of fractions collected during elution of
the S-Sepharose HP column used to purify rhTFPI from a polyethyleneimane
facilitated refold.
Figure 22 shows the absorbance at 280 nm during the loading and elution of
the Q-Sepharose FiP column used to purify rh°'I FFPI from a
S~~Sepharose pool prepared
from a polyethyleneimine-facilitated refold.
Figure 23 shows SDS PAGE analyst's of fractions collected during elution of
the (~-Sepharose FIP column used to purify rhTFPI from a S~Sepharose pool
prepared
from a polyethyleneinW e-facilitated refold.
Figure 24 shows thd canon exchange I'IPLC analysis of a ~.4 ~b
polyphosphate-facilitated rhTFPI refold in the absence of urea.
Figure 25 shows aesults of cation exchange HPLC analysis of an evaluation of
different levels of cysteine on a rhTFPI refold in 0.4 ~ golyphosphate, 50 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
cation
exchange IiPLC.
_ Figure 2°7 shows the effect of concentration of polyphosphate (Glass
I~ on the
refolding of rhTFl?I from inclusion bodies as monitored by catlon exchange
HPLC.
Figure 28 shows the canon exchangc HPLC analysis of polyethylenein~ine
and polyphosphate-facilitated refolding of purred and reduced rhTFPI.
CA 02450800 2003-12-19
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Summary of the Invention
It is an object of an aspect of the present in~ventior~ 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.
I0 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
IS 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 iai light of the
hydrophobic
20 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 compositio:r~s 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
25 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
30 (SDS) as well as charged polymers. In some compositions, TFPI may be
present in
concentrations of more than I mg/m~ 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, rnannitol, or
sorbitol.
CA 02450800 2003-12-19
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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 car.. increase the solubility by ;~. factor of I00. The
solubility is
very dependent on the concentration of arginine, as 300 mM is about 30 tunes
more
effective than 200 mM. ZJrea also is quite effective i:n soluhilization 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
I O 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/rnodifzed monomeric forms of TFPI are also
removed during this process. The separation e~~nploys hydrophobic interaction
I S chromatography. The oligomeric species of TFPI bind more tightly to a
hydrophobic
resin than does the active TFPI monomers. Resins such as PharmaciaTM octyl
sepharose and ToyopearlTM butyl 650-M have been successful. The process is
carried
out in the presence of Iugh salt, such as 1 M ammonium sulfate or 0.5 M sodium
citrate.
20 In accordance with one aspect of the present irmention, there is provided
an
aqueous formulation comprising TFPI and a charged polymer wherein the
concentration of 'fFPI is greater than 1 mg/ml.
Detailed Description of the Invention
25 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
30 solubility improved by masking one of the charged regions with polycations
or
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polyanions. harriers to conformational flexibility an ~. 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
solubiiize
and maintain solubiity during purification operations ~;an be solubilized and
processed
15
25
PffPrtivPlv neina mnlvinnc
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Iviany 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 interaction with ch'~rged polymeric
templates. 1'he
nature of the modification will depend on the specific pmtein structure, the
chain
length, charge, and charge density of the ionic polynraer.
~Ve have characterized refolding of pure TFPI in a guanidine or a urea based
refolding buffer and the results indicate that refolding cff ciencies and
kinetics can be
significantly improved by the addition of charged polymers including heparin,
dextran
sulfate, polyethyleneimine (PEI) and polyphosphates. 'These polymers increase
'T°FPI
solubility and enhance re9:olding through ionic interacaions with either the
I~-terminus
~ or the C-terminus. In addition to the polymer additives, refolding pure
1"FPI requires
a cysteinelcystine redox buffer where the refolding reaction can be completed
within
4g hours. Refolding yields are a strong function of pFI, a~edox 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 ~r aulfated polysaccharides such as, for example, heparin
and
dexttan sulfate, to a solution containing a denatured protein prior t~
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 Dlmi'A 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 c~li and several other expression :9yStems, 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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_g2_
conformation that charged residues of different parts of the amiho 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 Portion 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
Iimit the access of solve~at 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 wF~rk 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 :~~nic 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 Iength) of the charged
polymer may vary depending on the specific protein. Suitable polymers include:
24 sulfated polysaccharides" heparins, dextran sulfates, agaropectins,
carboxylic acid
polysaccharides,alginic acids, carboxymethyl celluloses, polyinorganics
,
polyphosphates, polyarninoacids, polyaspartates, polyglutamates,
polyhistidines,
polyorganics, polysaccharides,I~Er~E ~extrans, polyorganic amines,
polyethyIeneinimes, pc~lyethyleneinime celluloses, polyamines, polyamino
acids,
polylysines, and polyarginines.
>proteins with pI greater than 7 may bene;f t more from interactions with
negatively charged polymers, as these proteins will have a positive charge at
phi 7.
Proteins with pIs below 7 may interact more strongly with positively charged
polymers at neutral pli. Changing the solution pH will modify the total charge
and
charge distribution of agcy protein, and is another variable to be evaluated.
CA 02450800 2003-12-19
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_I3-
Recombinant L~I~1~. technology has allowed the high revel expression of many
proteins that could not normally be isolated from natural sources in any
appreciable
quantities. In ~. coli and several ether 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
andlor repulsion between Iike 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 1 ~ Lion in the primary sequence can still demonstrate areas
of
charge locaiization due to their secondary structure found a refolding
intermediates,
misfolds and improperly folded protein. Proteins which are particularly
suitable
according to the present invention include but are not Iirnited to TFPI, TPPI
muteins,
TFPI-2, tissue plasminogen activator, PST, PST. ile: 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, oligomerizecl, 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 "Y'FPI, 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.
CA 02450800 2003-12-19
W~ 9614784 PCT/US96/a9980
14-
Charged polymers can be used to modify the; charge, charge density, and
reduce or eliminate ionica:lly mediated limitations to c:onformation that may
arise in
the unfolded state. °L°he ,juxtaposition of charged groups that
are not norrru~lly 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 process9 second, the additicn of the charged polymer
may
enhance the solubility of ~:he unfolded protein, reducing or eliminating the
need for
chaotroges which have a negative effect on protein conformation.
Eecause 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 '~ will possess a net negative charge, and will
thus be rraore
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. ~ther variables, such as pH and solvent
ionic
strength, would also be evaluated. Initial screening would involve
polyethyleneimine,
I)EAE dextran, dextran sulfate, and polyphosphate at several different
concentrations
and molecular weights. ~~ork with rhTFPI has demonstrated the significant
impact
that polyphosphate chain length and concentration can have on the course of
the TFPI
refolding reaction. I~elatuvely short chain length (n~5) produces high levels
of
CA 02450800 2003-12-19
w0 96140784 PCTJUS96/09980
-15-
aggregate. The optimal polyphosphate chain length for refolding rh "PI was
approximately 25 repeating units. Longer chain length polyphosphates (n = 75)
also
produced more aggregate and less properly folded monomer.
atinn
lProteins consist of chains of amino acids, the exact compositeon and sequence
of which constitutes one; of the prirnary structure determinants of the
proteirm. The
secondary deternunant 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
p-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. 'I°he functional
form of a
protein is generally a moaiestly stable conformation held together by a
combination of
cysteine disulfide bonds, ionic bonds, and hydrophobic and 'Van tier VVaads
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 solutioru holypeptides 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 secondar~r 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.
CA 02450800 2003-12-19
W~ 96140786 PC'i"//~1S96I099~0
~ 16-
~.°he terns °~pr ssin" used herei~t refers to the steps
ins~lveet in the
purgfic~t~~n and prep lion of pharm~ceut~ca611y-accep 1e amounts of proteins.
CA 02450800 2003-12-19
~ l l -
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 onium.
The term "negatively charged polymer" as. used herein refers to polymers
containing chemical groups which carry, car carry, or can be modified t;o
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
Z 0 acids, formic and other carboxylic acids
Tlae term "polyethyleneimine" ac; used herein refers to polymers consisting of
repeating units of eth~rlene imine (H~N+-(CH2--CH2-NH2+)x-CH2-CH2-NH3+).
The molecular weight can vary from 5,000 to greater than X0,000.
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 (pyropl:aosphate) to several thousand.
Polyphosphate is frequently referred ~~ as sodium hexametaphosphate (SHMP).
~ther common names include Grahams salt, Cal'.gorirM, phosphate glas:9, sodium
tetrametaphosphate, and Glass HTM.
The term "refold" as used herein refers ~o 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 ,~ refolding step. A
folded
protein molecule is most stable in the conf~rmatic>n that has the least frc;e
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 trc,atments that cause a
polypeptide
to unfold, i.e. denature. .A folded proteins is the product of several types
of
interactions between the amino acids themselves and their environment,
including
ionic bonds, Van den VVaals interactions, hydroren bonds, disulfide bonds and
covalent bonds.
CA 02450800 2003-12-19
w~ 96!40784 PC°TILJS96I09980
!g
The term "denat~are~ as used herein refers to the i:reatment of a protein or
poI tide in which results in the disruption of the :ionic and covalent bonds
and the
clan der Wails interactions which exist in the molecule in its native or
renatured
state. I)enaturation of a protein be accomplished, for example, by treatment
with
S urea, reducing agents such as mercaptoethanol, heat, pI~, temperature and
other
oxidation of the -S-:li groups present on cysteine residues for the protein to
reform the
disulfide bonds.
The term "glycc~saminoglycan" as used hcrrein refers to polysaccharides
containing alternating residues of uronic acid and ;taexosamine and usually
contain
sulfate. The binding o~ a protein in a refolding reaction as described herein
to a
glycosaminoglycan is through ionic interactions.
The teran "dex n sulfate" as used herein refers a polyanionic derivative of
dex , ranging in molecular weight from S, to X00, tons. hex s are
polymers of glucose in which glucose residues are jcein ' y X1,6 ° ges.
The term °hepa ' " as a herein refers to 2 giucoaminoglycans or
h ' oids which are bash on a re ting disaccharide (-4vCalcA(p)~l, 4C~lc~TAc~1~
)m but are subject to extensive modification after assembly. I3eparin is
stored with
histamine in mast cell granules and is thus found in rr~ost connective
tissues. im general
heparins have shorter chains than heparin.
The term "~iIC" 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 aru other contaminants fcom
the
refolded product.A.
Negatively charged polymers include sulfated F~olysaccharides, such as
heparins,
dextrin sulfates, and agiropectins, as well as carbcixylic acid
polysaccharides such
isalginic acids and ca~o ethyl celluloses. Polyinorg; °es such as
polyphosphates are
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- i9 -
also included. Polyamino acids such as polyasparatate" polyglutamate, and
polyhistidine
can also be used.
Positively charged polymers include polysaccharides such as ~ dextran,
polyorgnic amines, such as polyethyleneimines, polyethyleneimine celluloses,
and
polyarnines, as well as the poly °no acids, polylysine: and
polyarginine. combinations
of polymers may be used, of either charge polarity. In addition, amphoteric co-
F>olymers
may also be used.
As used herein, " r' refers to mature 'Tisane Factor Pathway Inhibitor. As
noted above, I is also kno in the art as Lipoprotein Associated Coagulation
i0 Inhibitor (LA~I), extrinsic Pathway Inhibitor (API) and Tissue Factor
Inhibitor (TFI).
hduteins of I which retain the biological activity of I are encompassed in
this
definition. Further, I which has been slightly modified for production in
facterial
cells is encompassed in the definition as well. For example, a 1"FF9I analog
has arA aIanine
residue at the amino-terminal end of the I pollypeptide has been produced in
~scherica~ c~li. See LJ.:~. 5,212,091.
As used herein, "pharmaceutically acceptable composition" refers to a
composition that does not negate or reduce the biolol;ical activity of
f~rmulated °TFP'I,
and that does not have any adverse biological effects when foamulated 'TFI'I
is
administered to a patient.
As used herein, "patient" encompasses human and veterinary patients.
As used herein, the terrra "solubiIizer" refers to salts, ions, carbohydrates,
°no
acids and other organic molecules whibh, when present in solution, increase
the
solubility of I above 0.2 mg/ . Solubilizers ma:y also raise the
concentrations of
I above 1 mglmL, and above 10 mg/mL. It should tae 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
I molecule (e.g. by aciid catalyzed reactions).
As used herein, the term "secondary solubilizE;rs" refers to organic salts,
ions,
carbohydrates, amino acids and other organic molecule:e which, when present in
:solution
with a solubilizer, further increase the solubility of 1'FPI. Secondary
solubilizers may
CA 02450800 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 ~.s 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 ~n~lo.6,103,500, muteins of 'rFPI and TFPI-2, with
single or
multiple point mutations, and chimeric molecules of 7~FPI arid TFPI-2 can be
prepared.
For instance, the lysine residue in the P 1 site of the :first I~unitz-type
domain of TFPI
may be replaced with ;~rginine. Muteins, containing one to eve amino acid
substitutions, may be prepared by appropriate mutagenesi.s of the sequenf;,e
of the
recombinant cloning vehicle encoding TFPI or TFPI:-2. Techniques for
mt~tagenesis
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) Aazna~al Review orf Geneta'~s9 19:423, and
modifications of
I5 some of the techniques are°, described in 1VTETHODS 1N
EN~~iOLOGSc'', 154, part E,
(eds.) Wu and Grossman r~1987j, chapters 17, 18, I9, and L',0. 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
I~~°amer, et aL, in
chapter 17 of the Methods in Enzymology, above. .!mother technique for
generating
point mutations in a nucleic acid sequence is overlapping PCIZ. The procedure
for ,
using overlapping PCR to generate point mutations is described by Higuchi in
Chapter
22 of PCR P1ZOTOCOLS: A GUIDE TO METHODS APPLICATIONS, (eds.)
Innis, Gelfand, Sninsky and White (Academic Press, 1990).
Alternatively, hybrid proteins containing the first I~:ur~itz-type domain of
TFPI-2
and the second and third I~::unitz-type domains of TFPI could. be produced.
One skilled
in the art of DNA cloning in possession of the DIVA e;ncodir~g TFPI and TFPt-2
would
be able to prepare suitable DNA molecules for production of such a chimeric
protein
using known cloning pro~.edures. Alternatively, synthetic DNA molecules
encoding
part or all of each Kunitz-type domain anal peptide sequences linking the
I~unitz-type
domains can be prepared. As a f~u°ther alternative, the overlapping
PCIZ technique may
CA 02450800 2003-12-19
-21
also be used to prepare I~l~IA encoding chimeric molecules containing T'FPI
and
TFPI-2 sequences.
TFPI can be prepared in yeast expression sy;>tems as described in U.;S. Patent
Ieto. 6,103,500. Methods have also been disclosed for purification of TFPI
from
yeast cell culture rnediu~mn, such as in Petersen et al.., .L:~iol.Chem.
1:1334:4.-13351
(1993). In these cases, recombinant TFPI is secreted from the yeast cell. TFPI
recovered in such protocols is also frequently he;teroge:r~eous due to 1~1-
~terrninal
modification, proteolytic degradation, and variable p~lycosylation. 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 hornogeneovs.
TFPI can be produced in E. ~ol~ as described in U.S. Patent No. 5,212,091
which discloses a method of producing TFPI by expression of a non-
glyc;osylated
form of TFPI in an E. toll 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 ~f a denatured recombinantly produced protein product
using
polymers of sulfated polysaccharides which act a.s a templates for the
refolding
protein. Without being limited to any particular theory, the inventors believe
that
the interactions betwee-.~. the refolding protein aru the polymeric template
may
minimize aggregation of the refolding intermediates. and provide an
environment for
the protein to refold to its native conformation. T:~e 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-aggregated refolded protein, and generally
r~;sult in a
reduced overall yield o l° active refolded protein. The ~IaCI
concentration of the
refolding conditions is considered important and is selected to achieve the
maximum
efficiency of refolding by maximizing the interacticm between the template
polymer
and the refolding protein. For example, it has been found by the inventors
that
approximately 0.2 M cconcentration of ImlaCl or lo~,~Ter promotes binding cof
the C-
terminal and/or the third Kunitz domain of TFf I to heparin or other sulfated
polysaccharide polymer. The binding of polymer to the intermediate is presumed
to
CA 02450800 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.
Cameral Methods
TFPI may be prepared by recombinant methods as disclosed in IJ.S. 5,212,091.
Briefly, TFPI is expressed in Escherichi~ coil cells and the inclusion bodges
contammg
TFPI are isolated from ~,he rest of the cellular m,~ierial. The inclusion
bodies are
subjected to sulfitolysis, purified using ion exchange cl~~omatography,
refolded by
disulf de interchange reaction and the refolded, active; TFPI purified by
canon exchange
chromatography. TFPI may also be produced in ye;~st as disclosed in IJ.S.
Patent No.
6,103,500.
TFPI activity ma~7 be tested by the prothrombin time assay (P'fT assays).
Bioactivity of TFPI was measured by the prothrombin clotting time using a
anodel I2A4
1S Coag-A-MateTM from C)rganon Teknika Corpor-ation (C3klahoma City, C)1~).
TFPI
samples were first diluted to 9 to 24 ug/mL with a TBSA. buffer (SO Tris, 100
mM
NaCl, 1 mg/mL BSA, pI-I 7.5). Then 10 uL of Varif:y 1TM (pooled normal plasma
from
C)rganon Teknika Corp.) v~ras mixed with 90 uL of diluted T1~PI samples in a
;sample tray
and warned to 37 C in ~:he instrument. Finally SirnplastinTM Excel
(Thromboplastin
from ~rganon 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 Tl?PI standard curve.
The amount of soluble TFPI may also be quantified by measuring the area of the
main peak on a canon exs~hange chromatogram. FIP'LC analysis of TFPI sacrnples
was
performed using a Waters 62b LC system (Waters Corporation, Milford, MA.)
equipped
with a Water 717 plus heater/cooler autosampler . Data acquisition was
processed by a
Turbochrom system from I'erkin-Elmer.
The cation exchange (IE~) method used a hharmacia Mono S PIR. 5/5 glass
column. The column was equilibrated in 80% buffer t~, (20 mM sodium acetate
trihydrate:acetonitrile solu~:ion (70:3 0 v/v~ at pI~ 5.4) and 20% buffer B
(20 n~lVl sodium
acetate trihydrate - 1.0 M ammonium cl~.Ioride:acetcmitrile solution (70:30
v/v) at pPI
5.4). after a sample was injected, a gradient was applied to elute the TFPI at
a flow rate
CA 02450800 2003-12-19
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- 23
AlI reagents are gT.S.P. or A.C.S. grade. Suppliers include J.T. faker and
Sigma
Co. (St. Louis, ldlC)).
The present inv~:ntion will now be illustrated by reference to the f~llowing
examples, which set forth certain ernbodirnents. I3owever, it should be noted
that these
embodiments are illustra~:ive and are not to be construed as restricting the
invention in
any way.
EXAI\'IPLES
Example 1 - Refolding Denatured I
Z'he following example describes the making o:f stock solutions, the C; column
preparation, the initial recovery and purification of TF°l$I prior to
refolding, the ~refnlding
of I, and the recovery of active I.
The TFPI stock was prepared from refra,ctile bodies resulting from the
expression of recombinant I in bacteria. The refra~ctile bodies were
solubili::ed at 10
rnglml in 81V3 urea., 50 'iris pI~ 8.5 containing 10 I~TT, 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 T'FPI
was
prepared with S-Sepharo;~e beads mixed in 7.5 NI urea, 10 Tris and 10 i sodium
phosphate (pIi 6.5) containing 5 TT and 1 mlvi lEI)TA. The solubilized 7I at a
concentration of 5 rngJanl was then run over the S-Se;pllarose column and
eluted with a
sodium chloride gradient of 0 to 1 NI. The purii:~ed 'f~PI had an absorbency
at
wavelength 280 non of 3.2 (which is equivalent to .I nng/ml using an
extinction
coeclent of 0.78).
The dextran sulfate stock consisted of dextran sulfate of molecular weight
8000
daltons available frown Sigma, item number D-4811, rruade up at 50 mg/anl
(6.25 in
50 m11~ Tris (phi 8.8) in 0.1 1V1 sodium chloride, and stored at -20 degrees
centigrade
between uses.
CA 02450800 2003-12-19
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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,
M~),
item number H-3393, made up at 60 rnglml (3.33 mM) in 50 rnM 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 T:fPI
under
denaturing conditions ire 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/l TFPI, and to a final dexaran sulfate concentration of
0.6
mg/ml (75 MM) or a final heparin concentration of I.5 mglml (83 ~.1V1),
depending on
which was used to facilitate the refolding. Cystine was added to the refolding
solution to a final concentration edual to the final DTT concentration. The
refolding
solution vvas incubated at 4°C with gentle agitation for frozen 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 cor heparin.
To 610 ~.1 of TFPI stock either 60 ~,1 of dextran sulfate with 65 ~.1 of 50 mM
Tris (pH 8.8) in 0.1 M rTaCI, or I25 ~.I of heparin stock solution with 50 mM
Tris
(pH 8.8) or 0.1 M NaCl 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 tla~e refolding
solution and mixed. Finally, 61 ~1 of 50 mM Cysiine made up in 120 n~lVl
sodium
hydroxide was added and. the total solution was incubated at 4°C with
gentle agitation
for 4 days. The free sulfl~ydryl content was checked with Ellman's reagent
(also
called DTNB). Idoacetarnide was added, to 20 mM, made up at 1 M in
100°,io ethanol
for storage at .20°C.
The hydrophobic interaction column (HIC) was prepared from Eutyl-650M
Tosohaas ToyopearlTM resin particle size 40-90, part # 014.'702. 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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-25-
'The refolding samples, stored at -20QC remained in the standard refolding
buffer
containing 3 IvT urea, 50 ~ Tris, pFi ~., 1-4 redo~c, 0.5 mg/rrel I, and
0.2~0.6
NaCI depending on condition. Samples refolded with dextran or heparin had
0.21!
salt, and samples without d~xtran or heparin had 0.6 Pvl NaCI.
'The following steps were performed at room temperature to effect the further
purification of the refolded I. To 300 ~cl of refolded sample, an equal volume
of 2
NI opium sulfate, 3 i~ urea, 50 Tris, and 10 sodium phosphate (pFi 6.5)
was added. Next, 100 p1 of washed Butyl-650N! beads 'was added to the diluted
refolded sample. The solution with the beads was r,ncubated with gentle
rocking or
mixing for 30 minutes a~: room temperature. The rr~ix 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
ass not to
disturb the beads.
To wash the TFl'I-bound beads, 1 ml of wash buffer composed of 1 ammonium
sulfate, 3 ICI urea, g0 Tris, 10 sodium phosphate (pII 6.5) was added to the
wash and settling, the supernatant was removed with a flame~~pulled-tip
Pasteur pipette
very carefully.
To elute the refolded I, 300 p1 of elution buffer comfosed of 3 urea, 0.1 NI
ammonium sulfate, 50 Tris and 10 sodium phosphate (pI-I 6.5) was added t~
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
Tl~"PI was
recovered. To avoid contamination of the beads with the product, some of the
supernatant was left behind.
Image
CA 02450800 2003-12-19
W~ 96/40784 PCT/iJS96/09980
-2'~-
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 ~ absorbance. Figure I shows the results of these experiments. Solubility
of
TFPI increased greatly i.n 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 concentration of citrate ion in the presence df 10 mM Na phosphate
at pH
7. I solubility increases with increasing concentration of citrate. Figure 3
shows
the solubility of '1'FPI as a function of cancentration of NaCI at pH ?Ø
TFPI
solubility increases with increasing salt concentrations 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 'U~ absorbance after dialyzing 5 to 10 mglmL TFPI uito
these
buffer solutions.
CA 02450800 2003-12-19
O 96/407~d PC:'i°/LJS9fi/099~0
_2~_
Table 1
Salt effect _ Solubiiit
Content H c (mg/ml)
uv
lOmM NaPO4, 7 ~ ~ 0.21
t OmM NaP04, 150mM NaCi 7 0.72
20mM NaPO4, 150mM NaCI 7 0.85
20mM NaP04, 0.5M NaCi 7 6.71
20mM NaP04, 1 M NaCI 7 8.24.
pH effect
Content a m /ml
uv
20mM NaOAc, 150mM NaCI ~ 10.27 I
20mM NaOAc, 150mM NaCI 3.5 10.25
20mM NaOAc, 150mM NaCI 4 7.54
20mM NaOAc, 150mM NaCI 4.5 1 .75
20mM NaOAc, 150mM NaCI 5 1.15
~
20mM NaOAc, 15OmM NaCI 5.5 0.85
20mM NaP04, 150mM NaC! 5.5 0.89
20mM NaP04, 150mM NaCI _ 6 0.78
20mM NaP04, 150mM NaCI 6.5 0.79
20mM NaP04, 150mM NaCI 7 0.9
5
20mM NaP04, 150mM NaCI 7.5T _
0.82
___ 8 0.86
20mM NaPO4, i50mM NaCI
20mM NaCitrate, 150mM NaCP ~ 4 2.17
~
20mM NaCitrate, 150mM NaG1 4.5 1.19
20mM NaCitrate, 150mM NaCI . 5 1.1
~
20mM NaC~trate, 150mM NaCI 5.5 1 .84
20mM NaCitrate, 150mM NaCI 6 2.09
20mM NaCitrate, 150mM NaCI 6.5 2.12
20mM NaCitrate, 150mM NaCI 7 1 .92
20mM GI cine, 150mM NaC! 9 0.32
20mM Glycine, 150mM NaCI _ ~ 1 0 0.9
20mM GI tine, 150mM NaCB 1 t 13.94
20mMt--Glutamate, 150mM NaCI 4 9.07
20mM L-Glutamate, 150mM NaCi 5 1 .21
20mM Succinate, 150mM NaCI 4 8.62
20mM Succinate, 150mM NaCI ~ S 1 .21
20mM Succinate, 150mM NaC1 ~ 6 1 .07
Citrate
~
Content H c m !ml
uv
lOmM NaP04, 20mM NaCitrate j 7~ 1 .1 6
CA 02450800 2003-12-19
WO 96!40784 PC°TItJS46/09980
-29-
Tdb, a 1 (COflt
lOmM NaP04, 50mM NaCitrats 7 5.81
lOmM NaP04, 100mM NaCilrate 7 12.7
lOmM NaP04, 200mM NaCitrate 7 15.9
lOmM NaP04, 300mM NaCitrate 7 ! 8.36
NI 2+, Ca2+ and of hos hate
Content p c m Iml
lOmM NaP04, 150mM NaCI, 1 mM MgCf2 _ W uv
lOmM NaP04, 150mM NaCI, 1 OmM M CI2 7 0.66
7 ~ .02
lOmM NaP04, 150mM NaCI, 0.lmM CaCl2 7 0.67
10mM NaP04, 150mM NaCI, 1 mM CaCl2 . 7 0.71
lOmM NaP04, 150mM NaCI, lOmM tri hos hate 7 3.64
tOmM NaP04, 5% PEG-400
! 0.07
7
lOmM NaP04, lOmM EOTA i 0.36
7
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
tOmM NaP04, t00mA4 Tartaric acid 7 2.56
20mM NaP04, 100mM Malefic acid 7 0.17
20mM N 7 1.87
aP04, t00mM Malic acid
_ 7 0
tOmM NaP04, 100mM L- lutamic acid
_lOmM NaP04, 150mM NaCI 7 0.25
lOmM NaP04, 100mM isocitrate 7 10.83
_
NaOAc, NaP04 and NaC1 _
Content F! r. m ml
uv
lOmM NaOAc, 150mM NaCI 4.51.76
lOmM NaOAc 4.54.89
lOmM NaOAc 5.5~ 4.95
lOmM NaOAc 6.5~ 5.1
_tOmM NaOAc 7 5.87
_ 4.50.14
t OmM NaP04, 150mM NaC)
lOmM NaP04 4.54.97
lOmM NaP04 5.50.79
lOmM NaP04 6.50.091
lOmM NaP04 7 0_94
I
50mM NaOAc 5 5.24
5mM NaOAc 5.54.59
lOmM NaOAc _ 5.55.05
2OmM NaOAc 5.55.04
50mM NaOAc 5.55.71
100mM NaOAc 5.5i .4
200mM NaOAc j! 5.5i .32
CA 02450800 2003-12-19
WO 96/40784 P~T'/US96I09980
-30-
T~bl~ 1 (eont.)
5mM NaOAc. 5mM NaCI
~ 5:5 j 4.85
5mM NaOAc, tOmM NaCI I 5.5 5.04
5mM NaOAc, 50mM NaCI f 5.5 i 0.56
5mM NaOAc, 100mM NaCI 5.5 0.43
5mM NaOAc, 200mM NaCI A 5.5 j 0.8
5mM NaOAc 4.5 7.27
lOmM NaOAc 5 I 6.5
20mM NaOAc ~4.5 ( 8.32
50mM NaOAc I 4.5 t 9.17
5mM NaOAc ' S.5 8.98
t OmM NaOAc ' S.5 8.08
20mM NaOAc_ I 5.5 j 8.99
50mM NaOAc j 5.5 j 2.92
5mM NaOAc, 150mM. NaCI j 4.5 2.6
lOmM NaOAc. 150mM NaCI ~ 4.5 2.59
20mM NaOAc. 150mM NaC1 ~ 4.5 2.55
50mM NaOAc, 150mM NaCi j 4.5 ; 2. t
5mM NaOAc, 150mM NaCI 5.5 j 0.65
lOmM NaOAc, 150mM NaC) ~r j 5.5 0.69
20mM NaOAc; 150mM NaC1 _ ~ 5.5 0.7.1
50mM NaOAc, 150mM NaCt 5.5 0.91
j
H dro hobic chain ien th j
Content j H i c m Iml
uv
lOmM NaP04. 50mM Formic acid j 7 0,12
lOmM NaP04, 50mM Acetic acid ~ 7 0.16
t OmM NaPO4. 50mM Pro anoic acid ; 7 0.16
lOmM NaP04, 50mM Butanoic acid
I
lOmM NaP04. 50mM Pentanoic acid ~ I 7 0.14
lOmM NaP04. 50mM Hexanoic acid 7 0.1 t
i
_
~thers
Content ; pH I c m m!
uv
20mM NaOAc, 3% Mannitol, 2% Sucrose, 5% PEG-40~19.9
I 4
20mM Na Citrate, 3/~ Mannitol. 2/<> Sucrose, . 0.72
5% PEG-400 ~6.5
20mM Na Citrate, 150mM NaCI, 5l PEG-400 6.5 2.18
20mM NaOAc, 150mM NaCI. 5% PEG-400 4 t 9.8
20mM Na Citrate, 130mM NaCI, 1% Glycine. 0.25%1.48
Tweer~-80. 6.51
5% PEG-400 '
20mM Na Citrate, 130mM NaCI, 1 % GI tine, 7 .32
0.25% Tween-80 - 6.5 I
j Solubilily
Content ~I pli j a (mg/ml),uv
SmM N2Acetate ; 5.5 ! 8.9
5mM NaAcetate, 8% Sucrose j 5.5 ' t 1
5mM NaAcetate, 0.01 % Polvsorhate-80 j 5.5 7
j
CA 02450800 2003-12-19
b~0 96/40784 PC'!°ILJ596/09980
~3I
Tabl~ 1 (coat<)
5mM NaAcetate, 8/~ Sucrose, 0.01 % Podysorbate-80~ _ 1 2
1 omM NaAcetate 5.5 7.6
lOmM NaAcetate, 8i Sucrose ~ 1 0
1 OmM NaAcetate, 8% Sucrose, 0.01 % Pol~rsorbate-805.5 12.1
5.5
5.5
5mM NaAcetate, 5% Sorbitoi 5.5 7.8
5mM NaAcetate, 4.5% Mannitol 5.5 9.2
5mM Histidine 6 5.5
5mM Histidine 6.5 1
5mM NaCitrate 5.5 ~ 0.1
SmM NaCitrate 6 0.1
5mM NaCitrate 6.5 0.1
5mM NaSuccinate ' S.5 0.6
5rnM NaSuccinate 6 0.3
5mM NaSuccinate 6.5 0.2
lOmM dmidazole ~ 2.5, 10.8
6.5
t OmM Imidazole 7 0.8
iOmM Imidazole, 8/~ Sucrose 6.5 12.2
5mM NaAcetate 6 8.2
lOmM Imidazode. 5mM NaAcetate 6.5 12.8
t OmM NaCitrate 6 0.2
100mM NaCitrate 6 8.1
IOOmM NaCitrate 7 9.3
lOmM Naphosphate, 260mM Na2S~4 6 9.1
_IOmM Naphosphate, 100mM NaCitrate_ ~ 8 8.8
1 OmM NaGitrate, 1 % L- lutamic acid 6 4 .6
lOrnM NaCdtrate, 2% L-d Sine 6 1 .1
lOmM NaCitrate, 0.5/~ L-as artic acib! 6 0.4
t OmM NaCitrate, 0.1 /~ Phos hate lass ! 5.9
tOmM Tris, 100mM NaCitrate 8 8.5
lOmM NaCitrate, 1 M GI cine 6 0.3
lOmM NaCitrate, 300mM GI cine 6 0.3
lOmM NaCdtrate, 280mM Glycerol 6 0.3
lOmM NaCitrate, 0.5M NH4 2504 6 8.3
IOmM NaCitrate, 120mM NH4 2S~4 L 6 8.8
lOmM NaCitrate, 260mM Na2SC4 ~ 9.4
1 OmM NaP04, 0.1 % Phos hate lass ; 7 15. 8
1 OmM NaCatrate, 0.1 % SDS
~~ 6 11.2
lOmM NaCitrate. 0.02% SDS ~~ 6 7.8
t OmM NaAcetate, 8% PEG-400 I
_ ( 5.5 13.7
lOmM NaAcetate,.1~50riaM NaCI, 8% PEG-400 5.5 0.6
~
a OmM NaAcetate, 8% PEG-400 I' 6 16.2
~IOmM NaCitrate. 8% PEG-400 - ~~ 0.2
CA 02450800 2003-12-19
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- 32 -
The stability of ~'FPI stored at various pH conditions was tested. TFPI was
prepared by dialysis as above in 10 Na phosphate, 150 mM NaCI and 0.005
(w/v) polysorbat~e-80. S~.ability samples containing 1.50 mgJmL TFPI were
incubated
at 40'C for 20 days. Kinetic rate constant for the remaining soluble
°I~FPI was
analyzed by following decrease of the main on nation exchange chromatograms.
As can be sin in Fi re 5, the decay rate cons~,ant increases at pH above 6.0,
indicates more aggregation at higher pH conditions.
TFPI was also formulated at a concentration of 150 mglmL in 150 mM NaCI
and 0.005 % (wlv) polysorbate-80 at pH 7 with varying concentrations of
phosphate.
Figure 5A shows the percentage of remaining soluble TFl'I measured by tt~e
cation
exchange HPLC. Increasing concentrations of pho,~phate :ion in solution
resulted in
higher levels of soluble TFPI remaining after incubatioa~ at 4°C.
Higher levels of
phosphate ion also resul in higher levels of active TFPI as assayed by the
IS prothrombin time assay. These results are shown in Ffigure 5~.
Stability of TFPI at a concentration of 0.5 mg/mL and formulated in 10 mM
Na citrate, pH 6 and 150 mIVI NaCI was also tested at 40°C over a 40
day period.
As seen in Figure 6, canon-exchannge HPLC (triangle) shows the presence of
soluble
TFPI at levels greater than 60% initial, even after the 40 day incubation. In
life
manner, the prothrombln time assay (circle) show:9 the presence of active TFPI
at
levels greater than 60 % initial, even after the 40 da;~ incubation.
Figure 7 shows loss of soluble TFPI at ~#0°C rneasured 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 mlVd NaCI (circle).
Figure 8 shows loss of soluble 'TPPI at 40~C measured by botf cation-
exchange HPLC (open :symbol) and prothrombin rime assay (closed symbol) for
0.5
mglmL. TFPI formulated in 10 mRR Na acetate and ~~Ii 5.5 containing 150 nrNaCI
(triangle) o~ 8 % {wlv) sucrose (square) or 4.5 % (w/v) mannitol (circle).
CA 02450800 2003-12-19
w~ 96140'74 PC'd'/U~96/09980
_ 33
Figure 9 shows two non-reducing SDS gels for TFPI formulation samples in
30 mlVl lelaP~4, 150 mlvi IetaCl, and O.OOS l polysorbate-g0 at pH 4 to pH 9
stored at
40°C for 0 days (lower) and 20 days (upper). I~lo loss on TFPI is seen
at 0 days.
lElowever, at 20 days cleavage fragments of TFPI may be seen at the lower pH
range
(i.e. pH 4 and pH 5). ~Jithout being bound to a particular theory, it is
believed that
these fragments may resr~lt from an acid catalyzed r~.,action,.
Finally, Table 2 shows the half life of remaining soluble TFPI, at
~0°C for
various formulations. 0.S mg/mL PI was formulated in these formulation
° conditions and incubated at 40°C. Samples were withdrawn at
predetermined time
intervals and loss of soluble and active TFPI were exar~ir~ed by the IFX-HPLC
and
the PT assay. Half ~,ife 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 chromat~graphy resins using
polyionic compounds.
TFPI is first bound to a resin in a low salt buffer. I~Iext a buffer
containing
the polyionac compound used to elute TFPI in displacement mode, is pumped
through
the column. 'This compound 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 b N!: urea, 20
mlVl Tris, pH ~.0 was Ioaded onto a column packed with an anion exchange
resin, Q
Sepharase HP, to 20 mglmL resin. After loading, the column was washed with 6
NI
urea, 20 mN! Tris, pH 9Ø TFPI was eluted and 10 mglml bf filass H
(polyphosphate) in 6 urea, 10 mINI Tris, pH 9Ø.
Elution of TFPI from chromatography resin in aqueous buffer using polyionic
compounds.
CA 02450800 2003-12-19
~'~ 96140'8 1'C°y'/US9bI09980
_34~
Tav~ s 2
tll:P da at 40C
0.5 m ml TFP( formu9ated in: 9E:~--#~PLt>'PT assn
~
mP~l Na Acetate, 750 m~~! NaCI,10.8 1?.2
H 5.5
10 mM Na Citrate, 150 rraN' NaCl, 12.2 24.4
pH 5.5 ~
10 mM Na Acetate, S% vw'/V St~Cf~Se,4:3.2 42.2'.
ti 5.5
10 mM Na Acetate, 4.5/~ t~lannitot,47.7 4fi.6
pH 5.5
10 mtVl Na Succinate, 150 ~~M NaCI,7.F3 -1 ~ :0
pH fi.0
10 mM Na Citrate, 150 mt~l NaCi, 1 S.0 15.F3
H 6.0
10 mM Na Phos hate, 150 snM NaCt, ~.S 1 1.2
H 6.0
10 mP~ Na Phosphate, 500 mtv! NaCI,52.2 ~ SS.~
pH 6.0
10 mNf Na Citrate, 150 rnR~ NaCt,~H10.,0 I 14.5
6.5 ~
CA 02450800 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 used.
TFPI, in 3.5. M urea, 1 mg/ml polyphosphate, 50 mliil Tris, pH 5.9 was loaded
onto a cation exchange resin, SP Sepharose HP. After loading the column was
washed with anon-urea containing buffer, ZO mg/mI polyphosphate, 10 mM sodium
phosphate, pI-I 5Ø TFPI was eluted in the same buflE'er at p.~-I 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. V~hen 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/mI polyphosphate, 50 mM Tris, pH 5.9 eras loaded
onto a cation exchange resin, SP Sepharose . Af er Loading, the column is
washed
with 6 M urea, lrr~g/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/mI of polyphosphate.
Example 8
Neutralization oi' polyionic 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 th.e
charged
polymer with an oppositely Charged polymer, TFPI may bind to the resin.
In a buffer Containing polyphosphate (lass I-I), TFPI does not bind to Express
Ion STM (Whatman) and no purification is achieved. By mixing PEI into the
Column
load, TFPI now binds to the resin and TFPI can be purred.
CA 02450800 2003-12-19
w~ 96noas~ ~c~~s~sio~9so
-3b-
Refolding and purification of recombinant human TFPI (rhTFPT) using
Polyphosphate (Glass H) Facilitated Refolding Process.
Inclusion bodies containing about 40 g of rtd I 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. 'hhe thawed inclusion bodies 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 11~i urea, ~0 rn~i Tris-Cl, pH 10.5
buffer
containing 2 g/L Glass H contained in a I~ L polyethylene tank equfpped with
an
overhead stirrer. The contents were mixed for approximately 15 minutes, and
then
the absorbance of the solution is measured at 280 em. If the absorbance is
greater
than the mixture was diluted with sufficient dissolution buffer to obtain an
~bsorbance
at 280 em of 1.0-1.1. The solution was incubated with gentle agitation for 15-
30
minutes, and then sufficient cysteine was added tQ give a cysteine
concentration of
0.1 rnM. The solid L-cysteine was dissolved in approximately 50 ml of purified
water and added to the refold mixture. The pH was checkE;d and adjusted to pH
10.2
if necessary. The refold mixture was incubated with gentle agitation for 96-
1Z0
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 fltration process was used t~ remove
the
particulates that formed during previous steps and prepare the acidified
refold mixture
for SP-Sepharose chromatography. First the acidified refold mixture is passed
through A Cuno 60LP depth filter (filter housing model 8ZP1P) using a
peristaltic
pump ('/ - 3I~ 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. hack pressure was
maintained at a constant 20 PSI. Initial flow rate fbr a new filter was
approximately
3.0 5-6 L per minute. Filters were replaced when the flow rate dropped below 1
L per
CA 02450800 2003-12-19
!~O 96/40784 PCTAH.TS96t'o9980
-37-
minute in order to maintain the back pressure at 20 PSx. The second stage; of
the
filtration used a 0.45 micron filter cartridge (Sartorius Sartobran pH or
equivalent)
with a peristaltic pumping systern. After ~yltration, thn pH was checked, and
adjusted
to pH 5.9 if necessary.
'The acidified, filtered refold was loaded onto the esluilibrated SP epharos~
HP column at a flow rate of approximately 50.0 ml/min. Flow rate was adjusted
to
accommodate overnight loading of the acidified filter°ed refold
ra~ixture. °The column
was esluilibrated in 6 M urea, 20 rnM sodium phosphate buffer pH 5.9 prior to
loading. After loading, the column is washed with 2 CF of 6 M urea, 0.3 NI
NaCI,
20 mM sodium phosphate buffer, pH 5°9 prior to the gradient elution
step. The
column flow rate was incr to 1g0-200 ml/rnin 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 NaCI in 6 M urea, 20 mM
s~dium phosphate buffer pH 5.9. The gradient was formed by delivering 6 M
urea,
0.5 ~I NaCI, 20 mM sodium phosphate buffer into 6 M urea, 0.3 M NaCI, 20 mM
SI~S PAGE and pooled on the content of the correctly folded SC-59735 relative
to other anisfolds and irr~purlties. After ling the process s is referz'ed to
as
the S pool.
The pH of the S pool was next adjusted to pH S.0 with 2:5 N NaO. The S
2$ pool was concentrated 2-3 fold to approximately 2 1. using an Amicon DC-
10Y,
ultrafiltr-ation unit cc~n ' '.ng an Amicon ~'MIO spiral. cartridge (10, M.VV.
cut-off
membrane). After concentration, the concentrated S pool was diafiltered
against 7
volumes of fi M urea, 20 mM Tris-HCI buffer, pH 5Ø The diafiltration was
considered complete when the conductivity of the retentate was below 2 mS.
'The
30 diaftltered c~ncentrate was drained from the ultra.~xltratia~n unit and the
r,~nit was
CA 02450800 2003-12-19
w0 9614070 E'C"Ti'iJS96l099$0
_3g_
with approximateby I L of diafiltration buffer. The was is combined with the
concentrate to form the Q-load.
An Amicon column (7.0 cm diameter) was packed with approximately 700 ml
of Q-Sepharose high performance medium ~harrnacia ~Q-Sepharose ). The
column was packed with 20% ethanol at 20 psi. T'he bed height~after pacl~ing
was
approximately 18 cm. The column was equilibrated with 5 CF of 6 M urea, 0.02 M
Tris/FICI buffer, pH 8. 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 30-3S
ml/min
(50 cmihr). After loading, the c~lumr~ was washed with approximately 5 Cd of 5
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
I~'aCl in 6 M urea, 20 Tris6HCl buffer, pH 8.0 over ;~5 column volumes. The
first seven column volumes were collected as a single fraction, followed by 30
fractions of 0.25 column volume each.
1 ~ Fractions are routinely analyzed by reducing and non-reducing SDS-PAGE
and size exclusion chromatography. Fractions are pooled based on aggregate
content
( < 5 96 by SEC htPLC Method MSL 13929) and qualitative evaluation by SDS
PACaE
to assess puaty. The fractions are stored frozen at -l0~C until pooled.
Acceptable Q Sepharose fractions were pooled, and the pFi of the pool was
adjusted to 7.2 using 2 M HCl. The pool was then conccntt~ated approximately 5
fold
in an Amicon I5C-1 ultrafiltration system containing a S1Y1 Amicon YM-YO
cartridge (10,000 MW'CG spiral cartridge membrane). The concentra Q Pool was
then diafiltered against seven column volumes of 2 M urea, 0.15 M hl'aCl, 20
mM
sodium phosphate buffer, pH '~.2~ Following ultra~.ltratiori, the solution was
drained
from the ultrafiltration system. Approximately 100 ml of 2 M urea, 0.15 M
IviaCl,
20 mM sodium phosphate buffer, pH '~.2 was circulated through the
ultr'afiltration
system for approximately ~ min. The rinse solution was combined with the
original
concentrate and the solution was faltered through a 0.45 micron vacuum alter
unit
(l~Talgene).
CA 02450800 2003-12-19
dV0 96/40784 P~'rII~S96/09980
~3g~
Refolding and Purification of rhTFPI using Polyethyleneimine (PEI)
Facilitated Refolding Process.
Inclusi~n bodies containing about 40 g of rh PI 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 PT45/ff0) or until the inclusion bodies were then
added to 40 L of 6 urea 100 mlvl "Tris/I-ICl buffer pig 9.8 containing 300 mM
NaCl and 0.4 g/L PEI contain~i in a 100 L polyethylene tank equipped with an
overhead stirrer. . The bnixture was vigorously stiwed for 20-30 min. a pH was
monitored and adjusted to pFI 9.S as necessary. The absorbance of the
dissolved
inclusion body mixture was ured at 280 nrn, and if the absorbance was greater
than 2.1, the sample was diluted with 10 liters of the dissolution buffer
described
above to ob ' an A2S0 value ~f .0-2.1. Gentle agitation was continu for
another
IS-30 minutes. Next, the dissolved inclusion body solution was diluted with an
equal
volume of 1.0 Ivi a , 300 NaCI 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 I and added as a solution to the diluted relfold. °The phi was
checked and
adjusted, if necessary. The refold continued with gentle mixing for 95-120
hours
with periodic ch~ks of the phi, and adjustment to pF~ 9.5, of necessary. a
progress
of the refold was monitored by Mon-S radon exchange and prothrombin time
assays.
After approximately 96 h, the refolding process was terminated by adjusting
the pH of the refold to pFi 5.9 using glacial acetic acid. Stirring was
continued for
90 minutes and the pIi checked. More acid was added, if necessary to adjust
the plEi
to~.96-0.1.
A two-step filtration process was used to remove the particulates that formed
during previous steps and prepare the acidified refold for SP-Sepharose
chromatograph. First, the acidified refold is passed through a Cuno 60LP depth
filter
CA 02450800 2003-12-19
..40-
(filter housing model 8~P1P) 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 f lter 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, altered 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 was
then
1 S washed with S.5 column volumes of 6 M urea, 0.3 M NaCI, 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 = ~ 4'~ crnlhr). 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 NaCl, 20 mM sodium phosphate buffer into 6 M urea, 0.3 M NaCI, 20
mM sodium phosphate buffer into 6 M urea, 0.3 Ni NaCl, 20 mM sodium phosphate
buffer. Limit buffer was pumped with a MasterflexT~ 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 ParatrolTM A mixer from Parametrics (model 250210).
The
total volume of the gradient was 71.0 liters or 13.0 C~. 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 C~J) were collected. A Pharm~acia F~-ac-300 fraction
collector
was used with numbered, S00 ml polypropylene bottles. ~~J'hen the fraction
collection
was stopped, the remainder of the gradient was collected as a pool.
CA 02450800 2003-12-19
1~'~ 96/4074 P lIJS96/09980
-41 m
Column fractions were assayed by A280, size exclusion F~~'LC, and in
addition, for informational pu see, SI7S pEl~iE, reverse phase ~iLC, and
assays. Fractions were led if they rnet the pooling criteria of containing 20~
of
less aggregate as determined by the in process SE~~ fI~C. Pooled SP Sepharose
fractions are referred to as the S Pool.
The phi of the S- 1 eves next adjusted t~ phi S.0 ~rith 2.5 r1 ~TaCFt.
°I°he S
Fool was concentfa 2-3 fold to approximately 2 L using an Amicota I9C~lOL
ultration unit containing an Amicon ~'1dI10 spiral °dge (10; 1~T.. cut-
off
membrane). After concentration, the concentrated S Pool was diafiltered
against 7
volumes of 6 M ur , 20 m Tris-I-iCl buffer, 1-I 5Ø The diafil lion vvas
considered complete when the conductivity of the retentate was below 2 n~S.
'The
diafiltered concentrate was drained from the ultr~ultratioaa unit and the unit
was
ed with approximately I L of diafiltration buffer. The was is combin with the
concentrate to form the ~ 3-load.
An Amicon colurr~n (7. cm diameter) wa.<<; packed with approximately 7
Sepharose resin. 'fhe load was appli to the column at a flow rate 30-3gi
ml/min
(50 cm/hr). After loading, the column was washed r~ith approximately S C'~ of
6 ~i
urea, 20 Tri Cl buffer, pI3 5.0, or until the .a.bsorbance at 2S0 nm resumed
to
baseline. The product eras eluted using a sodium chloride gradient fr~m 0-0.15
lei
aCI in 6 1vI urea,, 20 mTrxsl~°ICl buffer, pIi 8.f1 over :25 column
volumes. a
f rst seven column volumes were coilec as a single faction, followed by 30
fractions of 0.25 column wolurne each.
Fractions are routinely analyzed by reducing d non-reducing SIBS-PAGE
and size exclusion chromatography. Fractions are pooled based on aggregate
content
(5l by SEC LC) and qualitative evaluation by S1~S PACE t~ assess purr,ty. The
fractions are stored frozen at -20°C until pooled.
CA 02450800 2003-12-19
W~ 96/4074 ~C'I°/tJ~9~1099~0
_42~
The Q-Sepharose fractions to be pooled were thawed by incubation at 2-8
° C,
pooled, and the pH of the pool was ad,~usted to 7.2 using 2 MHCI. The pool was
then concentrated approximately S fold in an Amicon ~C-1 ultrafiltration
system
containing a SlYl Amicon 'YM-10 cartridge (10,000 MWCO spiral cartridge
membrane). The concentrated Pool was then diafiltered against seven column
volumes of 2 M urea, 0.15 M ~taCl, 20 rnM sodium phosphate buffer, ~pH ~.2.
Following ultrafiltration, the solution was drained from the ultrafiltration
system.
Approximately 100 ml of 2 M urea, 0. i5 M IVaCI, 2t~ mM sodium phosphate
buffer,
pH 7.2 was circulatcd through the ultrafiltration system for approximatcly 5
min.
The rinse solution was combined with the original concentrate and filtered
tpirough a
0.15 micron vacuum filter unit {I~algene).
Solubilization, refolding, and purification of rhTFPI from inclusion bodies
was maintained at pH 10.5 with 1 ~T ~taf~H. The ref~ld solution was incubated
at 2-
8 ° C with gentle mixing for 72-96 h.
'The refold was next adjusted to pH 6 using glacial acetic acid and then
filtered
through a 0.2 micron filter. An aliquot of the filtered refold was applied to
a 200 and
column of SP-Sepharose HP (I'harmacia) previousl~~ equilibrated in 0.4 ~ Glass
H,
20 mM sodium phosphate pH ~ buffer after loading, the column was wished with 4
purified in this manner.
CA 02450800 2003-12-19
w~ 96/40784 PC'~'/tJ5961~9980
-4~-
Improved solubility of rhTFl3I in water by t:orrnation of a complex between
T~PI and polyphosphate (GIGS 5327046-47)
about 10 g of purified rh PI in about 1 liter of 2 Irk urea, 12S mhs sodium
chloride, 20 sodium phosphate p7.4 buffer was thawed by incubation at 2-8~C
for 1S-36 h. Sufficient: dry ur was added to make the solution 6 in urea. The
solution was then filtered through a .2 micron filter. dive g of polyphosphate
glass
(Glass ~I, FldlC) was dissolved in 50 ml of 6 1vI urea, adjusted to p 7 with 1
1't
removed from the u1 ~ltration unit. T'he ultraf lixution unit was washed with
about
150 ml of purified wata~r and the was added to the; protein c~ncentrate. 7."he
final
protein concentrate contained almost 10 g of proteiin in 400 rnl of water
(about 24
mgiml~protein). T'he normal solubility of rhTFPI in water is less than .5
nag/rral.
Lrse of cationic p~~lymers for removal of E. c:~lp contaminants from T'FPI
cell
lysates and refractile bodies.
T'he use of cationic polymers to precipitate and remove coli contaminants
from crude I rote ediates (lysates, refractlle bodies) signifi tly ianpr~ve
subsequence process operations (refolding, chroLatography etc.) random
screening of cationic polymers identified candidats~s which selectively
precipitate
bacterial con inants while PI remains in solution. Specifically, tz lpolYmer
624 precipitated substantial amounts of bacterial contaminants, while 1 virag
I in
solution in an aqueous environment.
Solubilized I refractile bodies (in 3.5 ; guanidine hydrochloride, 2 AZ
sodium chloride, 50 mlvi S, 50 mNI dithiothreitol, pI-I7.1) w the s °ng
material used for a polymer screening experiment. This material was diluted 10
fold
into a 0.5 % solution of various polymers. T°he precipitates from this
experiment
Image
CA 02450800 2003-12-19
Charged polymer facilitated refolding of bovine somatotropin from E. c~la
inclusion bodies
Ten grams (wet weight) of inclusion bodies containing 5 graans of bovine
somatotropin are added to about 1 Iiter of 1 % ~ilass H, 50 mM Tris buffer pH
10.5.
The mixture is thoroghly blended using a polytron (f3rinl~man~ 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
1Q.6 using 1 N Na~H. Solid cysteine (121 mg) is added to make the reaction 1
mM
cysteine, and the refolding reaction is mixed far 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 the art without departing
from
the spirit and the scope of the appended claims.
