CA2450795A1 - Method of refolding tissue factor pathway inhibitor - Google Patents

Abstract

A method of modifying protein solubility employs polyionic polymers. These facilitate the solubilization, formulation, purification and refolding of proteins especially incorrectly folded proteins and aggregated proteins. Compositions are described that are suitable for formulating TFPI. The compositions allow preparation of pharmaceutically acceptable compositions of TFPI at concentrations above 0.2 mg/mL and above 10 mg/mL.

Description

WO 96!40784 PCT/US96/09980 lVdETHOI) OF SOL~.T~yLiZII~TG, P~T>I~ING, A1~TD REFOLDII~TG PIROTEIN
Technical Field of the Invention The invention :relates to methods useful for refolding, solubilizing, formulating and purifying proteins. These methods are particularly usefi~l for proteins that; have been engineered by genetic x ecombination at~d produced irl 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 g~rne expression, it is as irrvportant to understand the proves>~ for the folding of the peptide chain into a biologically active protein as it is important to understand the synthesis of the primary sequt;nce. 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 integrioy of a protein can destroy its activity. Tsou et ~xl (1988) Biochemistry 27:1809- l 812.
Under the proper conditions, the in vitr~ 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 of unfolding and refc'Iding appears to be close to the native state in learns 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 i~VO 96/4074 PC'i'/TJS96/099~0 _2_ lowered reversibility, as described in Fischer and Schn~id, (1990) ~iocherttisiry 29:2205-2212.
In the case of suciently Large protein rnolec~ales, the nascent polypept:ide chain acquires its native three-dimensional structure by the modular assembly of rnicro-domains. Variables including temperature, and cosolvents such as polyols,'.~rea, and guanidiniuchloride, have been tested to dete~ine their role in stabilizing and destabilizing protevl conformations. The action of eos~olvents may be the result of direct binding or the alterations of the physical properties of water, as described in Jaenicke et cal. ( 1991 ) Biocher,~is3 0 ( 13 ):3147-3 I 61.
Experimental observations of how unfolded proteins uefold to their native three-dimensional structures contrast with many popular theories of protein folding mechanisms. Under conditions which allow for refolding, unfolded protein rr'olecules rapidly equilibrate between different conformafiions ~~rior to complete refolding. The rapid prefolding equilibrium favors certain compact conformations that have somewhat 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 thve native conformation. There appears to be a single transition through which essentially all molecules fold, as described in Creighton et al. (1!88) Pa~oc. Nat. ~cac~
S'ci. I,S~4 85:5082-5086.
Various methods of refolding of purified, recombinantly produced proteins have been used. For example., the protease encoded by th,e human immunodeficier~cy virus type I -I) can be produced in ~"sche~°ichia coli, yielding inclusion bodies harboring the recombinant 1'3:IV-I protease as described by Hiu:i ~~ al. (1993) J
~'t°ot. Chem.l2:
volumes of cold 0.1 IvI sodium acetate, pI-I 5.5, contaiong 5~/o ethylene glycol and 10%
glycerol. Exclusion of glycerol and ethylene glycol ledl to gradual loss of protein due to precipitation. About 85 ang of correctly folded I-IIV-a protease per liter of.E toll cell culture was obtained by this method, and the enzyme ha.d a high specific activity.

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. Biochetn. 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 Ii-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 Gustaf'son et al., (1994) Protein Expression and Purification 5: 23~-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 chromatograp=by and renaturation in 6M urea. The refolded mixture was then fractionated to yield a purifaed nonglycosylated TFPI possessing in vitro biological activity as measure in the Prothombin clotting time assay comparable to TFPI purified from mammalian cells.
A non-glycosyla~4ed form of TFPI has also been produced and isolated from Escherichia coli (E. coil) cells as disclosed in Lt.S. Patent No. 5,212,091.
The invention described in IJ.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. 5,212,092 has been shown to be active in the inhibition of bovine f~.ctor 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 hepato~na cells. However, TFPI
produced in E. coli cells is modified in ways that increase heterogeneity of the protein.

_Q_ 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 TFI'I. Presently the yields of recombinantly produced I
have been lower than desirable, and a need exists in the art of producing correctly folded TFPI. See for example Gustafuson et al. ( 1994) P'rotetn Expression and Pur~cation .5:
233-241.
TFPI inhibits the coagulation cascade in at Ieas~t two ways: preventing formation of factor VIIa/tissue factor complex and by binding to the active site of factor Xa. The primary sequence of TF'PI, deduced from cT3NA 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 VIIaltissue facts~r complex. The second Kunitz-type domain is needed for the inhibition of factor Xa. ~'he function of the third Kunitz-type domain is unknowns. 'fFPI 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 tl"'e active site of one protease molecule. The carboxy-ternainal end of TFPI is believed to have a role in cell surface localization via heparin binding and by interaction with pho5pholipid. 'I°FPI is also kruown as Lipoprotein Associated Coagulation Inhibitor (I~ACI), 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. 'T'FPI contains 1S
cysteine residues and forms 9 disulphide bridges v~rhen correctliy folded. The primary sequence also contains three Asn-X-Ser/Thr N-linked glycosylation consensus sites, the asparagine residues located at positions 145, 195 and 256. The carbohydrate component of mature I is appro~cimately 30~f~ 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 function.
TFPI has been isolated from human piasra~a and from human tissue culture cells including HepG2, Chang liver and SK hepatoma cells. Recombinant TFPI has been W~ 96/40784 PCTNS96/09980 -~-expressed in mouse C 127 cells, baby hamster kidney cells, Chinese hamster ovary cells and human SK hepatoma cells. Recombinant TFPI fi°om the mouse C 12.'~
cells has been shown in animal models to inhibit tissue-factor induced coagulation.
A non-glycosylated form of recombinant TF'PI has been produced and isolated fromEscherichia coli (~:. 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 in plasma. Methods have also been disclosed for purification of TFPI from yeast cell culture medium, such as in Petersen et al, J.Biol.Chem. 1i~:13344-13351 (1993).
Recently, another protein with a high degree of structural identity to TFPI
has been identified. Sprecher et al, Proc. Nat. Acad. Sri., USA 91:3353-3357 (1994). The predicted secondar~r structure of this protein, called TIWI-2, is virtually identical to TFPI
with 3 Kunitz-type domains, 9 cysteine-cysteine linkages, are 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 ICunitz-type domains 1, 2, and 3, respectively. Recombinant TFPI-2 strongly inhibit:. the arnidolytic activity of factor ~lIla/tissue factor. By contrast, I-2 is a weak inhibitor of factor Xa ainidolytic activity.
TFPI has been shown to prevent mortality in a lethal Escherichra Golf (E.
cola) septic shock baboon model. Creasey et al, J. Clin. Invest. 91:2850-2860 (1993).
Ad tion of TFPI a9: 6 mglkg body weight shortly after infusion of a lethal dose of ~ coli resulted in survival in all five TFl?I-treated animals with significant improvement in quality of life compared with a mean survival time fir the eve control animals of 39.9 hours. The administration of TFPI also resulted in significant attenuation of the coagulation response, off' various measures of cell ir'jtary 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, TI~PI may also be used to prevent thrombosis during microvascular surgery. For example, U.S. 5,276,015 discloses the use of TFPI in a method for reducing thrombogenicity of microvascular anastomoses wherein TFPI is administered at the site of the microvascular anastomoses contemporaneously with microvascular reconstruction.
TFPI is a hyd~-oph~bic protein and as such, has very limited solubility in S 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 SepharoseTMHIC 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 1 SO mM NaC 1. The concentration of remaining soluble TFPI after dialysis was measured by IJ~~ absorbance after filtering out the precipitates through 0.22 mm filter units.
Figure 6 shows the solubility of TFPI as a fiznction of concentration of citrate in the presence of 10 mM Na phosphate at pH f. TFPI 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 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 mIVI NaCl and 0.005% (wlv) polysorbate-80. Stability samples containing 1~0 mg/mL TFPI were incubated at 40°C for 20 days. Kinetic rate constant for the remaining soluble TFPI was analy~.ed by following decrease of the main peak on canon exchange chromatograms.

W~ 96/40784 P~:TIUS96109980 _'~_ Figure 9 shows the percentage of remaining soluble TFPI measured by canon exchange HPLC (A) and remaining active TFPI b;y prothrombin time assay (E) as a function of phosphate concentration. The formulation contains 150 mglmL TFPI
prepared in 150 mM l~ZaC1 and 0.005 ~o (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/rnL
TFPI
formulated in 10 ml~I hla citrate, pH 6 and 1~0 mM ~TaCI.
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 mglmL TFPI formulated in 10 mM ImTa phosphate, pH 6 and either 150 mM ~TaCl {triangle) or 500 mM I~aCI (circle).
Figure 12 shows loss of soluble TFPI at ~0°C measured by both cation-exchange HPLC (open symbol) and prothrombin dme assay (closed symbol) for 0.5 mglmL TFPI formulated in 10 mM Na acetate and pH 5.5 containing 150 mM IolaCl (triangle) or S %'o (w/v) sucrose (square) or 4.5 J m'u~nitol (circle).
Figure 13 shows two non-reducing SDS gels for TFPI formulation s<~mples at pH 4 to 9 stored at 40°(r for 0 and 20 days.
Figure i4 shows the time course of a polyphosphate-facilitated rhTFPI refold monitored using SDS PAGE.
Figure 15 shows the absorbance at 2g0 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 fracaions collected during elution of the S-Sepharose HP column used to purify rhTFPI from a polyphosphate-facilitated refold. Figure 1 ~' shows the absorbance at 280 nrn during the loading and elution of the Q-Sepharose IiP column used to purify rhTFPI from a S-Sepharose pool prepared from a polyphosphate-facilitated refold.

WO 96/40784 PC.'TfIJS96/09980 -g-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-Sepharose HP ~ column used to puri;Fy rhTFPI firom a polyethyleneimine-facilitated refold.
Figure 21 shows SDS PAGE analysis of fractions collected during elution of the S-Sepharose HP column used to purify rh1 FPI from a polyethyleneimine facilitated refold.
Figure 22 shows the absorbance at 280 nm duriatg the loading and elution of the t'~Sepharose HP column used to purify rhTFPI from a S-Sepharose pool prepared from a polyethyleneimine-facilitated refold.
Figure 23 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 polyethyleneimine-facilitated refold.
Figure 24 shows the canon exchange HPLC analysis of a 0.4.
polyphosphate-facilitate~~ rhTFPI refold in the absence of urea.
Figure 25 shows results of ration exchange HPLC analysis of an evaluation of different levels of cysteine on a rhTFPI refold in 0.4 l polyphosphate, SO
ml~i 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 b;~
canon exchange HPLC.
_ Figure 27 shows the effect of concentration of polyphosphate (Glass I-I) on the refolding of rhTFPI fro~r~ inclusion bodies as monitcored by radon exchange IEiPLC.
Figure 28 shows the ration exchange HPLC analysis of polyethyleneimine and polyphosphate-facilitated refolding of purified and reduced rhTFPI.

_g_ Summary of the Invention It is an object of an aspect of the present invention to describe a method of S 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 '~: FPI including the steps of adding charged polymers to a solution of denatured T:1~'PI prior to allowing the TF'PI 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 1 ~ column and passing a solution of denatured TFPI through the column and eluting the refolded TFPI after the ~~efolding 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 rCFPI. 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 re~.ates 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.

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 mc~nomeric forms of TFPI are also removed during this process: The separation employs hydrophobic interaction I5 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 earned out in the presence of high salt, such as 1 M ammonium sulfate or 0.5 M sodium citrate.
In accordance with one aspect of the present invention, there is provided a method of refolding an improperly folded or denatured protein comprising the step of adding charged polymers to a solution comprising the protein prior to allowing the protein to refold.
In accordance with another aspect of the present invention, there is provided a method of refolding TFPI comprising the step of adding a charged polymer to a solution comprising improperly folded or denatured TFPI prior to allowing the TFPI
to refold.
In accordance with a further aspect of the present invention, there is provided a method of refolding TFPI comprising the step of immobilizing polymers of sulfated polysaccharides on a column and passing a solution of denatured TFPI through the column and eluting the refolded TFPI after the refolding has occurred.

10a 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 nun ;emus 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.

~4'O 96/40784 ~'~'C1U~96/099~0 _1l_ 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 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.
We have characterized refolding of pure TFPI in a guanidine or a urea based refolding buffer and the results indicate that refolding efficiencies and I~netl.cs can be significantly improved by the addition of charged polymers including heparin, dextrin sulfate, polyethyleneimine (PEI) and polyphosphates. These polymers increase TFPI
solubility and enhance refolding through ionic interactions with either the hT-terminus or the C-terminus. In addition to the polymer additives, refolding pure TFPI
requires a cysteinelcystine redox buffer where the refolding reaction can be compleGrd within 4g hours. Refolding yields are a strong function of gI-I, redox concentration, and polyri~er 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 dextrin 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 I3I~.~ 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. cmla and several other expression systems, the protein is fr~"quently expressed in an inactive, denatured state where the primary amino acid uence 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 WO 96/40784 PC'TltTS96109980 conformation that charged residues of different parts of the amino acid bacl~bone 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. '.fhe protein in an inclusion body rr~ay 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 t~ the polar aqueous environment.
Such occurrences may work to prevent the dissolution of inclusion bodies in solvents other than strong chaot~~opic agents such as urea oI° guanidine or detergent.=. such as SI7S.
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 environaalent. "y"he charged polymers ntay 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 (chai:n 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, polyarrunoacids, polyaspartates, polygl~Itamates, polyhistidines, polyorganics, polysaccharides,DEAE l~extrans, 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 pI3. ~~hanging the solution pH will modify the total charge and charge distribution of any protein, and is another variable to be evaluated.

WO 96!40984 PCT/US96/09980 _13_ Be Recombinant I~1~IA technology has allowed the high level expression of rryany 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 inacti~re, denatured st~.te 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 sever~eby Iimit the refolding pathways available to the denatured protein and reduce the efficiency of the refolding process.
Some proteins leave specific areas of charge whose interactions may limit 1~ conformational flexibility or promote aggregation. I~iany 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 plasminngen 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 benef t from the present ~rnethods. Preferably the protein will have at least 2 and more preferably the protein will have at least 4 or 6 disulfide bonds.

w~ 96/40'84 PCTNS96/09980 l~ _ ~harge~l polyma:rs can be used to modify the charge, charge density, and reduce or eliminate ionically mediated limitations to confarmation that may 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: iarst 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 pI3 (pI) of the protein can serve as a s ' g point. At neutral pIi a 1 ~ 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 pI~ , and will trove a stronger tendency to bind a negatively charged polymer. However, it is well established that charges are unevenly distributed aroa~nd 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 requirear~ents, 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 pI3 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. ~Jork 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 W~ 96/4Q784 P~'~'/t3S96/09980 _ 1~ _ aggregate. The optimal polyphosphate chain length for refolding rhTFPI was approximately 25 re ting units. Longer chain length polyphosphates (n= 75) also produced more aggregate and less properly folded rr~onomer.
Proteins consist of chains of amino acids, thc; 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.
T'hirdiy, the specific amino acid sequence directs the formation of tertiary structure, such as ~i-sheets and «- helices. The three dimensional nature of protein conformatiion often brings into proximity amino acid residues that are not normally close to each other based on the direct sequence of the polypeptide chain. °rhe functional farm of a protein is generally a modestly stable conformation held together by a combination of cysteine disulfide bonds, ionic bonds, and hydirophobic and Van de:r Waals interactions.
In general, protein solubility can be related tc> the nuumber of charged and t~ a - 1~ -Complexation with charged polymers with relatively high charge density represents one approach to increasing the charge density of any protein. ~
protein with a small number of positively charged residues lysine or argininel can be complexed with a negatively charged polymer such as polyphosphate. Sonne of the negatively charged groups of the polymer will interact with the positively charged groups present in the protein. The remaining charg~;d groups on the polymer will be free to interact with the solvent, in most cases water, and effectively increase the charge density and s~lvation 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 confoi°mation, charged polymer charge density, charged polymer chain length, solution pI~, 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 ran be found that will improve the solubility characteristics of that protein in aqueous medium.
Definitiflns 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 stn~ctural 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 _ 17-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 g~°oups 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 (H3h1+-(CH2-CH2-hTH2+)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, CalgonTM, phosphate glass, sodium tetrametaphosphate, and Glass HT~.
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 amd their environment, including ionic bonds, Van der ~laals interactions, hydrogen bonds, disulfide bonds and covalent bonds.

WO 96/40784 pC'fIUS96/09980 _18_ The term xdenature" 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 fan der Waals interactions which exist in the molecule in its native or nenatured state. Denaturation of a protein can be accomplished,, for example, by treatment vvtith 8 M urea, reducing agents such as mercaptoethanol, heat, pH, temperature and other chemicals. Reagents such as Il IvI 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-1-3: groups. Refolding of proteins which contain disulfide iznkages in their native or refolded state may also involve the oxidation of the -S-:F3 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 dextaart, ranging in molecular weight from 8, to SO~,OnO daltons. Dex~rans are polymers of glucose in which glucose residues are joined by aI,6 linkages~
The term °heparin" as used herein refers to 2 glucoaminoglycans or heparinoids which are based on a repeating disaccharide (-4DGlcA(p)~31;
4GIcNAca1-)n but are subject to extensive modification ai~er assembly. Heparin is stored with histamine in mast cell granules and is thus found in mast connective tissues.
In general heparins have shorter chains than heparin.
The term "HIG" 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 aga;iopectins, as well as carbo:~ylic acid polysaccharides such asalginic acids and carboxyr~ethyl celluloses. Polyinorganics such as polyphosphates are W~ 9614074 ~'CTl1JS96/099~0 1g _ also included. Polyamino acids such as polyasparatate, polyglutamate, and polyhistidine can also be used.
Positively charg~°d polymers include polysaccharides such as I3EAE
dextran, polyorgnic amines, such as polyethyleneimines, polyethyleneimine celluloses, and polyamines, as well as ttie polyamino acids, polylysine and polyarginine.
Combinations of polymers may be used, of either charge polarity. In addition, amphoteric co-polymers may also be used.
As used herein, "TFPT" 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 (FPI) and Tissue Factor Inhibitor (TFI).
I~iuteins of TFPI which :retain the biological activity of TFPI are encompassed in this definition. Further, TFPI which has been slightly modified 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 Escherichaa c~la. See IJ.,~. 5,212,081.
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 effects 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 mgfmL and above 10 mg/mL. It should be noted that solubilizers may act as stabilizing agents. Stabilizing agents preserve the unit activity of I in storage and may act by preventing formation of aggregates, or by preventing degradation of the TFPI molecule (e.8. by acid catalyzed reactions).
As used herein, the term "secondary solubilizers" 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 have other effects as wall. For example, secondary stabilizers may be useful in adjusting tonicity (e.g. to aotonicity).
The amino acid sequence of TFPI is disclosed in U.S,. Patent No. 5,106,833 and Figure 4. Muteins of TFfI and TFPI-2 are disclosed in ZJ.S. Patent No.
6,10:3,500. As described in Il.S. Patent No.6,103,500, a=nuteins of TFPI and TFPI-2, with single or multiple point mutations, and chimeric molecules of 'hFPI and TFPI-2 can be prepared.
For instance, the lysine rE;sidue in the P 1 site of the first Kunitz-type domaia~ of TFPI
may be replaced with arginine. Muteins, cont<~ining 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 miztagenesis include, without limitation, site specific mutagenesis. Site specific mutagenesis can be carried out using army number of procedures known in the art. These techniques are described by Smith (1985) Annual Review of C;eneti~°~, 19:423, and ~nodifi~cations of some of the techniques ar° described in METHODS IN EN:~YMOLOCp~', 1 '_i4, part E, (eds.) Wu and tlrossman (1987), chapters 1'~, 18, lCe, and 20. A preferred procedure when using site specific mutagenesis is a modification of the Clapped Duplex site directed mutagenesis method. The general procedure: is described by Kramer, et al., in chapter 17 of the Methods in Enzymology, above. ~f~,rrlother 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 C~L1IDE TO METTIODS AND APPLICATIONS, (eds.) Innis, i~elfand, Sninsky and White (Academic Press, 1.980).
Alternatively, hybrid proteins containing the first Kunitz-type domain .of and the second and third hunitz-type domains of TFF'I could be produced. One skilled in the art of DNA cloning in possession of the DNA wracoding TFPI and TFPI-2 would be able to prepare suitable DNA molecules for production of such a chirneric 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 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. Patent No. 6,103,500. Methods have also been disclosed for purification of TFPI from yeast cell culture medium, such as in Petersen et al., 3.Biol.Chem. i x:13344-X1993). 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, proteolyti~ 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 ~J.S. Patent No. 5,212,091 which discloses a method of producing TFPI by expression of a non-glycosylated form of TFPI in an E. cc~li 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 sulfhte 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 betwee~l 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 fonmed, are generally less active tha~a non-aggregated 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 proteins. For example, it has been found by the inventors that approximately 0.2 M concentration of NaCI or louver 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 intermediate is presumed to facilitate the solubility of the intermediate and provide an environment for tree 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 Eschew-ichirx coil cells and the inclusion bodies containing TFPI are isolated from. the rest of the cellular material. The inclusion bodies are subj ected to sulfitolysis, purified using ion exchange clhromatography, :refolded by disulfide interchange reacaion and the refolded, active TFPI purified by ration exchange chromatography. TFPI may also be produced in yeast as disclosed in LJ.S.
Patent No.
6,103,500.
T FPI activity may be tested bgT the prothrombin time assay (PTT assays).
Bioactivity of TFPI was measured by the prothrombin clotting time using a model RA4 Coag-A-MateTU from Organon Teknika Corporation (Oklahoma City, OK). TFPI
samples were first dilutes: to 9 to 24 ug/mL with a 'fBSA buffer (50 mM Tris, 100 mM
NaCl, 1 mg/mL BSA, p1-1 7.5). Then 10 uL of ~arify 1'T'~ (pooled normal ~>lasma from ~rganon 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 (Thrornboplastin 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 TFPI standard curve.
The amount of soluble TFPI may also be quantified by measuring the area of the main peak on a ration exchange chromatogram. HI'LC analysis of TFPI samples was performed using a Waters 620 LC system (Waters Corporation, Milford, MA) equipped with as Water 717 plus heater/cooler autosampler . I7~ata acquisition was processed by a Turbochrom system from Perkin-Elmer.
The ration exchange (IEX) method used a pharmacia Mono S FIFZ 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 Ir~1 ammonium chloride:acetonitrile solution (70:30 vlv) at pH
5.4). After a sample was i~ajected, a gradient was applied to elute the TFPI
at a flow rate w~ 96140754 PC'°r//~JJS96/09980 ~23-All reagents are ~;r.S.P. or A.C.S. grade. Suppliers include J.T. Eaker and Sigma Co. (St. Louis, C3).
°The present invention will now be illustrated by reference to the :following examples, which set ford, certain embodiments. I-Iowever, it should be noted :hat these embodiments are illustr~.tivc and are not to be construed as restricting the invention in any way.
E hIPLES
Example 1 ~ Refolding denatured I
'The following ex~~mple describes the making of stock solutions, the I3Itr column preparation, the initial recovery and purification ofTFfI prior to refolding, the refolding of I, and the recovery of active I.
The TFPI stock was prepared fr~m refractile bodies resulting ):corn the expression of recombinant I in bacteria. The refi-a.ctile bodies were solubili,~ed at 10 mglml in 8 urea, 50 r~ Tris pl~i 8.5 containing 10 a 1.7TT, and this solution was clarified by centrifugation at 10,000 x g for 10 minutes.
The column prel>aration for the initial purification of the solubilize7:'~I
was prepared with S-Sepharose beads nixed in 7.5 ure<~, 10 Tris and 10 ~ sodium phosphate (pI-I 6.5) conta.ning 5 I)TT and 1 Fi7TA. The solubilized '. I at a concentration of 5 mgirr~l was then run over the S-Sepharose column and eluted with a sodium chloride gradierdt of 0 to 1 NI. The purified TFPI had an absorbency at wavelength 280 nm of 3.2 (which is equivalent to 4.1 mgJml using an extinction coecient of 0.78).
The dextran sulfave stock consisted of dextrar;~ sulfatc of molecular weight daltons available from Sil;ma, item number D-4911, made up at 50 mg/ml (6.2'.>
in 50 Tris (pI~ 8.8) in 0.1 Ie~: sodium chloride, and stored at -20 degrees c~:ntigrade between uses.

The heparin stock, if heparin was used to conduct the refolding, was of molecular weight 600t) 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-3x93, made up at 60 mg/ml (3.33 arzM) in 50 mM Tris (pT~:8.8) in 0.I
sodium chloride, and stored at -20°centigrade between uses.
To the S-Sepharose purified TFPI either dextran sulfate stock solution or heparin stock solution ~~an 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/rnl TFPI, and to a final dextran sulfate c~ncentration of 0.6 mg/ml (75 MM) or a fr;ial heparin concentration of 1.5 mg/ml (83 ~uM), depending on which was used to facilitate the refolding. Cystine was added to the refolding sohation to a final concentration equal to the anal DT'T 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 p1 of TFPI stock either 60 ~l of dextran sulfate with 65 p1 of SO mM
Tris (pH 8.8) in 0.1 M NaCl, or 125 pI of heparin stock solution with 50 mM
Tris (pH 8.8) or O.I M NaCl was added. The refolding ;>olution 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 ~l of 50 mM Cy:>tine made up in 120 m~~I
sodium hydroxide was added and the total solution was incubated at 4°C with gentle agitation for 4 days. The free sulfhydryl content was checl~ed with Ellman's reagent (also called DTNB). Idoacetamide was added, to 20 mM, made up at I M in I00% ethanol for storage at .20°C.
The hydrophabic interaction column (HIC) was prepared from Butyl-650M
Tosohaas ToyopearlTM rE;sin particle size 40-90, pai°t # 014702. The butyl resin was washed in 3 M urea, I M ammonium sulfate, 50 mM Tris, 10 mM sodium phosphate, pH 6.5 and resuspended at a 50% slurry.

w~ 96/40784 ~CTII~JS96A09980 _2S_ The refolding samples, stored at -20~C remained in the standard refolding buffer containing 3 M urea, SO mM Tris, pH 8.8; 1-4 mM redox, 0.5 mg/ml TFPI, and 0.2-0.6 M Na~l depending on condition. Samples refolded ,,pith dextran or heparin had 0.2 M
salt, and samples without dextran or heparin had 0.6 M Na~l.
S The following steps were performed at room temperature to effect the further purification of the refolded TFPI. To 300 pI of refolded sample, an equal volume of 2 ~I amrnoraium sulfate, 3 M urea, 50 mM Tris, and 1 d3 mM sodium phosphate (pH
6.5) was added. Next, 100 p1 of washed Butyl-6SOM beads was added to the diluted refolded sample. The solution with the beads was incubated with gentle rcPcking or mixing for 30 minutes at room temperature. The mix was then spun in an ependorf centrifuge for 5 second s, and put in a rack and allowed to sit for one nunute for the beads to settle flat in the tube. The supernatant was aspirated carefully, so as not to disturb the beads.
To wash the T'FPI-bound beads, 1 ml of wash bufl'er camposed of 1 M ammonium 1S sulfate, 3 M urea, SO mla~ Tsis, I0 nxM sodium phosphate (pH 6.S) was addsrd 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 there washed with the gash 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 I, 300 p1 of elution bufl;er composed of 3 M urea, 0.1 M
ammonium sulfate, S0 m.IVI Tris and 10 mM sodium phosphate (pH 6.S) was added to the slurry of beads and rocked for more than 10 nunutes. The beads were pe;lleted by spinning in an ependorf centrifuge, and the supemata~t containing refolded TFPI was recovered. To a~roid contamination of the beads with the product, somsr of the supernatant was left behind.

w~ 96/40784 ~'C°TlHJS96I09980 Example 2 - C of Dextran Sulfate Refold The sample of TFPI was renatured at a concentration of 0.5 mg/ml TFI'I, 0.6 mgJml Dextran sulfate, 3.0 M I:lrea, 200 NaCI and SO m~M Tris (pH ~.5). The HIfC
column was prepared from TosoHaas Butyl beads for HIC, ~.6 mmD/100mmL, in a 1.66 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 NH4S~, at a final pH of 5.68;

2 ml of sample was loaded. The gradient start was 33 MES/33 mM PE;S/33 mM
sodium acetate, 1.0 M NH~S04, and 3.0 M Urea, pH 6.0; the gradient end was 33 mM
MES/33 mM HEPES/33 mM sodium acetate, 3.0 e/ Urea at pH 6Ø The gradient volume was 5.0 CV. From this column, the recover5r of native TFPI was 68%. The results ofthis run are sl;~own 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 W NH4SC1~ and two rnl were loaded. The gradient start was mM MES/33 mM HEPESi33 mM sodium acetate, O.S M NH4S~~, and 3.O M Urea, pH
6.0; the gradient end was 33 MESf33 mM HEIPES/33 sodium acetate, 3.0M
Urea at pH 6Ø 'The g~°adient volume was 5.0 CV. "fhe recovery of native ~°FPI from this second column was 74~/°. The results of this ruri are shown in Figure ~.
The samples were analyzed by non-reducing SDS-PAGE as illustrated in Figure 1. Correctly refolded, active I species major bard) are seen on the gel.
Example 3 About 10 mglmL TFPI in 2M urea was dialyzed against one of the following: 20 mM acetate, 20 mM phosphate, 20 mM citrate, 20 rnM glycine, 20 I,-glutamate or 20 mM succinate in 150 mM NaCI as described above. 6-10 mglml~ ~"FPI bulls stock was loaded into SpecIPo:r 7 dialysis tubings cutoff 3,a ). Dialysis was carried out either at 4; C or ambient temperature. Three changes of buffer at a protein solution to buffer ratio: 1 to 50-lnU, were made during c~urse of dialysis over 12 to 24 hr time period. Aft~:r dialysis, TFPI solution was filtered by Cos 0.22 micron filter units to separate p>recipitated TFPI from soluible TFPI. The solubility of TFPI
was then measured by ~TVIVis absorbance assuming an absorptivity 0.68 ~mg/mL)'1 cm'' at 278 nm. The solutions were prepared at v;ous pH levels by titration with HCl or NaOH.

w0 96/40784 P~'T1U896/0'9980 After completion of dialysis, the precipitates were filtered through 0.22 ,um filter units. The concen;xation of remaining soluble m FPI after dialysis was measured by tJ~1 absorbance. Figure 1 shows the results of these experiments.
solubility of TFPI increased greatly :in solutions containing 20 r~iacetate, 20 m~I
phosphate, 20 S m~I L-glutamate and ~?0 mI~ succinate at pI-I levs~ls below 7 and particularly at or below pI~ 4.5. solubility of TFPI was also su~~stantially increased in solutions containing 20 mIvT glyc~,ne above pI~ 1~. Figure 2 shows the solubility of ',fFPI as a function of concentration of citrate ion ire the presence of 10 m11~ Na phosphate at pH
7. TFPI solubility incre~~ses with increasing concentration of citrate. Figure 3 shows the solubility of TFPI as a function of concentration of NaCI at p.I~ ~.U.
TFPI
solubility increases with increasing salt concentration, indicating salt.
promotes solubility of TFPI.
The solubility o:~ TFPI was studied using a number of different solubilizers and secondary solubilizers. Table 1 shows solubility of TFPI in varying buffer 1S solutions measured by IJ-V' absorbance after dialyzing 6 to 10 mg/mL TFPI
into these buffer solutions.

'6~'~ 96/4074 PCT'/LJS96/099~0 ~2~m Tabl 2 1 Satt effect _ Solubilit Content H c m /ml _ _ uv l OmM NaP04. 7 0.21 ~

iOmM NaP04, 150mM NaCJ 7 0.72 20mM NaP04, 150mM NaCI 7 0.85 20mM NaPO4. 0.5M NaCI 7 6.71 20mM NaP04, 1 M NaCI ~ 7 8.24 ~

_--pE-f effect Content N c m Imf uv 20mM NaOAc, 150mM NaCI 3 10.27 20mM NaOAc, 150mM NaG! 3.510.25 20mM Na0Ac,150mM NaCB 4 7.54 _ 4.5i .75 20mM NaOAc, 150mM NaCi 20mM NaOAc, i 50mM NaCI 5 1 .1 5 20mM NaOAc, 150mM NaC! i 5.50.85 20mM NaP04, 150mM NaCI 5.50.89 20mM NaP04, 150mM NaCI 6 0.78 20mM NaP04. 150mM NaCI ~ 6.50.79 20mM NaP04, 150mM NaCI g5 20mM NaP04, 150mM NaCI ~ ~5 ~:
A -- 8~

20mM NaP04, 150mM NaCB 8 0.86 ~

20mM NaCitrate, 150mM NaCI 4 2.1 7 20mM NaCitrate, 150mM NaCI 4.51 .19 20mM NaCitrate, 150mM NaCI . 5 1 .1 20mM NaCitrate, 150mM NaCI 5.51.84 20mM NaCitrate, 150mM NaCI 6 2.09 20mM NaCitrate, 150mM NaCI 6.S2.12 20mM NaCitrate, 150mM NaCI 7 1 .92 s 20mM GI tine, 150mM NaCI 9 0.32 ~ g 20mM Glycine, 150mM NaCI 1 0.9 ' 0 _ 1 20mM GI cine, 150mM NaCI 1 13.94 20mM'L-Glutamate, 150mM NaCI 4 9.07 20mM L-Glutamate, 150mM NaCI 5 .21 20mM Succinate, 150mM NaCI 4 8.62 20mM Succinate, 150mM NaCI 5 1.21 20mM Succinate, 150mM NaCI ~ 6 .07 Citrate Content o-i c m /ml uv lOmM NaP04, 20mM NaCitrate 7 .1 WO 96140984 PdCT/63S96109980 Table 1 (c0nt.) tOmM NaP04, 50mM NaCitrate 7 5.g1 tOmM NaP04, 100mM NaCitrate 7 12.7 ~

t 0mM NaP04, 200mM NaCitrate V _ ~ t 5.9 lOmM NaP04, 300mM NaCifrate 7 8.36 M 2+, Ca2+ and of hos hate Content _ H c m Iml uv lOmM NaPO~, 7 50mM NaCI, t mM MgCl2 7 0.66 lOmM NaP04, 150mM NaCi, tOmM IVi CI2 7 1.02 lOmM NaP04, 150mM NaCI, 0.7mM _CaCl2 7 0,67 tOmM NaP04, 150mM NaCI, 1mM CaCl2. 7 0,71 tOmM NaP04, 150mM NaCt, iOmM tri hos hate 7 3,6q tOmM NaP04, 5% PEG-400 ~ 0.07 tOmM NaP04, tOmM EDTA _ 0.36 ~

lOmM NaP04, 100mM Na2S04 7 5.08 lOmM NaP04, 100mM !_-as antic acid 7 0.4 lOmM NaP04, 100mM Succinic acid 7 2.33 tOmM NaP04, 100mA4 Tartaric acid _ 2.56 20mM NaP04, 100mM Malefic acid 7 p 2omM NaP04, t00mM Malic acid 7 1.87 lOmM NaP04, t OOmM l- lutamic acid 7 0 _tOmM NaP04, 150mM NaCI _ 7 0.25 tOmM NaP04, 100mM isocitrate ~ 7 10.83 NaOAc, NaP~4 and NaCi Content H r. m ml ' uv tOmM NaOxlc, t50mM NaCI 4.5t.76 lOmM NaOAc _ 4.54.89 t OmM NaOAc 5.54.95 tOmM NaOAc 6.5' S.1 t OmM NaOAc 7 5.87 lOmM NaP04, 150mM NaC! 4.50.14 tOmM NeP04 4.54.97 tOmM NaP04 5.50.79 1 OmM NaP04 6.5~ 0.09 - t lOmM NaP04 7 0.94 ~

a 50mM NaOAc 5 5.24 SmM NaOAc 5.54.59 t OmM NaOAc 5. 5:05 20mM NaOAc .5 5.04 _ _ 5.7't SOmM NaOAc _ - I 5.5 t00mM NaOAc 5,51.4 200mM NaOAc - -__- ~ 5.5 t.32 -W~ 96!40784 ~'C~'liJS96109980 ~30~
Table 1 (cont.) 5mM NaOAc, 150mM NaCI ~ 5.5 ~ 0.65 lOmM NaOAc, 150mM NaC!
f 5.5 0:69 20mM NaOAc, 150mM NaC1 5.5 0.7.1 ~

50mM NaOAc, 150mM NaCI 5.5 0.91 Fi dro hob j icchain !en ih _ ! H c Content ! mo/ml uV

lOmM NaP04, 50mM Formic acid 0 7 0,12 lOmM NaP04, 50mM Acetic acid 7 I 0.16 lOmM NaP04, 50mM Pro anoic ae:id 7 0.16 _ lOmM NaPO4. 50mM Butanoic a_cid_ ~ 7 0.13 lOmM NaP04, 50mM Pentanoic acid ~ 0.14 ~ 7 lOmM NaP04, 50mM Fiexanoic said ~ ~
~ ~ 0.1 7 I! 1 t ~thBfS

Content ! pf~l c (mg/ml~ uv ~
I

20mM NaOAc, 3% Mannitot, 2% Sucrose, 5% PEG-400~ 4 19.9 20mM Na Citrate, 3J Mannitol, 2/z~ Sucrose, ~ 6.5 0.72 5% PEG-400 20mM NaCitrate, 159mM NaCl, S.o PEG-X00 8.5 2.18 20mM NaOAc, 150mM NaCI, 5% PEG-~&00 ~4 t 9.8 20mM Na Citrate. 130mM NaCl, 1~% Gfycine, 0.25% ~ _ Tween-80. ' .48 _ _ 20mN~ Na Citrate, 130mM NaCi, 1 '/ Glycine, ~~ 6.5 ~ 1 .32 0.25% Tween-80 _~ Solubi8ity Content ) pEi c (mg/m!),uv j Smt~ NaAc_etate ; 5.5 8.9 I

5mM NaAcetate, e% Sucrose _ t 5.5 1 1 ~SmM NaAcetale, 0.01 /~ Polvsorbate-80 ~ 5.5 7 ~

W~ 96!40?84 ~CT/~IS96109980 Table 1 (cost.) 5mM NaAcetate, 8% Sucrose, 0.01 % 5 _ 1 2 Polysorbate-80 lOmM NaAcetate ~ 7.6 5.5 1 omM NaAceiate, 8! Sucrose ~ 5.5 1 0 1 OmM NaAcetate, 8% Sucrose, 0.01 ; 5.5 12.1 % Polysorbate-80 5mM NaAcetate, 5% Sorhitol 5.5 7.8 5mM NaAcetate, 4.5% Mannitol l 5.5 9.2 5mM Histidine 6 5.5 5mM Histidine 6.5 1 5mM NaCitrate I 5.5 0.1 5mM NaCitrate I 6 0.1 5mM NaCitrate 6.5 0.1 ~

5mM NaSuccinate - 5.5 0.6 5mM NaSuccinate 6 0.3 5rnM NaSuccinate 6.5 0.2 t OmM Imidazole j 6.5 2.5, 10.8 t OmM Imidazole , 3 0.8 iOmM Imidazole, 8/~ Sucrose _~ 6.5 12.2 5mM NaAcetate ~ 6 8.2 lOmM Imidazcte, 5mM NaAcetate i 6.5 12.8 t OmM NaCiirate i 6 0.2 1 OOmM NaCitrate ~ 6 8.1 100mM NaCitrate ~ 7 9.3 tOmM Naphosphate, 260mM Na2S04 6 9.1 lOmM NaPhosphate, 100mM NaCitrate 8 8.8 ~

lOmM NaCiirate, 1 % L- lutamic acid f 6 4.6 lOmM NaCitrate, 2! l_-I sine 6 1.1 iOmM NaCitrate, 0.5% L-as antic acid ~ 6 0.4 lOmM NaCitrate, 0.1 % Phos hate lass '7 5.9 lOmM Tris, 100mM NaCitrate 8 8.5 1 OmM NaGitrate, 1 M Gi cine 6 0.3 lOmM NaCitrate, 300mM GI cine ~& 0.3 lOmM NaCitrate, 280mM Glycerol 6 0.3 lOmM NaCitrate, 0.5M NH4 2504 6 8.3 lOmM NaCitrate, l2omM NH4 2504 6 8.8 lOmM NaCitrate, 260mM Na2S04 6 9.4 lOmM NaP04, 0.1% Phos hate lass 7 15.8 lOmM NaCitrate, 0.1% SOS 6 11.2 lOmM NaCitrate, 0.02! SOS 6 7.8 lOmM NaAcetate, 8% PEG-400 5.5 13.~

lOmM NaAcetate,.1~50raM NaCI, 8% PEG-4005.5 _0.6 lOmM NaAcetate, 8% PEG-400 6 16.2 lOmM NaCitrate, 8% PEG-400 - ~ 0.2 W~ 96/40784 PC'T/I,TS96/09980 ~'he stability of '1'FPI stored at various pH conditions was tested. '1"FPI
was prepared by dialysis as above fn 10 mM IeTa phosphate, 150 mM lVaCl and 0.005%
(w/o) polysorbate-80. Stability samples containing 150 mg/mL TFPI were incubated at 40iC for 20 days. Kinetic rate constant for the remaining soluble 7::~?PI
was analyzed by following decr of the main on ration exchange chromatogra>rcs.
As can be seen in Figure ~, the decay rate constant increases at pH above 6.0, indicates yore aggregation at higher pIi conditions.
TFPI was also fE~rrnulated at a concentration. of 150 mg/mL in 150 nnM NaCI
and 0.005% (wlv) polysorbate-80 at pII 7 with varying concentrations of phosphate.
~°igure SA shows the percentage of remaining soluble T'FPI measured by the ration exchange HPLC. IncrE;asing 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 "iCFPI as assayed by the prothrombin time assay. 'T'hese results are shown in F°igua°e 5B.
Stability of TFP:i at a concentration of 0.5 mglmL and formulated in 10 mM
I~Ta citrate, pH 6 and i50 mM l~aC1 was also tested at 40°C over a 40 day period.
As seen in Figub, ca~.ion-exchange ~IPLC (triangle) shows the presence of soluble TFPI at levels greater ~;han 60 % initial, even after' the 40 day incubation.
In like manner, the prothrombin time assay (circle) shows the presence of active TFPI
at levels greater than 60 % initial, even after the 0 day incubation.
Figure 7 shows loss of soluble T'FPI at 40°C measured by bond cation-exchange HPLC (open symbol) and prothrombin time assay (closed symbol) for 0.5 mglmL ~"FPI formulated in 10 mM Na phosphate, p~I 6 and either 1~0 mM NaCI
(triangle) or 500 mM bT6tCl (circle).
Figure 8 shows loss of soluble 1'FPI at 40°C measured by both cation-exchange HPLC (open symbol) and prothrombin tune assay (closed symbol) for 0.5 mgfmi, TFPI formulated in 10 mM IVa acetate and pI°I 5.5 containing 150 mhTaCl (triangle) or 8 % (wlv) sucrose (square) or 4.5 %~ (wl~r) mannitol (circle).

VV~ 96!40784 F°CT/~1S96/09980 - 3~
Figure 9 shows two non-reducing SIBS gels for TFPI formulation samples in mM NaPO~, 150 mM NaCl, and 0.005 % polysorbate-80 at pH 4 to pH 9 stored at 40~C for 0 days (lowers and z0 days (upper. No Ioss on TFPI is seen at 0 days.
However, at 20 days cleavage fragments of TFPI may be seen at the Iower pH
range 5 (i.e. pH 4 and pH 5). without being bound t~ a pa °culax 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 X10°C for various formulations. 0.5 mgt TFPI was formulated in these formulation ° conditions and incubated at 40°C. Samples were withdrawn at predetermined time 10 intervals and loss of soluble and active TFPI were examined by the IEX-H:PLC and the PT assay. Half life for remaining soluble TFPI was then calculated by performing a single exponential fitting to the IEX-HPL~ and PT assay results.
Elution of TFPI in displacement mode from chromatography resins using polyionic compounds.
TFPI 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 pum through the column. This compound binds stronger to the resin than TFPY and elisplaces TFPI. For a pasitiveiy charged resin (anion exchanger) a negatively charged compound is used and for a negatively charged resin (canon exchanger) a p~sitively charged compound is used.
Partially purified 1"FPI was used as starting material. TFPI, in 6 urea, 20 Tris, pH 8.0 was lo;~ded onto a column packed with an anion exchange resin, Q
Sepharose HP, to 20 mgfmL resin. After loading, the column was washed with 6 M
urea, ~ 20 mM Tris, pH 9Ø P1 was eluted d 10 mgiml of (lass H
(polyphosphate) in 6 M urea, 10 mM Tris, pH 9Ø
Elution of TFPI from chromatography resin ia~ aqueous buffer using polyionic compounds.

WO 96140°784 PC'><YfJS96/09980 -34~
Table 2 tll2 (da at 40~

0.5 m ml TFPI formulated in: iEX-HPLC PT assa ~

mM Na Acetate, 150 mM NaCI~H _'10.5 s 17.2 5.5 0 ~

10 mM Na Citrate, 150 mM NaCI, 12.2 24.4 p1-! 5.5 i0 mM Na Acetate, 8/~ v~lv) Sucrose,~H48.2 42.2 5.5 10 mM Na Acetate, 4.5/~ Mannitoi, 47.'7 46.C
pH 5.5 i0 mM Na Succinate, 150 mM NaCI, 7.8 7 1 .0 pH 6.0 10 mM Na Citrate, 150 mM NaCf, 13.0 18.8.
H 6.0 10 mAA Na Phosphate, 150 mM NaCI, 7.8 1 1 .2 H 6.0 10 mM Na.Phosphate, 500 mM NaCI, 52.2 58.9 pH 6.0 [ 10 mM Na CBtrate, 150 mM NaCI, 10.0 14.8 pH 6.5 r 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, I mg/ml polyphosphate, ~0 mM Tris, pH 5.9 was loaded onto a canon exchange resin, SP Sepharose HP. After loading the column was washed with a non-urea containing buffer, 10 mglmi polyphosphate, 10 rriM
sodium phosphate, pH 5Ø TFI'I was eluted in the same buffer at pH 7.5, without urea.
EXampIe 7 Selective eluticln of TFPI from ion exchange resins using polyionic compounds.
Because of thw charged ends of TFPT, oppositely charged polyionic compounds can bind to these ends. When the polyionic compound has a higher strength of binding to T FPI than does the resin, the TFPI may be selectively eluted from the chromatography resin.
TFPI, in 3.5. M urea, 1 mg/ml polyphosphate, 50 mM Tris, pH 5.9 was loaded onto a canon exchange ~°esin, SP Sepharose HP. After loading, the column is Washed with 6 M urea, Img/ml polyphosphate, 10 mM sodLium phosphate, pH 5.9. TFPI was eluted in a 25 column volume gradient up to 20 mg/ml of polyphosphate. 7CFPI
starts to elute at about 2-3 mg%ml of polyphosphate.
Example 8 Neutralization of 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 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 STM (Whatman) and no purification is achieved. By mixing PEI into the column load, TFPI now binds to the resin and TFPI can be purified.

~~ 96/40784 PC'T/L3~9o6/09980 -3~-Refolding arad purification of recornbinan,t human PI (rh °I) using polyphosphate ~Cilass Il:) Facilitated Refolding process.
Inclusion bodies containing about ~0 g of rh pI were thawed by r°emoving S the containers from the -20°C freezer and incubating them in a cold room at 4-1°C
overhead stirrer. The ~:ontents were mixed for apF~roximately 15 minutes, and then the absorbance of the solution is measured at 2$0 nm. If the absorbance is greater than the mixture was diluted with sufficient dissolution buffer to obtain an absorbance at 280 nm of I.0-1.1. 'The solution was incubated with gentle agitation for 15-minutes, and then sufficient cysteine was added to give a cysteine concentration of 0.1 . The solid I,~-cysteine was dissolved in approximately 50 ml of purified water and added to the rc;fold mixture. The p~I was checked and adjus to pI~
10m2 if necessary. The refold mixture was incubated 'with gentle agitation fo,r 96-hours.
after approximately 96 h, the refolding process was terminat by adjusting the pI-I of the refold mixture to pIi 5.9 using glacial acetic acid.
Stin°ing was continued for 90 minutes and the pI-I checked. More acid was added, if noes to adjust the pFi to ~.9 +~- 0.1. ~ two-step fil tion process was a to remove the °c t formed during previous steps and prepare tl~e acidified refold mixture for SP-Sepharose chromatographyo First the adidigied refold mixture i.s passed through A Cuno 60LP .depth filter falter housing rr~odel 8ZP1) using a peristaltic _ pump ('~ - 3/a inch inne~° diameter silicon tubing).
The filter system was washed with 8-10 I, of deionized b Ivi ur before use.
The filtrate was collec in a 1 I, polyethylene . clc 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 :~~ow ra.te dropped below 1 I, per W~ 96/40784 F~~°t°/L3S9~6/4D99~0 _37-minute in order to maintain the back pressure at 20 PSI. The second stage of the filtration used a 0.45 micron filter c °dge (Sartorius Sartobran pI3 ~r equivalent) with a peristaltic pumpir~h system. After filtration, tif~e pfd was checked, and adjusted to p~I 5.9 if neces The acidified, fi:~tered refold was loaded onto the equilibra SP S~epharose column at a flow rate of approximately 80.0 mlf~nin. Flour rate was adjusted to accommodate overnight loading of the acidified f lte:red refold mixture. e:
column eras equilibrated in 6 I~ urea, 20 mA~ sodium phosphate buffer pI~ 5.9 prior to loading. After loading, the column is gashed with 2 ~F of 6 Iii urea, 0.3 rvI
l~laCl, 20 msodium phosphate buffer, pIi :~.9 prior to the gradient elution st~:p. The column flow rate was increased to 190-2 mllmin for the wash step and all subsequent steps (fin velocity =- 47 c~n/hr~. °I'he product was eluted from the column using a linear s~dt gradient from 0.3 to 0. ii I~IaCI in 6 11~ urea, 20 m 250210). The total volc~me of the gradient was 71.0 liters or 13.0 C~. prhe p~T ~f the gradient buffers was 5.92 (+l- .02). Fractions are evaluated qualitativeay using SIBS PAGE and poaleti ba on the content of the correctly folded SC-59735 relative to other misfolds and impurities. After ling the process s is referred to as the 1.
'I°he pI-I of the 1 was next adjusted to pH S.0 with 2.S 1V a~i. The S
pool was concentrated 2-3 fold to approximately :~ L, using an Arnicon IBC-10I.
uI tration unit con ° ° ~g an Arr~icon ~NI10 spiral cartridge (10, NT.~J. cut-off membrane). After conc~;ntration, the concentrated S pool was diafaltered against 7 volumes of 6 urea, 20 mTris-~iCl buffer, ~a~i 5Ø a diafltraation was considered complete vrhe.n the conductivity of the r~etentate was below 2 rnS.
The diltered concentrate was drain from the ultrafnitration unit and the unit was W~ 96/40784 PC'r/I1~9G/09980 _38-washed with approximately 1 L of diafiltration buffer. The was is combined with the concentrate to form the ~-load.
An Amicon column (7.0 cm diaaa~eter) was packed with approximatel~r 700 ml of Q-Sepharose high performance medium (Pharrr~acia Q-Sepharose ). The column was packed with 20% ethanol at 20 si. T'he bed height after paclang was approximately 18 cm. The column was equilibrated with 5 CF of 6 M urea; 0,02 M
TrisIHCl buffer, pH 8. The target for protein loading is 8-10 mg ~rotein/ml Q
Sepharose resin. The Q load was applied to the column at a flow rate 30-35 ml/min (5Q cm/hr): After loading, the column was washed with approximately 5 Chi' of , 20 mM TrisIHCI 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
I~TaCl in 6 M urea, 20 n~M Tris/I3C1 buffer, pH 8.C) 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 roc.~tanely analyzed by reducing and non-reducing SDS-PAGE
and size exclusion chromatography. Fractions are pooled based on aggregate content ( < ~ % by SEC LC 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 Frool was adjusted to 7.2 using 2 M HCI. The pool was then concentrated approximately 5 fold in an Amicon DC-1 a~ltraf~ltration system contaiir~ing a SlYl Amicon I'NI-10 cartridge (I0,000 MVVCCI spiral cartridge membrane). The concentrated Q 1'001 was then diafiltered against seven column volumes of 2 M urea, 0.15 M NaCI; 20 mM
°um phosphate buffer, pH 7.. Following ultrafiltration, the solution 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 circniated thr~ugh the ultr~nltration system for approximately 5 min. The rinse solution was combined with the original concentrate and the solutaon was filtered through a 0.4~ micron vacuum alter unit (Nalgene).

VVO 96J40784 PCTII~JS96J09980 _3g_ 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 fzeezer and incubating them in a cold room at 4-10°C
for approximately 96 hours. The thawed inclusion bodies were then disperst;d with a high shear mixer t~ reduce the clumping that occurs during freezing. 'The inclusion body slurry urea vigorously blended for approximately 1 minute using a polytron homogenizer (Brinkman model PT45l80) or until the inclusion bodies were then added to 40 L of 6 M urea 100 mM TrislHCl buffer pH 9.8 containing 300 mM
NaCl and 0.4 g!L PEI contained in a 100 L polyethylene tank equipped with an overhead stirrer. The z:uxture was vigorously stirred for 20-30 min. a pH was monitored and adjusted to pH 9.8 as neces ,Che absorbance of the dissolved inclusion body mixture ~~as measured at 280 nm, and if the absorbance was greater IS 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 NaCI solution. Finally; L-cysteine was added to give a final concentration;: of 0.25 mM. The solid L-~cysteia~e was dissolved in 50 ml of WFI and added as a solution to the diluted refa~ld, The pH was checked and adjusted, if necessary. ~'he refold continued with gentle mixing for 96-120 hours with periodic checks of thc~ pH, and adjustment to pH 9.8, if' necessary. The progress of the refold was monitored by Mon-S ration exchange and prothrombin dine 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. Stirring was contin,aed for 90 minutes and the pH choked. More acid was added, if necessary to adjust the pH
to 5.9 !- 0.1.
A two-step ~ltraticsn process was used to remove the particulates that formed during previous steps and prepare the acidified refold for SP-Sepharose HP
chanmatograph. First, the acidified refold is passed through a Cuno 60LP depth filter (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.
5 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 lIl Order to maintain the back pressure at 2.0 PSI. The second stage of the filtration used a 0.45 micron filter cartridge (Sartorius Sartobran pH or equivalent) 10 with a peristaltic pumping system. After f Itration, 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 mllmm. Flow rate was adjusted to accommodate overnight loading of the acidified filtered refold. The column was then 15 washed with 5.5 column volumes of 6 M urea, 0.311~I 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 = ~ ~7 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 was formed by delivering fi M
20 urea, 0.5 M NaCl, 20 mM sodium phosphate buffer into 6 M urea, 0.3 M NaCl, rnM sodium phosphate buffer into 6 M urea, 0.3 M I~laCl, 20 mM sodium phosphate buffer. Limit buffer was pumped with a MasterflexT~ pump (model 7553-20) with a MasterflexT'~ head (model 7015.2I) at a flow rate of approximately 100 mi/mm with vigorous mixing using a ParatrolTM A mixer from Parametrics (model 250210).
The 25 total volume of the gradient was 71.0 liters or 13.0 Ctl. 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/crn as measured by the in-line Radiometer conductivity meter.
Forty 500 ml fractions (0.1 C ~I) were collected. A Pharma.cia Frac-300 fraction collector 30 was used with numbered, 500 ml polypropylene bottles. When the fraction collection was stopped, the remainder of the gradient was collected as a pool.

w~ 96/40784 PC~°IUS96I09980 Column fractions were assayed by A280, size exclusion ~iPLC, and in addition, for informational purposes, SIBS PAGE, reverse phase I~PLC, and P'I' 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 pI3 8.0 with 2.5 N Na~H. The S
Pool was concentrated 2-3 fold to approximately 2 L using an Amicon i~C-10L
ultrafiltration unit containing an Amicon 1'MI0 spiral cartridge (10,000 N..
cut-off membrane). After concentration, the concentrated S Pool was diafiltered against 7 volumes of 6 M urea, 20 mM Tris-~Cl 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 ultrafltration unit and the unit was washed with approximately 1 L of diai'~ltration buffet°. The was is combined with the concentrate to form the (~-load.
An Atnicon column (7.0o cm diameter) was packed with approximately 700 ml of Q-Sepharose high performance medium (Pharmacia Q-Sepharose I-IP). The column was packed in 20%~ ethanol at 20 psi. Ttae bed height after packing was approximately 18 c.m The column was equilibrated with 5 CV of 6 M urea, 0.02 M
TrisBHCI buffer, pI~ 8. The target for protein loading is 8-10 mg protein/ml Q
Sepharose resin. The Q load was applied t~ the column at a flow rate 30-35 ml/min (50 cmlhr). After loading, the column was washed with approximately 5 C~ of 6 M
urea, 20 mM Tris/~ICl buffer, pH 8.0, or until the absorbance at 280 nm returned to baseline. The product was eluted using a sodium chloride gradient from .15 M
NaCI in 6 M urea, 20 mM TrisB~ICI buffer, p~I 8.0 over 25 column volumes. 'The first seven column volumes were collected as a - single fraction, followed by fractions of 0.25 column volume each.
Fractions are routinely analyzed by reducing and non-reducing SI7S-PAGE
and size exclusion chromatography. Fractions are pooled based on aggregate content (5 % by SEC HPLC) and qualitative evaluation by SIBS PAtiE to assess purity:
The fractions are stored frozen at -20~C until pooled.

4 PC'FNS96/09980 The ~-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 IvIHCI. The pool was then concentrated approximately 5 fold in an Amicon DC-1 ultrafiltration system containing a S1Y1 Amicon -10 cartridge (10,000 M1~VC~ spiral cartridge membrane). The concentrated Pool was then diafrltered 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 ultrafiltration system.
Approximately 100 ml of 2 M urea, 0.15 M NaCI, 20 mM sodium phosphate buffer, pH 7.2 was circulated through the ultrafiltration system for approximately 5 min.
'The rinse solution was combined with the original concent~°ate and filtered through a 0.45 micron vacuum flier unit (Nalgene).
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 m/ inclusion body slurry containing 4~ mg/ml rhTFPI) was dissolved with mixing in 4 L of 50 mM; 'I'ris buffer, pH 10.5 containing 4 g/1 polyphosphate (Glass Ii, FMC Corporation) 2-8°C. Sufficient cysteine and cystine was added to male 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 8 ° C with gentle mixing for 72-96 h.
column volumes of 0.4 ~O Glass H, 20 mM sodium phosphate pH6 buffer. The column was eluted using a linear pH gradient Pram 0.4 9o Glass H, 20 mM sodium phosphate buffer pH 6 to 0.4 3o Glass H, 50 mM 'T'ris pH 8 buffer. Fractions were collected and analyzed by SDS PAGE. Relatively pure rhTFPI could be refolded and purified in this manner.

VVO 961~07~4 PC"Ti'~JS96/09980 improved solubility of rhTFPi in water by :formation of a complex between TFPI and polyphosphate (GDS 5327046-47) About 10 g of purified rhTFPi in about 1 litsmr of 2 IvI urea, 12S mr4 sodium S chloride, 20 sodiulr phosphate pI~ 7.4 buffer was thawed by incubation at 2-S ° G
for 1g-36 h. Sufficient dry urea was added to make the solution 6114 in urea.
The solution was then filtered through a 0.2 micron filter. Five g of polyphosphate glass (Glass Ii, FItIC) was dissolved in SO rnl of 6 r4 urea, adjusted to p 7 with 1 I~
removed from the ultrafiltration unit. The ultrafiltration unit was washed with about 1S0 ml of purified water and the was added to the protein concentrate. The fanal 15 protein concentrate contained almost 10 g of protein in 400 ml of water (about 24 mg/rnl protein). The normal solublllty of rhTFPI ire water is less than O.S
mg/ml.
I3se of cationic polymers for removal of ~'. cn~ri contaminants front TFPI ~Il lysates and refractile bodies.
20 The use of cationic polymers to precipitate a.nd remove E coli con inants - from crude I interanediates (lysates, refractile bodies) can signifi tly improve subsequence process operations (refolding, chronnatography etc.) A random screening of cationic polymers identified candidates which selectively precipitate bacterial contaminants while TFPI remains in solution. Specifically, tz polymer 2S 624 precipita substantial amounts of bacterial contaminants, while leaving I in solution in an aqueous environment.
Solubilized TFPi refractile bodies (in 3.S ltd guanidine hydrochloride, 2 sodium chloride, SO mlvl S, SO mIVI dithiothrnitol, pI-I7.1) was the starting material used for a polyrl~er screening experiment. This material was diluted 10 fold 30 into a 0.S%~ solution of various polymers. The prE:cipitates from this experiment W~ 9614084 PCT1US96I09980 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.
Fyam=1_34 .
The use of aqueous two phase extraction with a polyethylene glycol (PEG), polyphosphate, urea system offers processing advantages fcir TFPI
purification.
Typical aqueous two phase systems consist of two polymer systems (e.~., PEG
and dextran) or a polymer and salt (e.~., 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 s ific 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, 10 mM CAPS, 1 l monothioglycerol pIilO. Polyphosphate and PEG of different chain lengths were added to form two phases. Upon 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 rccombinant tissue plasminogen activator (t-PA) from ~ crab 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 9' Glass gI, 50 mM Tras buffer pkI 10.8 containing I mM reduced glutathione (GS~I) and 0.2 mM glutthione disulfide (GSSG). The mixture is thoroughly blended using a polytron (l3rinkman) horr~ogenizer 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 pI~ is maintained at 10.5-10.9 using I N Na~PI. The mixture is then gently mixed for 48-~2 hours at 2-BoC.
Examp Charged polyrme~- facilitated refolding of bovine somatotropin from ~. coli inclusion bodies Ten grams (wet weight) of inclusion bodies containing 5 grams of bovine somatotropin are added to about 1 liter of 1~/o Glass H, 5(1 mI~ Tris buffer pH 10.5.
The mixture is thoroghly blended using a polytron (~rinkman) homogenizes for 2-minutes to thoroughly disperse the inclusion bodies>. The mixture is incubated with mixing using an overhead stirrer for 25 minutes while the pH is maintained at 10.4 10.6 using 1 N Na~H. solid cysteine (121 mg) is added to make the reaction 1 mM
cysteine, and the refolding reaction is mixed for 48-;7? hours The present invention has been descriir,ed with reference to specific embodiments. However, this application is intended to cover those changes and substitutions which may be made by those spilled in the art without departing from the spirit and the scope of the appended claims.

Claims (14)

1. A method of refolding an improperly folded or denatured protein comprising the step of adding charged polymers to a solution comprising said protein prior to allowing said protein to refold.

2. The method of claim 1, wherein said polymer is a sulfated polysaccharide.

3. The method of claim 2, wherein said sulfated polysaccharide is dextran sulfate.

4. The method of claim 2, wherein said sulfated polysaccharide is heparin.

5. A method of refolding TFPI comprising the step of adding a charged polymer to a solution comprising improperly folded or denatured TFPI prior to allowing said TFPI to refold.

6. The method of claim 5, wherein said TFPI is ala-TFPI.

7. The method of claim 5, wherein the polymer is dextran sulfate.

8. The method of claim 5, wherein the polymer is heparin.

9. The method according to claim 7, wherein the heparin is added in solution.

10. The method according to claim 5 further comprising the steps of:
incubating said solution to allow said TFPI to refold; adding salt to disassociate the polymer from the TFPI, passing the solution over an HIC
column, and recovering the TFPI.

11. A method of refolding TFPI comprising the step of immobilizing polymers of sulfated polysaccharides on a column and passing a solution of denatured TFPI
through the column and eluting the refolded TFPI after the refolding has occurred.

12. The method of claim 11, wherein said TFPI is ala-TFPI.

13. The method of claim 11, wherein the sulfated polysaccharide is dextran sulfate.

14. The method of claim 11, wherein the sulfated polysaccharide is heparin.