Purification of vitamin K-dependent polypeptides using preparative reverse phase chromatography (RPC)

Fl ELD OF TH E I NVENTI ON The present relation relates to novel chromatographic methods suitable for purification of vitamin K-dependent polypeptides, in particular Factor VII polypeptides.

BACKGROUND OF THE INVENTION

In the pharmaceutical industry, purification is an integrated part of producing molecules for medical needs. Both biopharmaceutical molecules derived from recombinant tech- niques and more traditional smaller molecules derived from organic synthesis are generated, and in both cases chromatographic purification techniques play an essential role in production methods. Chromatographic techniques include separation based on ion- exchange, hydrophobic interaction etc. In reversed phase chromatography (RPC) a molecule in solution binds to the hydrophobic surface or hydrophobic ligand of a chroma- tographic resin. The partitioning of the molecule between the solution and the resin occurs as a result of hydrophobic interactions between the molecule with hydrophobic patches at its surface and the hydrophobic surface on the resin. A solvent of increasing hydrophobicity is subsequently used to dissociate or elute the bound molecule at a point at which the hydrophobic interaction between the exposed patches and the resin is less favourable than the interaction between the bound molecule and the solvent. The molecule then releases from the resin and elutes. Separation of different molecules in the same solution occurs if the molecules have different hydrophobicity and therefore elute at different point in time when the hydrophobicity of the eluting solvent is increased.

In general, RPC is capable of distinguishing between molecules with very small differences in hydrophobicity and it is thus regarded as very powerful separation tool and the preferred method in analytical chromatography. RPC is applied for preparative use as well; however, RPC is usually used for purification of smaller molecules and peptides that can withstand the harsh operating conditions including organic solvents. Larger mole- cules, such as proteins (also called polypeptides) denature more easily and preparative RPC is therefore generally considered to be unsuitable for native proteins ["Reversed Phase Chromatography. Principles and Methods", Amersham Pharmacia Biotech; and "Conformation of polypeptides and protein in reversed phase and lipophilic environments" MTW Hearn in "Biochromatography. Theory and Practice", Taylor & Francis, 2002].

Some disclosures of the application of preparative RPC on larger polypeptides do exist. In general these applications are, however, performed in fairly small scale, and with a relatively low polypeptide load, on a RPC column with a small diameter and with a column material with relatively small particles size (to be regarded as semi-preparative, non- industrial scale/load). Solvent system used for purification is typically acetonitrile with TFA at very low pH (see Wang, YM et al. {Biochem., 354, 161-168, 2001) directed to the use of RPC to purify proteases from snake venom; Chlenov, MA et al. (J Chromatogr. 1993, 631(1-2), 261-267) directed to the use of RPC at neutral pH to purify biological active thyroid stimulating hormone, luteinizing hormone and chorionic gonaditropin; Te- shima G and Canova-Davis E (J Chromatogr. 1992, 625(2), 207-215) directed to the purification of H ₂ O ₂ -treated human growth hormone).

Vitamin K-dependent coagulation factors, requiring gamma-carboxylation of glutamic acid residues in the so-called Gla-domain for activity, include factor VII, factor IX, factor X, prothrombin, Protein C and Protein S, all plasma proteins that are components of the coagulation system; Protein Z, also found in plasma, pulmonary surfactant-associated proteins (Rannels et al. Proc. Natl. Acad. Sci. USA 84: 5952-56, 1987), and the bone proteins osteocalcin (also known as bone gla-protein) and matrix gla-protein. Polypep- tides containing the amino acid γ-carboxyglutamic acid (GIa) are variously referred to as "Vitamin K-dependent polypeptides", "gla-proteins", or "gamma-carboxylated polypeptides." The plasma vitamin K-dependent polypeptides are dependent on gla-mediated binding to calcium and membrane phospholipids for their biological activity.

Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually gives rise to a fibrin clot. Generally, the blood components which participate in what has been referred to as the coagulation "cascade" are proenzymes or zymogens, enzymatically inactive polypeptides which are converted to proteolytic enzymes by the action of an activator, itself an activated clotting factor. Coagulation factors that have undergone such a conversion and generally referred to as "active factors," and are designated by the addition of a lower case "a" suffix (e.g., activated factor VII (FVIIa)).

The zymogens are generally single-chain, catalytically inactive polypeptides that are cleaved upon activation into two-chain (activated) molecules; for example, FVII circulates in the blood stream as a trace plasma glycoprotein in the form of a single-chain zymogen of 406

amino acids. The zymogen is catalytically inactive. Single-chain Factor VII is converted into catalytically active two-chain Factor Vila by cleavage of the internal Argi ₅₂ -Ilei53 peptide bond. This conversion of zymogen Factor VII into the activated two-chain Factor Vila is catalysed in vitro by Factor Xa, Factor XIIa, Factor IXa, Factor Vila, kallikrein and thrombin. Factor Xa is believed to be the major physiological activator of Factor VII.

Human recombinant Factor Vila (NovoSeven®; Novo Nordisk A/S, Denmark) is widely used as a therapeutic protein to control bleedings in, e.g., persons with haemophilia, persons undergoing surgery, and trauma victims.

Activated factor IX (FIXa) is a plasma coagulation factor required to convert FX into its activated form, FXa. The activated plasma coagulation factor FX (FXa) is required to convert prothrombin to thrombin, which then converts fibrinogen to fibrin as a final stage in forming a fibrin clot.

Protein C is a naturally occurring serine protease anticoagulant that plays a role in the regulation of homeostasis by inactivating factors Va and Villa in the coagulation cascade.

Protein S also exhibits anticoagulant activity in in vitro clotting assays. Protein S demon- strates anticoagulant cofactor activity for activated protein C.

Osteocalcin is composed of 49 amino acid residues which include three GIa residues. The function of this polypeptide is thought to be to suppress excessive mineralization.

Matrix GIa Protein (MGP) is composed of 79 amino acids including 5 GIa residues. This polypeptide is usually found in demineralized matrix and believed to have a certain function in the initiation of bone formation.

Vitamin K-dependent polypeptides are serine proteases and their hydrolytic activity does not necessarily stop after cleavage of their respective substrate. Auto-activation and auto-degradation may occur, i.e. where e.g. the FVIIa protease not only cleaves the Argl52 bond of a "neighbouring" FVII molecule (auto-activatson) but also hydrolyses other bonds adjacent to basic amino acids in the FVII sequence such as Lys38-Leu39, Arg290-Gly291 and Arg315-Lys316 (auto-degradation).

Vitamin K-dependent polypeptides, including FVII and FVIIa, are sensible to conformational changes as this is highly likely to disturb the enzymatic activity of the molecule. The polypeptides are large molecules (e.g. 406 amino acid sequence of FVII) with a highly complicated three-dimensional structure of both the single-chain and the two- chain molecules. The molecules have a complicated interaction within the coagulation cascade where they may act as substrate in one enzymatic reaction (e.g. activation of zymogen) and as catalyst in another; thus, a correct three-dimensional structure is crucial for correct interactions between the molecules and thereof following enzymatic activity.

Thus, it is important to possess methods for purification which are not only powerful separation tools but wherein also a substantial part of the bioactivity of the polypeptides subjected to purification is maintained. Surprisingly, the present inventor has found that Factor VII withstands the harsh conditions in RPC. It is thus possible to apply Factor VII to a reverse phase column and elute it from the column without substantial loss in the enzymatic activity e.g. as measured by a clotting assay. Furthermore, an acceptable yield is obtained.

Because of the resolving power of RPC and the need to purify native polypeptides with high purity, e.g. for therapeutic use, it is desirable to have methods which provide improved or alternative ways of applying RPC in the field of polypeptide separation in industrial scale, including using industrially suitable column loads and retaining substantial levels of bioactivity of the purified polypeptide.

Mollerup et al. describe the use of RP-HPLC for analysis of FVII solutions under denaturing conditions, where the solution is heated to 70 °C in a column oven prior to application (Mollerup et al. ; Biotech. Bioeng.; 1995; 48; 501-505)

SUM MARY OF TH E I NVENTI ON The present inventors have surprisingly found that reverse phase chromatography (RPC) can be used for purifying vitamin K-dependent polypeptides.

Accordingly, the present invention relates to a method for purifying vitamin K-dependent polypeptides, in particular Factor VII polypeptides, by reverse phase chromatography. The invention in particular relates to a method for purifying a vitamin K-dependent poly-

peptide from a composition comprising said polypeptide and at least one undesired impurity, the method comprising :

(a) loading a solution of said composition onto a reversed phase liquid chromatography column; and

(b) eluting said polypeptide from the column with a solvent;

(i) wherein : at least 30% of the polypeptide eluted in step b) has retained its bioactivity, or

(ii) wherein at least 30% of the product of step b) is bioactive after having been subjected to an activation process.

DESCRI PTI ON OF TH E I NVENTI ON

The present invention provides a method for industrial-scale polypeptide separation by reverse phase chromatography. In one aspect the method applies column loads in the range of 0.1-200 mg/mL of column material. The method further provides a gentle way of purifying vitamin K-dependent polypeptides, in particular Factor VII polypeptides, in industrial-scale, i.e. a method wherein a substantial amount of the loaded polypeptide survives the operating conditions and retains its bioactivity.

The term "purifying" a polypeptide from a composition comprising the polypeptide and one or more contaminants means increasing the degree of purity of the polypeptide in the composition by reducing the contents of at least one contaminant from the composition. The contaminants may be non-related impurities, such as e.g. host cell proteins (HCP), or they may be related impurities. The term "related impurity" as used herein means an impurity that has a structural resemblance to the target polypeptide but has different chemical or physical structure compared to the target polypeptide. Related im- purities may include, without limitation, truncated forms (such as, e.g., heavy chain degradation products), extended forms (extra amino acids, various derivatives, etc.), deami- dated forms, incorrectly folded forms, forms with undesired glycosylation including sialy- lation, oxidated forms, forms resulting from racemization, forms lacking amino acids in the intra-polypeptide chain, forms having extra amino acids in the intra-polypeptide chain, and forms having replacements of amino acids in the intra-polypeptide chain.

The term "derivative" as used herein in relation to a parent polypeptide means a chemically modified parent polypeptide or an analogue thereof, wherein at least one substitu-

ent is not present in the parent polypeptide or an analogue thereof, i.e. a parent polypeptide which has been covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, pegylations and the like.

The term "truncated forms" as used herein in relation to a polypeptide means any fragment of the polypeptide having at least 20% of the amino acids of the parent polypeptide, such as 35%, 50%, or 75%. Thus, for human serum albumin a fragment would comprise at least 117 amino acids as human serum albumin has 585 amino acids.

The term "industrial scale" is meant to include processes, wherein the RPC columns used are at least 0.1 I, such as at least 0.2 I, at least 0.5 I, at least 1 I, at least 2 I, at least 5 I, at least 20 I, at least 50 I, or such as at least 100 I. "Industrial scale" is also meant to include processes wherein the amount of polypeptide applied to the column is at least 0.01 g, such as at least 0.02 g, at least 0.05 g, at least 0.1 g, at least 0.2 g, at least 0.5 g, at least 1 g, at least 2 g, at least 5 g, at least 10 g, at least 20 g, at least 50 g, at least 100 g, at least 100 g, at least 200 g, at least 500 g, at least 1000 g, at least 2000 g, at least 5000 g, or such as at least 10000 g.

The solvent used to elute the polypeptide is an aqueous solvent comprising from 0-96% of an organic compound. Non-limiting examples of organic compounds include: Acetoni- trile; mono-alcohols, i.e. alcohols comprising only one alcohol group, such as, without limitation, methanol, ethanol, 1-propanol and 2-propanol, and mixtures of two or more thereof; di-alcohols i.e. alcohols comprising two alcohol groups, such as, without limitation, 1,5-pentanediol, 1,6-hexanediol, and 1,7-heptanediol. Included are also mixtures of two or more mono-alcohols; mixtures of two or more di-alcohols; mixtures of one or more mono-alcohol(s) with one or more di-alcohol(s); and mixtures of one or more mono-alcohol(s) and/or di-alcohol(s) with acetonitrile. In one embodiment of the invention the organic compound is acetonitrile. In another embodiment of the invention the organic compound is a mono-alcohol such as ethanol, 1-propanol, 2-propanol, or a mix- ture thereof; preferably the mono-alcohol is 2-propanol.

In one embodiment, the organic component is an alcohol, and in one embodiment, the solvent is a mixture of water and an alcohol. Particular mentioning is made of mono- alcohols, i.e. alcohols comprising only one alcohol group. Examples of mono-alcohols which can be used in the methods of the present invention include methanol, ethanol, 1- propanol and 2-propanol, and mixtures of two or more thereof. It is regarded as an addi-

tional advantage to use alcohols rather than acetonitrile due to the well-established environmental and occupational health problems connected to the use of acetonitrile.

The solvent used to elute the polypeptide may further comprise a salt in solution. The term salt is used for ionic compounds composed of positively charged cations (X) and negatively charged anions (Y), so that the product is neutral and without a net charge. Both X and Y may be multiply charged so that the ratio X:Y may be different from 1 : 1. Examples of salts which can be applied in the present invention include halides, such as chlorides, bromides, iodines; sulphates; borates; lactates; and citrates, and mixtures of two or more thereof. Examples of the positively charged counter ion include sodium; potassium; magnesium; calcium; and ammonium. Specific examples of salts include potassium chloride; sodium chloride; ammonia chloride and potassium lactate.

Typical salt concentrations to be used in the present invention are between 0.02 and 30 (w/w) %, such as between 0.05 and 10 (w/w) %, such as between 0.16 and 1.1 (w/w) %.

The solvent used to elute the polypeptide may also comprise a buffer. A buffer is a mix- ture of an acid (HA) and its conjugated base (A ^" ). A buffer is capable of resisting changes in pH as the result of addition of acid or base. This resistance (buffer capacity) is largest when pH is close to the pKa of the acid HA. In practical life, a mixture of an acid and the conjugated base is regarded as a buffer if the pH of the solution is within two pH units, such as within one pH unit from the pKa value of the acid. Examples of buffers which can be applied in the present invention include trifluoro acetic acid (TFA), acetate buffers, phosphate buffers, citric acid buffers, lactic acid buffers, TRIS buffers, CHAPS buffers, borate buffers, HEPES buffers, carbonate buffers, histidine buffers, MES buffers, ascorbic buffers, and mixtures of two or more of these. In one embodiment of the invention the buffer is trifluoro acetic acid (TFA).

Typical buffer concentrations to be used in the present invention are between 0.02 and 20 (w/w) %, such as between 0.05 and 5 (w/w) %, such as between 0.1 and 0.2 (w/w) %.

Various factors may influence the choice of the pH at which to purify a given polypeptide according to the method of the present invention, and in particular, the pi of the poly-

peptide is important. When pH of a solution is the same as the pi of a dissolved polypeptide, the solubility of the polypeptide is lowest and the risk of precipitation is highest. Normally it is desirable to use a pH which is at least one or two pH units away from the pi of the polypeptide to be purified, although pH close to or at the pi of the polypeptide to be purified can be used if solubility of the polypeptide is not a problem. This, of course, also influences the choice of buffer in that a given conjugated acid-base pair is only effective as a buffer when the pH is close to the pKa of the acid in the given solvent. Typically, pH of loading solvents used in the present invention is in the range of 4-10, such as e.g. 6-10, 5.5-9, or 5.5-8.7, and pH of elution solvents are typically in the range of 1-10, such as e.g. 1-8, 1-6, or 1-4.

The method of the present invention may be run at a range of temperatures depending on e.g. the type of polypeptide to be purified. If the temperature is too high, the polypeptide may denature irreversible, and if the temperature is too low, mechanical prob- lems may arise due to increased viscosity of the solvent. An adjustment of the temperature within these limits may be used to increase the separation of two polypeptides if the hydrophobicity of the two polypeptides has different temperature dependence. Generally, the methods of the present invention may be run at temperatures from 0-50°C, such as from 0-10 °C, 10-50 °C, 20-50 °C, 20-40°C, or 30-40 °C. In the preferred embodiment the method is run at 20-30 °C.

In one embodiment, the solvent used for elution comprises a refolding agent. Non- limiting examples of refolding agents include arginine, guanidine, and ethylene glycol. In one embodiment the refolding agent is arginine in a concentration from about 0.5M to about 5M. In another embodiment the refolding agent is ethylene glycol in a concentration from about 0.5M to about 1OM.

In another embodiment, the purification of said FVII/FVIIa polypeptides is performed using protein stabilizers such as sugars, e.g. sucrose, In one embodiment the stabilizer is sucrose in a concentration of from about 0,SM to about 5M,

In one embodiment the elution is performed into a solution comprising a refolding and/or a stabilizing agent. In another embodiment the refolding agent and/or stabilizing agent is/are added to the elution buffer.

The polypeptides are eluted with an increasing hydrophobicity of the solvent, i.e. by increasing the concentration of the organic compound. The concentration of the solvent used to load the polypeptide on to the column depends on the nature of the polypeptide and the hydrophobicity of the organic compound. This solvent is often referred to as the equilibration solvent as the column has typically been washed or equilibrated with one or more column volumes of this solvent prior to the loading of the polypeptide to the column. A typical concentration of the organic compound in the equilibrating solvents is from 0-90%, such as e.g. 0-80%, 0-50%, 0-20%, 0-10%, or 0-5%. The concentration is upward limited by the denaturing effect of the organic component. If the concentration is too high, there is a risk that the polypeptide may denature irreversibly. During elution of the polypeptide, the concentration of the organic component in the solvent is raised, typically to concentrations from 5-96%, such as 10-95%, 20-90%, 30-90%, or 40-80%.

The equilibration and/or loading solvent for Vitamin K-dependent polypeptides may con- tain divalent ions such Ca ²⁺ ions, preferably as CaCI ₂ . Vitamin K-dependent polypeptides containing Gla-residues are more hydrophobic in the presence of divalent ions and in the most preferable embodiment of the invention the equilibration and/or loading solvent contains 0.5-40, 0.5-30, 0.5-20, 0.5-10, 0.5-5 mM CaCI ₂ . The equilibration and/or loading solvent may also contain a buffer, such as, without limitation, trifluoro acetic acid (TFA), acetate buffers, phosphate buffers, citric acid buffers, lactic acid buffers, TRIS buffers, CHAPS buffers, borate buffers, HEPES buffers, carbonate buffers, histidine buffers, MES buffers, ascorbic buffers, and mixtures of two or more of these. In one embodiment of the invention the buffer is a TRiS buffer,

The method may comprise one or more steps of washing prior to elution. The washing solvent may be the same as equilibration solvent or a new solvent comprising an organic component. Washing may be performed by linear or stepped gradient. The washing solvent may also contain a buffer, such as those that may be used in the equilibration and/or loading solvent(s), and/or divalent cations, such as, e.g., calcium ions. In one embodiment the washing buffer is trifluoro acetic acid (TFA),

The organic solvent in the washing solvent is selected from acetonitrile, mono-alcohols, i.e. alcohols comprising only one alcohol group, such as, without limitation, methanol, ethanol, 1-propanol and 2-propanol, and mixtures of two or more thereof; di-alcohols i.e. alcohols comprising two alcohol groups, such as, without limitation, 1,5-pentanediol, 1,6- hexanediol, and 1,7-heptanediol. It is characteristic for the organic solvent in the wash-

ing buffer that it is not able to elute the polypeptide of interest at the concentration of the organic solvent used during washing.

Elution derived from the increase in the concentration of the organic component in the solvent (often referred to as the gradient) may be brought about in a number of ways. The gradient may be linear, stepped comprising one or more steps, isocratic or curved. Elution may also be performed in isocratic mode, that is, by constant organic component concentration. The elution scheme may also be applied in any combination of the above gradients and isocratic elution mode, e.g. an elution scheme may be a linear gradient followed by an isocratic elution followed by a step and followed by a linear gradient again, or it may be a linear gradient followed by another linear gradient.

Reversed phase column material is made of a resin to which hydrophobic material may be attached. Typical resin materials are silica and polystyrene; hydrophobic ligands may optionally be attached. In case of substituted resins, the resin is substituted with a hy- drophobic ligand, typically selected from (but not limited to) aliphates, such as C ₂ , C ₄ , C ₆ , Cs, Cio, Ci2, Ci ₄ , Ci6, or Ci ₈ or derivatives of these, e.g. cyanopropyl (CN-propyl) or branched aliphates, or benzene-based aromates, such as phenyl or other polar or non- polar ligands. The ligand may be a mixture of two or more of these ligands. A preferred resin is substituted with C ₂ -C ₈ ligands or derivatives of these. Suitable polystyrene based resins include, without limitation, resins supplied by Rohm Haas (e.g. Amberlite XAD or Amberchrom CG), Polymer Labs (e.g. PLRP-S), GE Healthcare (e.g. Source RPC), Applied Biosystems (e.g. Poros R).

The manufacturing processes for and optimal features of the column material often re- quire that a linking group also called a spacer is inserted between the resin and the ligand.

Other parameters in the methods of the present invention include load, i.e. amount of polypeptide which is loaded to the column and flow rate. These parameters may be opti- mised through experiments which are known to the person skilled in the art. The polypeptide is typically loaded onto the column in a concentration of at least about 0.1 mg per mL of resin, such as, e.g., at least about 0.2 mg, 0.5 mg, 1 mg , 2 mg, 5 mg, 10, or 20 mg per mL of resin; or in the range of 0.1-200 mg, such as, e.g., 0.1-100 mg, 0.5- 100 mg, 1-50 mg, or 2-30 mg per mL of resin; preferably the load is at least 1 mg per mL resin. Measurement of packed resin volume is typically done in suspension or similar mode.

The polypeptide is typically applied at a flow of 1-200 column volumes per hour (CV/h), such as at least 1 CV/h, such as at least 2 CV/h, such as at least 3 CV/h, such as at least 4 CV/h, such as at least 5 CV/h, such as at least 6 CV/h, such as at least 8 CV/h, such as at least 10 CV/h, such as at least 12 CV/h, e.g. at least 20 CV/h or at least 40 CV/h or at least 80 CV/h, e.g. 80-120 CV/h.

When the polypeptide is eluted from the column it is dissolved in a solvent with a relatively high concentration of the organic component, which may harm the polypeptide over time due to its denaturing effects. In one embodiment of the invention, the polypeptide is therefore transferred to another (second) solvent/medium immediately after elu- tion, e.g. by dilution, diafiltration, ultrafiltration, precipitation, crystallisation, desalting, gel filtration, or by binding the polypeptide onto another chromatographic medium (ion exchange-, hydrophobic interaction-, affinity or metal chelate medium), washing out the denaturing solvent and eluting the polypeptide. In one embodiment, this second solvent is an aqueous buffer with pH in the range of 5-9, preferably 6-9.

In one embodiment, the method of the present invention comprises the steps of: a) Loading the vitamin K-dependent protein dissolved in a equilibrating solvent comprising water, a buffer, a divalent cation such as Ca ²⁺ , pH 6-9, onto a RPC column at 2-10 g/l column material; b) Washing the column with the equilibrating solvent; c) Eluting the vitamin K-dependent protein in a linear gradient of 0 to 100% of an eluting solvent comprising a mono-alcohol; and d) Collecting the vitamin K-dependent protein-containing fractions.

In a preferred embodiment thereof, the eluting solvent comprises propanol, preferably 2- propanol; in an even more preferred embodiment, the eluting solvent comprises 70% 2- propanol.

In another embodiment, the method of the present invention comprises the steps of:

a) Loading the vitamin K-dependent protein dissolved in an equilibrating solvent comprising water, a buffer, a divalent cation such as Ca ²⁺ , pH 6-9, onto a RPC column at 2-10 g/l column material; b) Washing the column with a washing solvent comprising acetonitrile and optionally a buffer;

c) Eluting the vitamin K-dependent protein in a step gradient with an eluting solvent comprising a mono-alcohol; and d) Collecting the vitamin K-dependent protein-containing fractions.

In a preferred embodiment thereof, the eluting solvent comprises propanol, preferably 2- propanol; in an even more preferred embodiment, the eluting solvent comprises 70% 2- propanol.

In a preferred embodiment, the column is washed (in step b) with a linear gradient of 0 to 100% of a solvent comprising 80% acetonitrile. In one embodiment thereof, the sol- vent comprising 80% acetonitrile further comprises 0.09% (w/w) TFA.

In yet another embodiment, the method of the present invention comprises the steps of:

a) Loading the vitamin K-dependent protein dissolved in an equilibrating solvent comprising water, a buffer, a divalent cation such as Ca ²⁺ , pH 6-9, onto a RPC column at 2-10 g/l column material; b) Washing the column with the equilibrating solvent or a washing solvent comprising acetonitrile and optionally a buffer; c) Eluting the vitamin K-dependent protein in (i) a step gradient with an eluting solvent comprising a mono-alcohol; and, subsequently, (ii) in the washing buffer of step b); and d) Collecting the vitamin K-dependent protein-containing fractions.

Polypeptides

The term "vitamin K-dependent polypeptide", as used herein, means any polypeptide that is gamma-carboxylated on glutamic acid residues. Typical vitamin K-dependent polypeptides includes but are not limited to the procoagulant factors thrombin, factor VII, IX, and X; the anticoagulants protein C and protein S; and other polypeptides such as osteocalcin (bone GIa protein), matrix GIa protein, and proline-rich GIa protein 1.

Factor VII polypeptides

As used herein, the terms "Factor VII polypeptide " or "FVII polypeptide" means any polypeptide comprising the amino acid sequence 1-406 of wild-type human Factor Vila (i.e., a polypeptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950), variants thereof as well as Factor VII-related polypeptides, Factor VII deriva-

tives and Factor VII conjugates. This includes FVII variants, Factor VII-related polypeptides, Factor VII derivatives and Factor VII conjugates exhibiting substantially the same or improved biological activity relative to wild-type human Factor Vila.

The term "Factor VII" is intended to encompass Factor VII polypeptides in their un- cleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated Factor Vila. Typically, Factor VII is cleaved between residues 152 and 153 to yield Factor Vila. Such variants of Factor VII may exhibit different properties relative to human Factor VII, including stabil- ity, phospholipid binding, altered specific activity, and the like.

As used herein, "wild type human FVIIa" is a polypeptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950.

As used herein, "Factor VII-related polypeptides" encompasses polypeptides, including variants, in which the Factor Vila biological activity has been substantially modified, such as reduced, relative to the activity of wild-type Factor Vila. These polypeptides include, without limitation, Factor VII or Factor Vila into which specific amino acid sequence alterations have been introduced that modify or disrupt the bioactivity of the polypeptide.

The term "Factor VII derivative" as used herein, is intended to designate a FVII polypeptide exhibiting substantially the same or improved biological activity relative to wild-type Factor VII, in which one or more of the amino acids of the parent peptide have been genetically and/or chemically and/or enzymatically modified, e.g. by alkylation, glycosyla- tion, PEGylation, acylation, ester formation or amide formation or the like. This includes but is not limited to PEGylated human Factor Vila, cysteine-PEGylated human Factor Vila and variants thereof. Non-limiting examples of Factor VII derivatives includes GlycoPegy- lated FVII derivatives as disclosed in WO 03/31464 and US Patent applications US 20040043446, US 20040063911, US 20040142856, US 20040137557, and US 20040132640 (Neose Technologies, Inc.); FVII conjugates as disclosed in WO 01/04287, US patent application 20030165996, WO 01/58935, WO 03/93465 (Maxygen ApS) and WO 02/02764, US patent application 20030211094 (University of Minnesota).

The term "improved biological activity" refers to FVII polypeptides with i) substantially the same or increased proteolytic activity compared to recombinant wild type human

Factor Vila or ii) to FVII polypeptides with substantially the same or increased TF binding

activity compared to recombinant wild type human Factor Vila or iii) to FVII polypeptides with substantially the same or increased half life in blood plasma compared to recombinant wild type human Factor Vila. The term "PEGylated human Factor Vila" means human Factor Vila, having a PEG molecule conjugated to a human Factor Vila polypeptide. It is to be understood, that the PEG molecule may be attached to any part of the Factor Vila polypeptide including any amino acid residue or carbohydrate moiety of the Factor Vila polypeptide. The term "cysteine-PEGylated human Factor Vila" means Factor Vila having a PEG molecule conjugated to a sulfhydryl group of a cysteine introduced in human Factor Vila.

Non-limiting examples of Factor VII variants having substantially the same or increased proteolytic activity compared to recombinant wild type human Factor Vila include S52A- FVIIa, S60A-FVIIa ( Lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); FVIIa variants exhibiting increased proteolytic stability as disclosed in U.S. Patent No. 5,580,560; Factor Vila that has been proteolytically cleaved between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48: 501-505, 1995); oxidized forms of Factor Vila (Kornfelt et al., Arch. Biochem. Biophys. 363 :43-54, 1999); FVII variants as disclosed in PCT/DK02/00189 (corresponding to WO 02/077218); and FVII variants exhibiting increased proteolytic stability as disclosed in WO 02/38162 (Scripps Research Institute); FVII variants having a modified Gla-domain and exhibiting an enhanced membrane binding as disclosed in WO 99/20767, US patents US 6017882 and US 6747003, US patent application 20030100506 (University of Minnesota) and WO 00/66753, US patent applications US 20010018414, US 2004220106, and US 200131005, US patents US 6762286 and US 6693075 (University of Minnesota); and FVII variants as disclosed in WO 01/58935, US patent US 6806063, US patent application 20030096338 (Maxygen ApS), WO 03/93465 (Maxygen ApS), WO 04/029091 (Maxygen ApS), WO 04/083361 (Maxygen ApS), and WO 04/111242 (Maxygen ApS), as well as in WO 04/108763 (Canadian Blood Services).

Non-limiting examples of FVII variants having increased biological activity compared to wild-type FVIIa include FVII variants as disclosed in WO 01/83725, WO 02/22776, WO 02/077218, PCT/DK02/00635 (corresponding to WO 03/027147), Danish patent application PA 2002 01423 (corresponding to WO 04/029090), Danish patent application PA 2001 01627 (corresponding to WO 03/027147); WO 02/38162 (Scripps Research Insti- tute); and FVIIa variants with enhanced activity as disclosed in JP 2001061479 (Chemo- Sero-Therapeutic Res Inst.).

Examples of variants of factor VII include, without limitation, PlOQ-FVII, K32E-FVII, P10Q/K32E-FVII, L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII,

V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, K157A- FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII, and S336G-FVII, L305V/K337A-FVII, L305V/V158D-FVII, L305V/E296V-FVII, L305V/M298Q- FVII, L305V/V158T-FVII, L305V/K337A/V158T-FVII, L305V/K337A/M298Q-FVII, L305V/K337A/E296V-FVII, L305V/K337A/V158D-FVII, L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVII, L305V/V158T/M298Q-FVII, L305V/V158T/E296V-FVII, L305V/E296V/M298Q-FVII, L305V/V158D/E296V/M298Q-FVII, L305V/V158T/E296V/M298Q-FVII, L305V/V158T/K337A/M298Q-FVII, L305V/V158T/E296V/K337A-FVII, L305V/V158D/K337A/M298Q-FVII, L305V/V158D/E296V/K337A-FVII, L305V/V158D/E296V/M298Q/K337A-FVII,

L305V/V158T/E296V/M298Q/K337A-FVII, S314E/K316H-FVII, S314E/K316Q-FVII, S314E/L305V-FVII, S314E/K337A-FVII, S314E/V158D-FVII, S314E/E296V-FVII, S314E/M298Q-FVII, S314E/V158T-FVII, K316H/L305V-FVII, K316H/K337A-FVII, K316H/V158D-FVII, K316H/E296V-FVII, K316H/M298Q-FVII, K316H/V158T-FVII, K316Q/L305V-FVII, K316Q/K337A-FVII, K316Q/V158D-FVII, K316Q/E296V-FVII, K316Q/M298Q-FVII, K316Q/V158T-FVII, S314E/L305V/K337A-FVII, S314E/L305V/V158D-FVII, S314E/L305V/E296V-FVII, S314E/L305V/M298Q-FVII, S314E/L305V/V158T-FVII, S314E/L305V/K337A/V158T-FVII, S314E/L305V/K337A/M298Q-FVII, S314E/L305V/K337A/E296V-FVII, S314E/L305V/K337A/V158D-FVII, S314E/L305V/V158D/M298Q-FVII, S314E/L305V/V158D/E296V-FVII, S314E/L305V/V158T/M298Q-FVII, S314E/L305V/V158T/E296V-FVII, S314E/L305V/E296V/M298Q-FVII, S314E/L305V/V158D/E296V/M298Q-FVII, S314E/L305V/V158T/E296V/M298Q-FVII, S314E/L305V/V158T/K337A/M298Q-FVII, S314E/L305V/V158T/E296V/K337A-FVII, S314E/L305V/V158D/K337A/M298Q-FVII, S314E/L305V/V158D/E296V/K337A-FVII, S314E/L305V/V158D/E296V/M298Q/K337A-FVII,

S314E/L305V/V158T/E296V/M298Q/K337A-FVII, K316H/L305V/K337A-FVII, K316H/L305V/V158D-FVII, K316H/L305V/E296V-FVII, K316H/L305V/M298Q-FVII, K316H/L305V/V158T-FVII, K316H/L305V/K337A/V158T-FVII, K316H/L305V/K337A/M298Q-FVII, K316H/L305V/K337A/E296V-FVII, K316H/L305V/K337A/V158D-FVII, K316H/L305V/V158D/M298Q-FVII,

K316H/L305V/V158D/E296V-FVII, K316H/L305V/V158T/M298Q-FVII, K316H/L305V/V158T7ε296V-FVII, K316H/L305V/E296V/M298Q-FVII, K316H/L305V/V158D/E296V/M298Q-FVII, K316H/L305V/V158T/E296V/M298Q-FVII, K316H/L305V/V158T/K337A/M298Q-FVII, K316H/L305V/V158T/E296V/K337A-FVII, K316H/L305V/V158D/K337A/M298Q-FVII, K316H/L305V/V158D/E296V/K337A -FVII, K316H/L305V/V158D/E296V/M298Q/K337A-FVII,

K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/L305V/K337A-FVII, K316Q/L305V/V158D-FVII, K316Q/L305V/E296V-FVII, K316Q/L305V/M298Q-FVII, K316Q/L305V/V158T-FVII, K316Q/L305V/K337A/V158T-FVII, K316Q/L305V/K337A/M298Q-FVII, K316Q/L305V/K337A/E296V-FVII, K316Q/L305V/K337A/V158D-FVII, K316Q/L305V/V158D/M298Q-FVII, K316Q/L305V/V158D/E296V-FVII, K316Q/L305V/V158T/M298Q-FVII, K316Q/L305V/V158T/E296V-FVII, K316Q/L305V/E296V/M298Q-FVII, K316Q/L305V/V158D/E296V/M298Q-FVII, K316Q/L305V/V158T/E296V/M298Q-FVII, K316Q/L305V/V158T/K337A/M298Q-FVII, K316Q/L305V/V158T/E296V/K337A-FVII, K316Q/L305V/V158D/K337A/M298Q-FVII, K316Q/L305V/V158D/E296V/K337A -FVII, K316Q/L305V/V158D/E296V/M298Q/K337A-FVII,

K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII, F374Y/V158D-FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII, F374Y/V158T-FVII, F374Y/S314E-FVII, F374Y/L305V-FVII, F374Y/L305V/K337A-FVII, F374Y/L305V/V158D-FVII,

F374Y/L305V/E296V-FVII, F374Y/L305V/M298Q-FVII, F374Y/L305V/V158T-FVII, F374Y/L305V/S314E-FVII, F374Y/K337A/S314E-FVII, F374Y/K337A/V158T-FVII, F374Y/K337A/M298Q-FVII, F374Y/K337A/E296V-FVII, F374Y/K337A/V158D-FVII, F374Y/V158D/S314E-FVII, F374Y/V158D/M298Q-FVII, F374Y/V158D/E296V-FVII, F374Y/V158T/S314E-FVII, F374Y/V158T/M298Q-FVII, F374Y/V158T/E296V-FVII, F374Y/E296V/S314E-FVII, F374Y/S314E/M298Q-FVII, F374Y/E296V/M298Q-FVII, F374Y/L305V/K337A/V158D-FVII, F374Y/L305V/K337A/E296V-FVII, F374Y/L305V/K337A/M298Q-FVII, F374Y/L305V/K337A/V158T-FVII, F374Y/L305V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V-FVII, F374Y/L305V/V158D/M298Q-FVII, F374Y/L305V/V158D/S314E-FVII, F374Y/L305V/E296V/M298Q-FVII, F374Y/L305V/E296V/V158T-FVII, F374Y/L305V/E296V/S314E-FVII, F374Y/L305V/M298Q/V158T-FVII, F374Y/L305V/M298Q/S314E-FVII, F374Y/L305V/V158T/S314E-FVII, F374Y/K337A/S314E/V158T-FVII, F374Y/K337A/S314E/M298Q-FVII, F374Y/K337A/S314E/E296V-FVII, F374Y/K337A/S314E/V158D-FVII, F374Y/K337A/V158T/M298Q-FVII, F374Y/K337A/V158T/E296V-FVII,

F374Y/K337A/M298Q/E296V-FVII, F374Y/K337A/M298Q/V158D-FVII, F374Y/K337A/E296V/V158D-FVII, F374Y/V158D/S314E/M298Q-FVII, F374Y/V158D/S314E/E296V-FVII, F374Y/V158D/M298Q/E296V-FVII, F374Y/V158T/S314E/E296V-FVII, F374Y/V158T/S314E/M298Q-FVII, F374Y/V158T/M298Q/E296V-FVII, F374Y/E296V/S314E/M298Q-FVII,

F374Y/L305V/M298Q/K337A/S314E-FVII, F374Y/L305V/E296V/K337A/S314E-FVII, F374Y/E296V/M298Q/K337A/S314E-FVII, F374Y/L305V/E296V/M298Q/K337A -FVII, F374Y/L305V/E296V/M298Q/S314E-FVII, F374Y/V158D/E296V/M298Q/K337A-FVII, F374Y/V158D/E296V/M298Q/S314E-FVII, F374Y/L305V/V158D/K337A/S314E-FVII, F374Y/V158D/M298Q/K337A/S314E-FVII, F374Y/V158D/E296V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q-FVII, F374Y/L305V/V158D/M298Q/K337A-FVII, F374Y/L305V/V158D/E296V/K337A-FVII, F374Y/L305V/V158D/M298Q/S314E-FVII, F374Y/L305V/V158D/E296V/S314E-FVII, F374Y/V158T/E296V/M298Q/K337A-FVII, F374Y/V158T/E296V/M298Q/S314E-FVII, F374Y/L305V/V158T/K337A/S314E-FVII, F374Y/V158T/M298Q/K337A/S314E-FVII, F374Y/V158T/E296V/K337A/S314E-FVII, F374Y/L305V/V158T/E296V/M298Q-FVII, F374Y/L305V/V158T/M298Q/K337A-FVII, F374Y/L305V/V158T/E296V/K337A-FVII, F374Y/L305V/V158T/M298Q/S314E-FVII, F374Y/L305V/V158T/E296V/S314E-FVII, F374Y/E296V/M298Q/K337A/V158T/S314E- FVII, F374Y/V158D/E296V/M298Q/K337A/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q/S314E-FVII, F374Y/L305V/E296V/M298Q/V158T/S314E-FVII, F374Y/L305V/E296V/M298Q/K337A/V158T-FVII, F374Y/L305V/E296V/K337A/V158T/S314E-FVII, F374Y/L305V/M298Q/K337A/V158T/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q/K337A-FVII, F374Y/L305V/V158D/E296V/K337A/S314E-FVII, F374Y/L305V/V158D/M298Q/K337A/S314E-FVII, F374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII, S52A-Factor VII, S60A-Factor VII; R152E-Factor VII, S344A-Factor VII, T106N-FVII, K143N/N 145T-FVII, V253N-FVII, R290N/A292T-FVII, G291N-FVII, R315N/V317T-FVII, K143N/N 145T/R315N/V317T-FVII; FVII having substitutions, additions or deletions in the amino acid sequence from 233Thr to 240Asn; FVII having substitutions, additions or deletions in the amino acid sequence from 304Arg to 329Cys; and FVII having substitutions, additions or deletions in the amino acid sequence from 1531Ie to 223Arg.

Thus, substitution variants in a factor VII polypeptide include, without limitation substitutions in positions PlO, K32, L305, M306, D309, L305, L305, F374, V158, M298, V158, E296, K337, M298, M298, S336, S314, K316, K316, F374, S52, S60, R152, S344, T106, K143, N145, V253, R290, A292, G291, R315, V317, and substitutions, additions or dele- tions in the amino acid sequence from T233 to N240 or from R304 to C329; or from 1153 to R223, or combinations thereof, in particular variants such as PlOQ, K32E, L305V, M306D, D309S, L305I, L305T, F374P, V158T, M298Q, V158D, E296V, K337A, M298Q, M298K, S336G, S314E, K316H, K316Q, F374Y, S52A, S60A, R152E, S344A, T106N, K143N, N 145T, V253N, R290N, A292T, G291N, R315N, V317T, and substitutions, addi- tions or deletions in the amino acid sequence from T233 to N240, or from R304 to C329, or from 1153 to R223, or combinations thereof.

Bioactivity

The bioactivity of vitamin K-dependent polypeptides derives from their ability to partici- pate in the blood coagulation cascade. Methods and assays for quantifying bioactivity of said polypeptides are known to the skilled person.

The bioactivity or biological activity of Factor Vila in particular in blood clotting derives from its ability to (i) bind to tissue factor (TF) and (ii) catalyze the proteolytic cleavage of Factor IX or Factor X to produce activated Factor IX or X (Factor IXa or Xa, respectively). For purposes of the invention, Factor Vila biological activity may be quantified by measuring the ability of a preparation to promote blood clotting using Factor VII-deficient plasma and thromboplastin, as described, e.g., in U.S. Patent No. 5,997,864. In this assay, biological activity is expressed as the reduction in clotting time relative to a control sample and is converted to "Factor VII units" by comparison with a pooled human serum standard containing 1 unit/ml Factor VII activity. Alternatively, Factor Vila biological activity may be quantified by (i) measuring the ability of Factor Vila to produce of Factor Xa in a system comprising TF embedded in a lipid membrane and Factor X. (Persson et al., J. Biol. Chem. 272: 19919-19924, 1997); (ii) measuring Factor X hydrolysis in an aqueous system (see Experimentals section below); (iii) measuring its physical binding to TF using an instrument based on surface plasmon resonance (Persson, FEBS Letts. 413:359-363, 1997) (iv) measuring hydrolysis of a synthetic substrate (see Experimentals section below); and (v) measuring generation of thrombin in a TF-independent in vitro system (see Experimentals section, below). Specific activity of FVIIa is defined as the measured biological activity (using one of the above mentioned assays) divided by the amount of FVIIa. Specific activity is usually expressed in IU/mg or IU/ microgram.

Activation of polypeptides

The term "activation process", as used herein, represents any process which can convert a catalytically inactive form of a vitamin K-dependent polypeptide into its catalytically ac- tive form. Typical processes include the cleavage of a zymogen Factor VII polypeptide, including native FVII, into the corresponding two-chain, activated Factor VII polypeptide, including native FVIIa. In the case of native FVII, this cleavage takes place at the Argl52 bond. Agents suitable for activation of the vitamin K-dependent polypeptides are known to the skilled person. In the case of FVII, activation agents include FX (which is believed to be the major physiological activator of FVII) and trypsin.

In one embodiment, the vitamin K-dependent polypeptide to be purified according to the method of the present invention is selected from the list of: human Factor VII, human Factor Vila, human recombinant Factor VII, human recombinant Factor Vila, human Fac- tor IX, human Factor IXa, human recombinant Factor IX, human recombinant Factor IXa, human Factor X, human Factor Xa, human recombinant Factor X, human recombinant Factor Xa, human Protein C, human activated Protein C, human recombinant Protein C, and human recombinant activated Protein C.

In one embodiment, the Factor VII polypeptide to be purified according to the method of the present invention is selected from the list of: human Factor VII, human Factor Vila, human recombinant Factor VII, human recombinant Factor Vila, FVII variants as disclosed in WO 01/58935, US patent US 6806063, US patent application 20030096338 (Maxygen ApS), WO 03/93465 (Maxygen ApS), WO 04/029091 (Maxygen ApS), WO 04/083361 (Maxygen ApS), and WO 04/111242 (Maxygen ApS). In a preferred embodiment, the Factor VII polypeptide is selected from the list of: human Factor VII, human Factor Vila, human recombinant Factor VII, and human recombinant Factor Vila

In one embodiment, the Factor VII polypeptide has a glycosylation different from wild- type human Factor Vila. In some embodiments, the Factor VII polypeptide has substantial the same or increased half life in blood plasma compared to recombinant wild-type human Factor Vila.

In one embodiment, the Factor VII polypeptide to be purified according to the method of the present invention is selected from the list of: L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII,

K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII, and S336G-FVII, L305V/K337A-FVII, L305V/V158D-FVII, L305V/E296V-FVII, L305V/M298Q-FVII, L305V/V158T-FVII, L305V/K337A/V158T-FVII, L305V/K337A/M298Q-FVII, L305V/K337A/E296V-FVII, L305V/K337A/V158D-FVII, L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVII, L305V/V158T/M298Q-FVII, L305V/V158T/E296V-FVII, L305V/E296V/M298Q-FVII, L305V/V158D/E296V/M298Q-FVII, L305V/V158T/E296V/M298Q-FVII, L305V/V158T/K337A/M298Q-FVII, L305V/V158T/E296V/K337A-FVII, L305V/V158D/K337A/M298Q-FVII, L305V/V158D/E296V/K337A-FVII, L305V/V158D/E296V/M298Q/K337A-FVII, L305V/V158T/E296V/M298Q/K337A-FVII, S314E/K316H-FVII, S314E/K316Q-FVII, S314E/L305V-FVII, S314E/K337A-FVII, S314E/V158D-FVII, S314E/E296V-FVII, S314E/M298Q-FVII, S314E/V158T-FVII, K316H/L305V-FVII, K316H/K337A-FVII, K316H/V158D-FVII, K316H/E296V-FVII, K316H/M298Q-FVII, K316H/V158T-FVII, K316Q/L305V-FVII, K316Q/K337A-FVII, K316Q/V158D-FVII, K316Q/E296V-FVII, K316Q/M298Q-FVII, K316Q/V158T-FVII, S314E/L305V/K337A-FVII, S314E/L305V/V158D-FVII, S314E/L305V/E296V-FVII, S314E/L305V/M298Q-FVII, S314E/L305V/V158T-FVII, S314E/L305V/K337A/V158T-FVII, S314E/L305V/K337A/M298Q-FVII, S314E/L305V/K337A/E296V-FVII, S314E/L305V/K337A/V158D-FVII, S314E/L305V/V158D/M298Q-FVII, S314E/L305V/V158D/E296V-FVII, S314E/L305V/V158T/M298Q-FVII, S314E/L305V/V158T/E296V-FVII, S314E/L305V/E296V/M298Q-FVII, S314E/L305V/V158D/E296V/M298Q-FVII, S314E/L305V/V158T/E296V/M298Q-FVII, S314E/L305V/V158T/K337A/M298Q-FVII, S314E/L305V/V158T/E296V/K337A-FVII, S314E/L305V/V158D/K337A/M298Q-FVII, S314E/L305V/V158D/E296V/K337A-FVII, S314E/L305V/V158D/E296V/M298Q/K337A-FVII,

S314E/L305V/V158T/E296V/M298Q/K337A-FVII, K316H/L305V/K337A-FVII, K316H/L305V/V158D-FVII, K316H/L305V/E296V-FVII, K316H/L305V/M298Q-FVII, K316H/L305V/V158T-FVII, K316H/L305V/K337A/V158T-FVII,

K316H/L305V/K337A/M298Q-FVII, K316H/L305V/K337A/E296V-FVII, K316H/L305V/K337A/V158D-FVII, K316H/L305V/V158D/M298Q-FVII, K316H/L305V/V158D/E296V-FVII, K316H/L305V/V158T/M298Q-FVII, K316H/L305V/V158T/E296V-FVII, K316H/L305V/E296V/M298Q-FVII, K316H/L305V/V158D/E296V/M298Q-FVII, K316H/L305V/V158T/E296V/M298Q-FVII, K316H/L305V/V158T/K337A/M298Q-FVII, K316H/L305V/V158T/E296V/K337A-FVII,

K316H/L305V/V158D/K337A/M298Q-FVII, K316H/L305V/V158D/E296V/K337A -FVII, K316H/L305V/V158D/E296V/M298Q/K337A-FVII,

K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/L305V/K337A-FVII, K316Q/L305V/V158D-FVII, K316Q/L305V/E296V-FVII, K316Q/L305V/M298Q-FVII, K316Q/L305V/V158T-FVII, K316Q/L305V/K337A/V158T-FVII,

K316Q/L305V/K337A/M298Q-FVII, K316Q/L305V/K337A/E296V-FVII, K316Q/L305V/K337A/V158D-FVII, K316Q/L305V/V158D/M298Q-FVII, K316Q/L305V/V158D/E296V-FVII, K316Q/L305V/V158T/M298Q-FVII, K316Q/L305V/V158T/E296V-FVII, K316Q/L305V/E296V/M298Q-FVII, K316Q/L305V/V158D/E296V/M298Q-FVII, K316Q/L305V/V158T/E296V/M298Q-FVII, K316Q/L305V/V158T/K337A/M298Q-FVII, K316Q/L305V/V158T/E296V/K337A-FVII, K316Q/L305V/V158D/K337A/M298Q-FVII, K316Q/L305V/V158D/E296V/K337A -FVII, K316Q/L305V/V158D/E296V/M298Q/K337A-FVII, K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII, F374Y/V158D-FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII, F374Y/V158T-FVII, F374Y/S314E-FVII, F374Y/L305V-FVII, F374Y/L305V/K337A-FVII, F374Y/L305V/V158D-FVII, F374Y/L305V/E296V-FVII, F374Y/L305V/M298Q-FVII, F374Y/L305V/V158T-FVII, F374Y/L305V/S314E-FVII, F374Y/K337A/S314E-FVII, F374Y/K337A/V158T-FVII, F374Y/K337A/M298Q-FVII, F374Y/K337A/E296V-FVII, F374Y/K337A/V158D-FVII, F374Y/V158D/S314E-FVII, F374Y/V158D/M298Q-FVII, F374Y/V158D/E296V-FVII, F374Y/V158T/S314E-FVII, F374Y/V158T/M298Q-FVII, F374Y/V158T/E296V-FVII, F374Y/E296V/S314E-FVII, F374Y/S314E/M298Q-FVII, F374Y/E296V/M298Q-FVII, F374Y/L305V/K337A/V158D-FVII, F374Y/L305V/K337A/E296V-FVII, F374Y/L305V/K337A/M298Q-FVII, F374Y/L305V/K337A/V158T-FVII, F374Y/L305V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V-FVII, F374Y/L305V/V158D/M298Q-FVII, F374Y/L305V/V158D/S314E-FVII, F374Y/L305V/E296V/M298Q-FVII, F374Y/L305V/E296V/V158T-FVII, F374Y/L305V/E296V/S314E-FVII, F374Y/L305V/M298Q/V158T-FVII, F374Y/L305V/M298Q/S314E-FVII, F374Y/L305V/V158T/S314E-FVII, F374Y/K337A/S314E/V158T-FVII, F374Y/K337A/S314E/M298Q-FVII, F374Y/K337A/S314E/E296V-FVII, F374Y/K337A/S314E/V158D-FVII, F374Y/K337A/V158T/M298Q-FVII, F374Y/K337A/V158T/E296V-FVII, F374Y/K337A/M298Q/E296V-FVII, F374Y/K337A/M298Q/V158D-FVII, F374Y/K337A/E296V/V158D-FVII, F374Y/V158D/S314E/M298Q-FVII, F374Y/V158D/S314E/E296V-FVII, F374Y/V158D/M298Q/E296V-FVII, F374Y/V158T/S314E/E296V-FVII, F374Y/V158T/S314E/M298Q-FVII,

F374Y/V158T/M298Q/E296V-FVII, F374Y/E296V/S314E/M298Q-FVII, F374Y/L305V/M298Q/K337A/S314E-FVII, F374Y/L305V/E296V/K337A/S314E-FVII, F374Y/E296V/M298Q/K337A/S314E-FVII, F374Y/L305V/E296V/M298Q/K337A -FVII, F374Y/L305V/E296V/M298Q/S314E-FVII, F374Y/V158D/E296V/M298Q/K337A-FVII, F374Y/V158D/E296V/M298Q/S314E-FVII, F374Y/L305V/V158D/K337A/S314E-FVII, F374Y/V158D/M298Q/K337A/S314E-FVII, F374Y/V158D/E296V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q-FVII, F374Y/L305V/V158D/M298Q/K337A-FVII, F374Y/L305V/V158D/E296V/K337A-FVII, F374Y/L305V/V158D/M298Q/S314E-FVII, F374Y/L305V/V158D/E296V/S314E-FVII, F374Y/V158T/E296V/M298Q/K337A-FVII, F374Y/V158T/E296V/M298Q/S314E-FVII, F374Y/L305V/V158T/K337A/S314E-FVII, F374Y/V158T/M298Q/K337A/S314E-FVII, F374Y/V158T/E296V/K337A/S314E-FVII, F374Y/L305V/V158T/E296V/M298Q-FVII, F374Y/L305V/V158T/M298Q/K337A-FVII, F374Y/L305V/V158T/E296V/K337A-FVII, F374Y/L305V/V158T/M298Q/S314E-FVII, F374Y/L305V/V158T/E296V/S314E-FVII, F374Y/E296V/M298Q/K337A/V158T/S314E- FVII, F374Y/V158D/E296V/M298Q/K337A/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q/S314E-FVII, F374Y/L305V/E296V/M298Q/V158T/S314E-FVII, F374Y/L305V/E296V/M298Q/K337A/V158T-FVII, F374Y/L305V/E296V/K337A/V158T/S314E-FVII, F374Y/L305V/M298Q/K337A/V158T/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q/K337A-FVII, F374Y/L305V/V158D/E296V/K337A/S314E-FVII, F374Y/L305V/V158D/M298Q/K337A/S314E-FVII, F374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII, S52A-Factor VII, S60A-Factor VII; R152E-Factor VII, S344A-Factor VII, T106N-FVII, K143N/N145T-FVII, V253N-FVII, R290N/A292T-FVII, G291N-FVII, R315N/V317T-FVII, K143N/N145T/R315N/V317T-FVII; PlOQ-FVII, K32E-FVII, P10Q/K32E-FVII, FVII having substitutions, additions or deletions in the amino acid sequence of the Gla-domain; FVII having substitutions, additions or deletions in the amino acid sequence from 233Thr to 240Asn; FVII having substitutions, additions or deletions in the amino acid sequence from 304Arg to 329Cys; and FVII having substitutions, additions or deletions in the amino acid sequence from 1531Ie to 223Arg.

In another aspect the present invention provides a FVIIa polypeptide product manufactured by a process comprising the steps:

a) purifying the single-chain FVII polypeptide corresponding to said FVIIa polypeptide using the method according to the present invention, and b) activating the purified single-chain FVII polypeptide to the corresponding FVIIa polypeptide, and c) isolating the FVIIa polypeptide to give the resulting FVIIa polypeptide product, which has retained at least 30% of its specific biological activity.

Industrial-scale production and purification

The present invention is particular useful for industrial-scale production and purification of polypeptides. In such processes, a polypeptide is typically produced by means of a cell culture.

Thus, the present invention also provides an industrial-scale process for the production and purification of a desired polypeptide, said process including the steps of: (i) producing a crude bulk of a desired polypeptide in a cell culture; and

(ii) purifying said crude bulk by a purification sequence utilizing one or more reverse phase chromatography (RPC) processes; wherein at least one of such reverse phase chromatography processes is conducted as defined hereinabove.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein.

The use of the terms "a" and "an" and "the" and similar referents in the context of de- scribing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. For example, the phrase "the compound" is to be understood as referring to various "compounds" of the invention or particular described aspect, unless otherwise indicated.

Unless otherwise indicated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a

particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

The description herein of any aspect or aspect of the invention using terms such as "comprising", "having," "including," or "containing" with reference to an element or elements is intended to provide support for a similar aspect or aspect of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

EXPERI M ENTALS

General Methods

Preparation of chromatographic solvents

The desired amount of buffer (w/w) % and salt (w/w) % is weighted out. Water is added to dissolve the buffer and salt. The desired amount of alcohol (w/w) % (100 % ethanol, X % 1-propanol, Y % 2-propanol) is added. Water is added to 95-98 % of total amount of buffer. pH is adjusted in the solvent at room temperature when solvents are used at room temperature and 3O ⁰ C. Buffers used at 40 ⁰ C and 5O ⁰ C are pH-adjusted at 40 ⁰ C and 5O ⁰ C respectively. Prior to use the pH-meter is calibrated with IUPAC standard solutions at room temperature (20-25 ⁰ C). Water is added to 100 %.

Assays suitable for determining biological activity of Factor VII polypeptides Factor VII polypeptides useful in accordance with the present invention may be selected by suitable assays that can be performed as simple preliminary in vitro tests. Thus, the present specification discloses a simple test (entitled "In Vitro Hydrolysis Assay") for the activity of Factor VII polypeptides.

In Vitro Hydrolysis Assay (Assay 1) Native (wild-type) Factor Vila and Factor VII polypeptide (both hereinafter referred to as "Factor Vila") may be assayed for specific activities. They may also be assayed in parallel

to directly compare their specific activities. The assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S- 2288, Chromogenix, Sweden), final concentration 1 mM, is added to Factor Vila (final concentration 100 nM) in 50 mM HEPES, pH 7.4, containing 0.1 M NaCI, 5 mM CaCI2 and 1 mg/mL bovine serum albumin. The absorbance at 405 nm is measured continuously in a SpectraMax™ 340 plate reader (Molecular Devices, USA). The absorbance developed during a 20-minute incubation, after subtraction of the absorbance in a blank well containing no enzyme, is used for calculating the ratio between the activities of Factor VII polypeptide and wild-type Factor Vila : Ratio = (A405 nm Factor VII polypeptide)/ (A405 nm Factor Vila wild-type).

Based thereon, Factor VII polypeptides with an activity lower than, comparable to, or higher than native Factor Vila may be identified, such as, for example, Factor VII polypeptides where the ratio between the activity of the Factor VII polypeptide and the activity of native Factor VII (wild-type FVII) is about 1.0 versus above 1.0.

The activity of the Factor VII polypeptides may also be measured using a physiological substrate such as Factor X ("In Vitro Proteolysis Assay"), suitably at a concentration of 100-1000 nM, where the Factor Xa generated is measured after the addition of a suitable chromogenic substrate (eg. S-2765). In addition, the activity assay may be run at physiological temperature.

In Vitro Proteolysis Assay (Assay 2)

Native (wild-type) Factor Vila and Factor VII polypeptide (both hereinafter referred to as

"Factor Vila") are assayed in parallel to directly compare their specific activities. The as- say is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). Factor Vila (10 nM) and Factor X (0.8 microM) in 100 μl_ 50 mM HEPES, pH 7.4, containing 0.1 M NaCI, 5 mM CaCI ₂ and 1 mg/mL bovine serum albumin, are incubated for 15 min. Factor X cleavage is then stopped by the addition of 50 μl_ 50 mM HEPES, pH 7.4, containing 0.1 M NaCI, 20 mM EDTA and 1 mg/mL bovine serum albumin. The amount of Factor Xa generated is measured by the addition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final concentration 0.5 mM. The absorbance at 405 nm is measured continuously in a SpectraMax™ 340 plate reader (Molecular Devices, USA). The absorbance developed during 10 minutes, after subtraction of the absorbance in a blank well containing no FVIIa, is used for calculating the ratio between the proteolytic activities of Factor VII polypeptide and wild-type Factor Vila :

Ratio = (A405 nm Factor VII polypeptide)/(A405 nm Factor Vila wild-type).

Based thereon, Factor VII polypeptide with an activity lower than, comparable to, or higher than native Factor Vila may be identified, such as, for example, Factor VII polypeptides where the ratio between the activity of the Factor VII polypeptide and the activ- ity of native Factor VII (wild-type FVII) is about 1.0 versus above 1.0.

Thrombin generation Assay (Assay 3)

The ability of Factor Vila or Factor VII polypeptides to generate thrombin can also be measured in an assay (Assay 3) comprising all relevant coagulation Factors and inhibitors at physiological concentrations (minus Factor VIII when mimicking hemophilia A conditions) and activated platelets (as described on p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547, which is hereby incorporated herein as reference).

One-stage Coagulation Assay (Clot Assay ^" ) (Assay 4) Factor VII polypeptides may also be assayed for specific activities ("clot activity") by using a a one-stage coagulation assay (Assay 4). For this purpose, the sample to be tested is diluted in 50 mM PIPES-buffer (pH 7.5), 0.1% BSA and 40 μl is incubated with 40 μl of Factor VII deficient plasma and 80 μl of human recombinant tissue factor containing 10 mM Ca2+ and synthetic phospholipids. Coagulation times (clotting times) are measured and compared to a standard curve using a reference standard in a parallel line assay.

Assays suitable for determining concentration of FVII-polypeptides and contents of degradation products of FVII-polypeptides (Assay 5)

Concentration of FVII-polypeptides and content of heavy chain degradation products is determined by RP-HPLC as described in the following :

Reverse phase HPLC was run on a proprietary 4.5x250 mm butyl-bonded silica column with a particle size of 5 μm and pore size 3OθA. Column temperature: 70 ⁰ C. A-buffer: 0.1% v/v trifluoracetic acid. B-buffer: 0.09% v/v trifluoracetic acid, 80% v/v acetonitrile. The column was eluted with a linear gradient from X to (X+13)% B in 30 minutes. X was adjusted so that FVIIa elutes with a retention time of approximately 26 minutes. Flow rate: 1.0 mL/min. Detection : 214 nm. Load : 25 μg FVIIa.

Content of aggregates is determined by non-denaturing size exclusion HPLC. The content of oxidized forms is determined by RP-HPLC. The content of enzymatic degradation forms is determined by RP-HPLC (assay 5).

Non-denaturing size exclusion chromatography was run on a Waters Protein Pak 300 SW column, 7,5x300 mm using 0.2 M ammoniumsulfat, 5% 2-propanol pH 7,0 as mobile phase. Flow rate :0.5 ml/min. Detection : 215 nm. Load : 25μg FVIIa.

Assay 6:

Content of desGla-Factor VII polypeptide structures relative to the full length Factor VII polypeptide structures is determined by SDS-PAGE. 150 μl of sample is added 50 μl of sample buffer (non reducing, NuPAGE) and boiled for 5 mins. A 10 μl sample is loaded onto a 12% BisTris NuPAGE Gel (Invitrogen). The gel is run at 200 Volts, 120 mA for 55 mins. The gel is stained using coomassie brilliant blue solution, destained and dried. The relative desGla-Factor VII polypeptide content is calculated as the area of the desGla- Factor VII polypeptide band divided by the areas of the Factor VII polypeptide band at approx. 50 kDa and desGla-Factor VII polypeptide band at approx. 45 kDa.

EXAM PLES

Example 1 : Purification of Factor Vila using a silica-based C4 column at room tempera- ture r020 ¹ )

A solution of FVIIa containing 4.6 mg FVIIa was loaded onto a C4-YMC column (300 A, 15 micron, CV: 1.6 mL) pre-equilibrated with buffer A (10 mM Tris, 25 mM CaCI ₂ , pH 8.6). The column was washed with 3 CV of the equilibration buffer. Elution was per- formed with linear gradient from 0- 100% with a solvent comprising 70% 2-propanol over 5 CV, then with an aqueous solution containing 80% acetonitrile with 0.09% TFA over 5 CV at 0.33 mL / min. FVIIa began to elute after about 4 CV from the start of gradient elution and the elution was completed after about 1.5 CV from the start of elution with solvent comprising acetonitrile. 4 ml of a 1 M argiπiπe solution was added to the collecting tubes prior to collection. The fractions containing FVIIa were pooled and will be analysed by RP-HPLC and clot assay.

Example 2: Purification of Factor Vila using a silica-based C4 column at room tempera- ture 022 ¹ )

A solution of FVIIa containing 4.6 mg FVIIa was loaded onto a C4-YMC column (300 A, 15 micron, CV: 1,6 ml) pre-equilibrated with buffer A (10 mM Tris, 25 mM CaCI ₂ , pH 8.6). The column was washed with 3 CV of the equilibration buffer followed by washing with a linear gradient of 0 to 100% with an aqueous solvent containing 80% acetonitrile with 0.09% TFA over 5 CV. Elution was performed by step elution with 70% 2-propanol over 5 CV. Flow was 0.33 ml_ /min. 4 mL of a i M arginme solution was added to the collecting tubes prior to collection. The fractions containing FVII were pooled and analysed by RP-HPLC and clot assay.

Concentration of FVIIa in the pool (analysed by RP-HPLC, assay 5) : 0.38 mg/mL

Specific activity of FVIIa in the pool: 41842 IU/mg

Specific activity of FVIIa in the application (load) measured by assay 4: 60083 IU/mg

Example 3 : Purification of Factor Vila using a polystyrene/divinylbenzene column at room temperature (023)

A solution of FVIIa containing 4.6 mg FVIIa was loaded onto a PLRP-S column (Polymer Laboratories, 300 A, 15 micron, CV: 1.6 mL) pre-equilibrated with 10 mM Tπs and 25 mM CaCI?, pH 8,6. The column was washed with 3 CV of the equilibration buffer followed by washing with a linear gradient of 0 to 100% with an aqueous solvent comrising 80% acetonitrile with 0.09% TFA over 5 CV. Elution was performed by step elution with 70% 2-propanol over 5 CV. Flow rate was 0.33 ml / mm. 4 mL of a 1 M argimne solution was added to the collecting tubes prior to collection, Fractions of I mL were collected and the fractions containing FVIIa were pooled and analysed by RP-HPLC and dot assay.

Concentration of FVIIa in the pool (analysed by RP-HPLC, assay 5) : 0.38 mg/mL

Yield (measured by RP-HPLC, assay 5) : 66%

Specific activity in the pool (measured by Assay 4 as described above): 51290 IU/mg Specific activity of FVIIa in the application (load) measured by assay 4: 60083 IU/mg