RU2325181C2 - Glycoforms of vii factor - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns glycoforms of VII factor and compositions of VII factor, characterized by modified configurations on basis of asparaginic oligosaccharide chains. In addition invention includes detection method applied for polypeptide glycoforms of VII factor, receiving method and disease treatment method as well.

EFFECT: identification of biologically active forms of recombinant VII factor.

53 cl, 5ex

Description

Field of Invention

The present invention relates to compositions containing factor VII and other factors of the blood coagulation system having altered configurations of asparagine-linked glycosylation.

BACKGROUND OF THE INVENTION

Proteins involved in the coagulation system, including, for example, factor VII, factor VIII, factor IX, factor X and protein C, are shown to be therapeutic agents useful in treating a variety of pathological conditions. Accordingly, there is an increasing need for formulations containing these proteins that are pharmaceutically acceptable and exhibit uniform and predictable clinical efficacy.

Due to the many disadvantages associated with the use of human blood plasma as a source of pharmaceutical products, it is preferable to obtain these proteins in recombinant systems. Proteins of the blood coagulation system, however, undergo many co- and post-translational modifications, including, for example, asparagine-bound (N-linked) glycosylation; O-linked glycosylation; and γ-carboxylation of glu residues. These modifications can differ qualitatively or quantitatively when heterologous cells are used as hosts for the industrial production of proteins. In particular, production in heterologous cells often results in a number of different glycoforms, which are identical polypeptides containing various covalently linked oligosaccharide configurations.

In various systems, differences in the configuration of oligosaccharides of therapeutic proteins are, inter alia, associated with changes in immunogenicity and in vivo clearance. Thus, in this area there is a need for compositions and methods that provide preparations of proteins of the blood coagulation system, in particular, preparations containing recombinant human factor VII, modified factor VII, or polypeptides related to factor VII that contain predetermined glycoform configurations.

SUMMARY OF THE INVENTION

The present invention relates to preparations containing factor VII polypeptides or factor VII related polypeptides that exhibit predetermined glycoform configurations. As used herein, a factor VII preparation or a factor VII related drug refers to a variety of factor VII polypeptides or factor VII related polypeptides, including variants and chemically modified forms, as well as forms that are proteolytically activated (e.g., factor VIIa) that are secreted from the cell in which they are synthesized. The glycoform configuration refers to the distribution of oligosaccharide chains in a preparation having various configurations that are covalently linked to factor VII polypeptides or polypeptides related to factor VII.

In one aspect, the present invention provides a preparation comprising a plurality of factor VII polypeptides or factor VII related polypeptides, wherein the polypeptides contain asparagine-linked oligosaccharide chains and where one or more of the following conditions are true: (i) within about 94-100% of the oligosaccharide chains contain at least one sialic acid residue; (ii) within about 0-7% of the oligosaccharide chains have a neutral charge; (iii) less than about 16%, for example, in the range of about 6-16%, of the oligosaccharide chains contain at least one final galactose residue; (iv) less than about 25%, for example, in the range of about 6-9%, of the oligosaccharide chains, contain at least one final N-acetylgalactosamine residue; or (v) less than about 30%, for example, in the range of about 11-23%, the oligosaccharide chains contain at least one final residue of galactose or N-acetylgalactosamine. In some embodiments, in addition to one or more of the conditions (i) to (v): all sialic acid residues in the oligosaccharide chains are linked to galactose via an α2-> 3 bond; at least some of the sialic acid residues contain N-glycolylneuraminic acid (Neu5Gc) in addition to N-acetylneuraminic acid (Neu5Ac); and / or oligosaccharide chains contain fucose residues bound α1-> 6 to the N-acetylglucosamine framework. In one embodiment, the present invention encompasses a preparation comprising wild-type factor VIIa in which at least one sialic acid residue is present in the range of about 94-100% oligosaccharide chains and all sialic acid residues are linked to galactose via an α2-> bond 3. In another embodiment, the present invention encompasses a preparation comprising wild-type factor VIIa in which at least one sialic acid residue is present in the range of about 94-100% oligosaccharide chains, and at least some of the sialic acid residues are N- glycolylneuraminic acid. In yet another embodiment, the present invention encompasses a preparation comprising wild-type factor VIIa, in which at least 94-100% of the oligosaccharide chains contain at least one sialic acid residue and at least some of the chains contain N-acetylgalactosamine. The preparations of the present invention, therefore, do not cover wild-type factor VII or factor VIIa, which is released from human plasma and is not modified ex vivo by treatment with glycosidase.

In another aspect, the present invention provides a preparation comprising a plurality of factor VII polypeptides or factor VII-related polypeptides, wherein the polypeptides contain asparagine-linked oligosaccharide chains and where at least about 2% of the oligosaccharide chains contain at least one fucose bound α1- > 3 with the antennary residue of N-acetylglucosamine (i.e., with the residue of N-acetylglucosamine, which is linked β1-> 2, 4 or 6 to the residue Man). Preferably, at least about 5% of the oligosaccharide chains contain at least one such antennae fucose residue; more preferably at least about 10% or 20%; and most preferably at least about 40%.

The preparations in accordance with the present invention may contain one or more of the following compounds: wild-type factor VII; wild-type factor VII that has undergone chemical and / or enzymatic modification; and variants of factor VII having one or more changes in the amino acid sequence with respect to wild-type factor VII. The preparations of the present invention can be obtained from human cells expressing factor VII from the endogenous factor VII gene, or from cells programmed to express factor VII or a factor VII related polypeptide from a recombinant gene.

In another aspect, the present invention provides preparations comprising factor VII or factor VII related polypeptides that exhibit one or more improved functional properties, including, without limitation, increased storage stability, bioavailability and / or half life.

In another aspect, the present invention encompasses methods for determining and / or optimizing the configuration of factor VII glycoforms and factor VII related polypeptides, which are carried out using the steps:

(a) culturing a cell expressing factor VII or factor VII related polypeptides in a first set of predetermined culture conditions;

(b) recovering factor VII or factor VII related polypeptides from the culture to produce a preparation containing the polypeptides; and

(c) analyzing the configuration of oligosaccharides associated with the polypeptides to determine the glycoform configuration of the drug.

The methods may further include changing the culturing conditions of step (a) to obtain a second set of predetermined culturing conditions; and repeating the steps until the desired glycoform configuration is obtained. Alternatively, the methods may further include chemically or enzymatically treating the preparation to alter the configuration of the oligosaccharides; and repeating the steps until the desired glycoform configuration is obtained. In addition, the methods may include additional steps in which preparations having predetermined glycoform configurations are subjected to at least one study of biological activity or other functionality (e.g., pharmacokinetic profile or stability) and correlations between specific glycoform structures and specific profiles of biological activity or functionality.

In another aspect, the present invention provides methods for preparing a preparation comprising factor VII polypeptides or factor VII related polypeptides having a predetermined N-linked glycosylation configuration. In some embodiments, the methods are carried out by culturing a cell expressing the polypeptides, under conditions in which at least about 94% of the asparagine-linked oligosaccharides associated with factor VII polypeptides or with factor VII-related polypeptides contain at least one sialic residue acids, for example, one, two, three or four sialic acid residues. In some embodiments, the methods are carried out by culturing a cell expressing the polypeptides under conditions in which at least about 5% of the oligosaccharide chains contain at least one fucose bound α1-> 3 to the antennae residue of N-acetylglucosamine. In some embodiments, factor VII polypeptides or factor VII-related polypeptides are enzymatically processed depending on their origin to obtain the desired glycoform configurations.

In another aspect, the present invention provides pharmaceutical preparations comprising the preparations of the present invention and methods for preventing and / or treating syndromes that are sensitive to factor VII polypeptides or to factor VII related polypeptides. The methods include administering pharmaceuticals to a patient in need of treatment under conditions leading to either increased or inhibited blood coagulation. In one series of embodiments, factor VII preparations are administered when enhanced coagulation is desired, for example, with hemophilia A, hemophilia B, factor XI deficiency, factor VII deficiency, thrombocytopenia, or von Willebrand disease; in syndromes accompanied by the presence of a coagulation factor inhibitor; before, during, or after surgery or therapeutic treatment with anticoagulants; or after an injury. In another series of embodiments, preparations of polypeptides related to factor VII (i.e., preparations having a reduced or modified biological activity with respect to wild-type factor VII) are administered to reduce blood coagulation, for example, in patients undergoing angioplasty or in those suffering deep vein thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIG), deposition of fibrin in the lungs and kidneys associated with gram-negative endotoxemia, or myocardial infarction. In accordance with the present invention, preparations of factor VII-related polypeptides can also be administered when it is desired to modify, for example, reduce intracellular signaling via a pathway mediated by tissue factor (TF), for the treatment of conditions such as acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), hemolytic uremia syndrome (HUS), multiple organ failure (MOF), and thrombocytopenic purpura (TTP).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that coagulant protein preparations having predetermined glycoform configurations exhibit improved functional properties. Accordingly, the present invention relates to methods and compositions that provide for the preparation of these protein preparations. In particular, the present invention relates to preparations containing factor VII polypeptides and factor VII related polypeptides having specific predetermined configurations of asparagine-linked (N-linked) oligosaccharides. The formulations of the present invention exhibit altered properties, including, without limitation, improved pharmacokinetic properties and improved clinical efficacy. The present invention also encompasses pharmaceutical compositions that contain these drugs, as well as therapeutic methods that use these drugs.

Factor VII Polypeptides and Factor VII Related Polypeptides

The present invention encompasses human factor VII polypeptides, for example, such as those having the amino acid sequence described in US Pat. No. 4,784,950 (wild-type factor VII). As used herein, “factor VII” or “factor VII polypeptide” encompasses wild-type factor VII as well as factor VII variants exhibiting substantially the same or improved biological activity compared to wild-type factor VII. The term "factor VII" is intended to encompass factor VII polypeptides in their unsplit (zymogenic) form, as well as those which are processed proteolytically, to obtain their corresponding biologically active forms, which can be designated as factor VIIa. Typically, factor VII is cleaved between residues 152 and 153 to produce factor VIIa.

As used here, the term "factor VII related polypeptides" encompasses polypeptides, including variants in which the biological activity of factor VIIa significantly changes or decreases when compared with the activity of wild-type factor VIIa. These polypeptides include, without limitation, factor VII or factor VIIa, which are chemically modified, and variants of factor VII, which are introduced specific changes in the amino acid sequence that modify or destroy the biological activity of the polypeptide.

The biological activity of factor VIIa in blood coagulation comes from its ability (i) to 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 the purposes of the present invention, the biological activity of factor VIIa can be quantified by measuring the ability of the drug to promote blood coagulation using plasma deficient in factor VII and thromboplastin, as described, for example, in US Pat. No. 5,997,864. In this assay, biological activity is expressed as a decrease coagulation time compared to a control sample and converted to “factor VII units” by comparison with a stored standard human serum containing 1 unit / ml factor VII activity. Alternatively, the biological activity of factor VIIa can be quantified by (i) measuring the ability of factor VIIa to produce factor Xa in a system containing TF incorporated into the lipid membrane and factor X. (Persson et al., J. Biol. Chem. 272: 19919-19924, 1997); (ii) measuring the hydrolysis of factor X in an aqueous system (see example 5, below); (iii) measuring its physical binding to TF using surface plasmon resonance (Persson, FEBS Letts. 413: 359-363, 1997); (iv) measuring the hydrolysis of the synthetic substrate (see example 4, below); and (v) measuring thrombin generation in a TF-independent in vitro system.

Variants of factor VII having substantially the same or improved biological activity compared to wild-type factor VIIa encompass those compounds that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75% and, most preferably, at least about 90% of the specific activity of wild-type factor VIIa, which is produced in the same type of cells when they are examined in one or more of the assays using coagulation assays analysis, proteolysis, or TF binding assays as described above. Variants of factor VII having significantly reduced biological activity compared to wild-type factor VIIa are those that exhibit less than about 25%, preferably less than about 10%, more preferably less than about 5%, and most preferably less than about 1% of the specific activity of wild-type factor VIIa that is produced in cells of the same type when examined in one or more assays from a coagulation assay, proteolysis assay, or TF binding assay described above. Variants of factor VII having significantly modified biological activity compared to wild-type factor VII include, without limitation, variants of factor VII that exhibit TF-independent proteolytic activity of factor X, and those that bind TF but do not cleave factor X .

Variants of factor VII, either exhibiting substantially the same or better biological activity than wild-type factor VII, or alternatively exhibiting significantly modified or reduced biological activity compared to wild-type factor VII, include, without limitation, polypeptides having an amino acid sequence that differs from the wild-type factor VII sequence by the insertion, removal or substitution of one or more amino acids. Non-limiting examples of factor VII variants having substantially the same biological activity as wild-type factor VII include S52A-FVIIa, S60A-FVIIa (lino et al., Arch. Biochem. Biophys. 352: 182- 192, 1998); FVIIa variants exhibiting enhanced proteolytic stability are described in US Pat. No. 5,580,560; factor VIIa, which is proteolytically cleaved between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48: 501-505, 1995); and oxidized forms of factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363: 43-54, 1999). Non-limiting examples of factor VII variants having significantly reduced or modified biological activity compared to wild-type factor VII include R152E-FVIIa (Wildgoose et al., Biochem 29: 3413-3420, 1990), S344A-FVIIa (Kazama et al., J. Biol. Chem. 270: 66-72, 1995), FFR-FVIIa (Holst et al., Eur. J. Vasc. Endovasc. Surg. 15: 515-520, 1998), and factor VIIa, where the Gla domain is missing (Nicolaisen et al., FEBS Letts. 317: 245-249, 1993). Non-limiting examples of polypeptides and sequence variants of chemically modified factor VII are described, for example, in US patent No. 5997864.

Asparagine Bound Glycosylation

The present invention provides preparations of factor VII polypeptides or factor VII related polypeptides that contain a specific spectrum of factor VII glycoforms, i.e., factor VII polypeptides or factor VII related polypeptides having predetermined configurations of asparagine-linked (N-linked) oligosaccharide chains.

As used here, the "configuration" of N-linked glycosylation or the "configuration" of glycoforms, the "distribution" or "spectrum" refers to the representation of specific oligosaccharide configurations in a given population of factor VII polypeptides or factor VII related polypeptides. Non-limiting examples of such configurations include the relative proportion of oligosaccharide chains that (i) contain at least one sialic acid residue; (ii) have no sialic acid residues (i.e., are charge neutral); (iii) contain at least one final galactose residue; (iv) contain at least one final residue of N-acetylgalactosamine; (v) contain at least one “unblocked” antenna, that is, contain at least one final residue of galactose or N-acetylgalactosamine; or (vi) contain at least one fucose bound α1-> 3 to the antennae residue of N-acetylglucosamine.

As used here, the term oligosaccharide chain refers to the entire oligosaccharide configuration, which is covalently linked to one single asparagine residue. Factor VII is usually glycosylated on Asn 145 and Asn 322. The N-linked oligosaccharide chain present on factor VII, produced in humans in situ, can be bi-, three, or tetraantennar, with each antenna having the Neu5Ac configuration (α2-> 3 or α2-> 6) Gal (β1-> 4) GlcNAc, binds (β1-> 2, 4 or 6) to the remainder of Man, which is bound (α1-> 3 or 6) to Man (β1-> 4) GlcNAc (β1-> 4) GlcNAc-Asn. (Neu5Ac is N-acetylneuraminic acid (sialic acid), Gal is galactose, GlcNAc is N-acetylglucosamine, and Man is mannose). Oligosaccharide chains can also contain fucose residues, which can be linked α1-> 6 to GlcNAc. when factor VII is produced in humans in situ, some oligosaccharide chains lack fucose residues in the framework; all chains lack antennae fucose residues; and all chains are almost completely sialylated, that is, the final sugar of each antenna is N-acetylneuraminic acid linked to galactose via an α2-> 3 or α2-> 6 bond.

However, when it is produced in other circumstances, factor VII may contain oligosaccharide chains having different final configurations on one or more of their antennas, such as those lacking sialic acid residues; containing residues of N-glycolylneuraminic acid (Neu5Gc); containing a final N-acetylgalactosamine residue (GalNAc) instead of galactose; etc. When they are produced, for example, in BHK cells cultured in the presence of calf serum, factor VII preparations exhibit the following oligosaccharide configurations:

- 87-93% of oligosaccharide chains contain at least one sialic acid residue;

- 7-13% are neutral (sialic acid is absent);

- 9-16% contain at least one final galactose residue;

- 19-29% contain at least one final residue of N-acetylgalactosamine; and

- 30-39% contain at least one unblocked antenna, that is, contain at least one final residue of galactose or N-acetylgalactosamine.

The authors of the present invention have created factor VII preparations containing specific predetermined configurations of oligosaccharides that differ from those described previously. The present invention encompasses preparations containing factor VII polypeptides or factor VII related polypeptides that exhibit one or more of the following glycoform configurations:

(i) Within about 94-100% of the oligosaccharide chains contain at least one sialic acid residue, for example, in the range of about 94-99%, in the range of about 95-98%, or in the range of about 96-97%. In various embodiments, at least about 94%, 95%, 96%, or 97% of the oligosaccharide chains contain at least one sialic acid residue.

(ii) 6% or less of the oligosaccharide chains are neutral, for example, in the range of about 1.5-6%, or in the range of about 2-4%.

(iii) Less than about 16%, preferably less than about 10% of the oligosaccharide chains contain at least one terminal galactose, for example, in the range of about 6-10% or in the range of about 8-9%;

(iv) Less than about 25%, preferably less than about 10% of the oligosaccharide chains contain at least one final GalNAc residue, for example, in the range of about 6-9% or in the range of about 7-8%;

(v) Less than about 30%, preferably less than about 25% of the oligosaccharide chains contain at least one non-blocked antenna, for example, in the range of about 11-23% or in the range of about 12-18%; and

(vi) at least about 2%, preferably at least about 5%, more preferably at least about 10% or 20%, and most preferably at least about 40% of the oligosaccharide chains contain, at least one fucose bound α1-> 3 to the antennae residue of N-acetylglucosamine (i.e., to the residue of N-acetylglucosamine, which is linked β1-> 2, 4 or 6 to the residue Man).

It will be understood that each of conditions (i) to (vi) may represent a different glycoform configuration that is encompassed by the present invention, that is, a preparation according to the present invention can be described by only one of (i) - (vi). Alternatively, depending on the particular configuration of the glycoform, the preparation encompassed by the present invention may be described by more than one condition from (i) to (vi).

In addition, the preparation covered by the present invention can be described by one or more of the conditions from (i) to (vi) in combination with one or more other structural features. For example, the present invention encompasses preparations containing factor VII polypeptides or factor VII related polypeptides in which sialic acid residues (Neu5Ac or Neu5Gc) are associated with galactose exclusively in the configuration α2-> 3. The present invention also encompasses preparations containing factor VII polypeptides or factor VII-related polypeptides that contain fucose bound α1-> 6 with N-acetylglucosamine in the scaffold and / or fucose bound α1-> 3 with antennae N-acetylglucosamine. In one embodiment, the preparations of the present invention encompass factor VII polypeptides or factor VII-related polypeptides in which more than 99% of the oligosaccharide chains contain at least one sialic acid residue, and (a) sialic acid residues bind exclusively in the α2 configuration -> 3, and / or (b) there are fucose residues associated with N-acetylglucosamines in the framework, and / or (c) the detected number of antennas terminate with N-acetylgalactosamine. In one embodiment, the present invention encompasses preparations containing wild-type factor VIIa in which more than 99% of the oligosaccharide chains contain at least one sialic acid residue and the sialic acid residues are associated with galactose exclusively in the configuration α2-> 3. In another embodiment, the present invention encompasses preparations containing wild-type factor VIIa in which more than 99% of the oligosaccharide chains contain at least one sialic acid residue and at least some oligosaccharide chains contain N-acetylgalactosamine. The present invention does not cover wild-type factor VII or wild-type factor VIIa that is isolated from human plasma and is not ex vivo modified by treatment with glycosidases.

In one embodiment, the factor VIIa preparation contains oligosaccharide chains having one fucose linked to α1-> 3 with one antennae N-acetylglucosamine. In another embodiment, the factor VIIa preparation contains oligosaccharide chains having fucose residues associated with α1-> 3 with each antennae N-acetylglucosamine of the biannennary oligosaccharide (Lewis Xialyl structure). In another embodiment, the factor VIIa preparation contains oligosaccharide chains having (i) a fucose linked by N-acetylglucosamine in the backbone, and (ii) one fucose linked by α1-> 3 with one antennae N-acetylglucosamine. In another embodiment, the factor VIIa preparation contains oligosaccharide chains having (i) fucose bound to N-acetylglucosamine in the backbone, and (ii) fucose residues bound α1-> 3 to each antennae N-acetylglucosamine of the biannennary oligosaccharide.

In carrying out the present invention, the configuration of N-linked oligosaccharides can be determined using any method known in the art, including but not limited to: high performance liquid chromatography (HPLC); capillary electrophoresis (CE); nuclear magnetic resonance (NMR); mass spectrometry (MS) using ionization techniques such as fast atom bombardment, electrospray or laser desorption from an auxiliary matrix (MALDI); gas chromatography (GC); and treatment with exoglycosidases in combination with anion exchange (AO) -HPLC, size exclusion chromatography (BX) or MS. See, for example, Weber et al., Anal. Biochem. 225: 135 (1995); Klausen et al., J. Chromatog. 718: 195 (1995); Morris et al., In Mass spectrometry of biological Materials, McEwen et al., Eds., Marcel Dekker, (1990), pp. 137-167; Conboy et al., Biol. Mass Spectrom. 21: 397, 1992; Hellerqvist, Meth. Enzymol. 193: 554 (1990); Sutton et al., Anal. Biochem. 318: 34 (1994); Harvey et al. Organic Mass Spectrometry 29: 752 (1994).

After determining oligosaccharide chains obtained from factor VII using any of the above methods (or any other method that identifies oligosaccharide chains having different configurations), certain types are compared, for example, with one of groups (i) - (v). The relative content of each of (i) - (v) is calculated as the sum of the oligosaccharides compared with this group relative to the total content of oligosaccharide chains in the sample.

For example, using AO-HPLC, 13 or more peaks of N-linked oligosaccharides from a recombinant factor VII preparation produced by BHK cells can be detected. See, for example, Klausen et al., Mol. Biotechnol. 9: 195, 1998. Five of the peaks (designated 1-5 in Klausen et al.) Do not contain sialic acid, while eight of the peaks (designated 6, 7, and 10-15) contain sialic acid.

It will become clear that in this analysis, the number and distribution of chains containing sialic acid and not containing sialic acid may depend on (a) the polypeptide that is expressed; (b) cell type and culture conditions; and (c) the analysis method that is used, and that the resulting configurations may vary accordingly.

In any case, after the determination of oligosaccharides containing sialic acid and oligosaccharides not containing sialic acid, conventional data analysis programs are used to calculate the area under each peak; the total peak area and the percentage of the total peak area represented by a particular peak. Thus, for a given preparation, the sum of the peak areas containing sialic acid / total peak area × 100 gives the% sialylation value for the preparation of the present invention (i.e., the fraction of oligosaccharide chains having at least one sialic acid residue). Likewise,% chains containing no sialic acid or containing at least one galactose or N-acetylglucosamine can be calculated.

Methods for preparing factor VII preparations having a predetermined configuration of N-linked oligosaccharides

Factor VII preparations, factor VII variants or factor VII-related polypeptides each having a predetermined configuration of N-linked oligosaccharides can be obtained using an appropriate host cell that expresses factor VII or factor VII-related polypeptides.

Host Cells: In some embodiments, the host cells are human cells expressing the endogenous factor VII gene. In these cells, the endogenous gene may be intact or may be modified in situ, or some sequence outside the factor VII gene may be modified in situ to alter the expression of the endogenous factor VII gene. A human cell capable of expressing the endogenous factor VII gene can be used.

In other embodiments, heterologous host cells are programmed to express human factor VII from a recombinant gene. Host cells can be vertebrate, insect or fungal cells. Preferably, the cells are mammalian cells capable of the entire spectrum of N-linked glycosylation; O-linked glycosylation and γ-carboxylation of mammals. See, for example, US Patent No. 4,784,950. Preferred mammalian cell lines include CHO (ATCC CCL 61), COS-1 (ATCC CRL 1650), Hamster Baby Kidney (BHK) and HEK293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72, 1977). A preferred BHK cell line ^{is a} tH ^- ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79: 1106-1110, 1982), hereinafter referred to as BHK 570 cells. BHK 570 cell line is available from American type culture collection, 12301 Parklawn Dr., Rockville, MD 20852, accession number ATCC CRL 10314. The BHK tk ^- ts13 cell line is also available from ATCC under accession number CRL 1632. In addition, a number of other cell lines can be used, including Rat Hep I (rat hepatoma; ATCC CRL 1600), Rat Hep II (rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), human lungs (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1) and DU cells KX (CHO cell line) (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-4220, 1980), (DUKX cells are also referred to as CXB11 cells) and DG44 (CHO cell line) (Cell, 33 : 405, 1983, and Somatic Cell and Molecular Genetics 12: 555, 1986). Also suitable are 3T3 cells, Namalwa cells, myelomas, and fusion products of myelomas with other cells. In a particularly preferred embodiment, the host cells are BHK 21 cells that are adapted for growth in the absence of serum and are programmed for expression of factor VII. In some embodiments, the cells may be mutant or recombinant cells that express a qualitatively or quantitatively different spectrum of glycosylation enzymes (such as, for example, glycosyltransferases and / or glycosidases) than the type of cells from which they are derived. Cells can also be programmed to express other heterologous peptides or proteins, including, for example, processed forms of factor VII.

In one embodiment, the host cells are CHO cells that are programmed to coexpress both the factor VII polypeptide of interest (i.e., factor VII or factor VII related polypeptide) and another heterologous peptide or polypeptide, such as, for example, a modifying enzyme or a fragment of factor VII.

Methods: The present invention encompasses methods for preparing a preparation containing any of the glycoform configurations described above as (i) to (vi), and in a further embodiment, methods for optimizing the distribution of factor VII glycoforms and factor VII related polypeptides. These methods are carried out using the stages:

(a) culturing a cell expressing factor VII or factor VII related polypeptides in a first set of predetermined culture conditions;

(b) recovering factor VII or factor VII related polypeptides from the culture to obtain a preparation containing the polypeptides; and

(c) analyzing the configuration of oligosaccharides associated with the polypeptides to determine the configuration of glycoforms.

The methods may further include:

(d1) changing the cultivation conditions in step (a) to obtain a second set of predetermined culturing conditions;

(e1) repeating steps (b) to (d1) until the desired glycoform configuration is obtained.

Alternatively, the methods may further include:

(d2) chemical and / or enzymatic treatment of the drug to change the configuration of oligosaccharides; and

(e2) repeating steps (b) to (d2) until the desired glycoform configuration is obtained.

These methods may further include a step in which preparations having predetermined glycoform configurations are subjected to at least one biological activity test (including, for example, coagulation, factor X proteolysis or TF binding) or other functionality (such as as a pharmacokinetic profile or stability) and correlations of specific glycoform configurations with specific profiles of biological activity or functionality in accordance with the identification of the desired conf guration glycoforms.

The parameters of cultivation conditions that can be changed in step (d1) include, but are not limited to: a cell of origin, such as, for example, a cell obtained from species other than those originally used; or a mutant or recombinant cell having changes in one or more glycosyltransferases or glycosidases, or in other components of the glycosylation apparatus (see Grabenhorst et al., Glycoconjugate J. 16:81, 1999; Bragonzi et al., Biochem. Biophys. Acta 1474 : 273, 2000; Weikert, Nature Biotechnol. 17: 1116, 1999); the level of expression of the polypeptide; metabolic conditions, such as, for example, glucose or glutamine concentration; absence or presence of serum; vitamin K concentration; hydrolysates of proteins, hormones, traces of metals, salts, as well as process parameters like temperature, level of dissolved oxygen and pH value.

Enzymatic treatments that can be used in step (d2) to modify the configuration of drug oligosaccharides include, but are not limited to, treating one or more enzymes from sialidase (neuraminidase), galactosidase, fucosidase; galactosyltransferases, fucosyltransferases and / or sialyltransferases sequentially and under conditions that give the desired modification in the distribution of oligosaccharide chains having specific final structures. Glycosyl transferases are commercially available from Calbiochem (La Jolla, CA), and glycosidases are commercially available from Glyko, Inc., (Novato, CA).

In one set of embodiments, host cells expressing factor VII or a related polypeptide are exposed to specific culture conditions under which they secrete glycosylated factor VII polypeptides having the desired configuration of the oligosaccharide configurations described above as any of (i) to (vi). Such culture conditions include, without limitation, recovery in serum or in its complete absence. Preferably, the host cells are adapted for growth in the absence of serum and are cultured in the absence of serum both in the growth phase and in the production phase. Such adaptation procedures are described, for example, in Scharfenberg, et al., Animal Cell Technology Developments towards the 21 ^st Century, EC Beuvery et al. (Eds.), Kluwer Academic Publishers, pp. 619-623, 1995 (BHK and CHO cells); Cruz, Biotechnol. Tech. 11: 117-120, 1997 (insect cells); Keen, Cytotechnol. 17: 203-211, 1995 (myeloma cells); Berg et al., Biotechniques 14: 972-978, 1993 (293 human kidney cells). In a preferred embodiment, the growth medium that is added to the cells does not contain a protein or other component that is released from animal tissue or animal cell culture. See, for example, example 1, below. Typically, in addition to the usual components, a medium suitable for the production of factor VII contains vitamin K at a concentration in the range of 0.1-50 mg / liter, which is required for the γ-carboxylation of glutamine residues in factor VII.

In another series of embodiments, the glycoforms of the present invention are produced by exposing the preparation of factor VII or factor VII-related polypeptides to enzymatically and / or chemically modify the N-linked oligosaccharides contained therein.

Factor VII preparations

As used herein, the term “factor VII preparation” refers to a variety of factor VII polypeptides, factor VIIa polypeptides or factor VII related polypeptides, including variants and chemically modified forms that are isolated from the cell in which they are synthesized.

Isolation of polypeptides from cells of their origin can be achieved using any method known in the art, including, without limitation, removing the cell culture medium containing the desired product from the cell monolayer culture; centrifugation or filtration to remove non-adherent cells; etc.

Optionally, factor VII polypeptides may be further purified. Purification can be performed using any method known in the art, including, but not limited to, affinity chromatography, such as, for example, an anti-factor VII antibody column (see, for example, Wakabayashi et al., J. Biol. Chem. 261: 11097, 1986; and Thim et al., Biochem. 27: 7785, 1988); hydrophobic interaction chromatography; ion exchange chromatography; size exclusion chromatography; electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), differences in solubility (e.g., precipitation with ammonium sulfate) or extraction, and the like. See, in general, Scopes, Protein Purification, Springer-Verlag, New York, 1982; and Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989. After purification, the preparation preferably contains less than about 10 wt.%, More preferably less than about 5% and, most preferably, less than about 1% proteins, non-factor VII derived from the host cell.

Factor VII polypeptides and factor VII-related polypeptides can be activated by proteolytic cleavage using factor XIIa or other proteases having trypsin-specificity, such as factor IXa, kallikrein, factor Xa and thrombin. See, for example, Osterud et al., Biochem. 11: 2853 (1972); Thomas, US patent No. 4456591; and Hedner et al., J. Clin. Invest. 71: 1836 (1983). Alternatively, factor VII can be activated by passing it through an ion exchange chromatographic column, such as Mono Q® (Pharmacia), or the like. The resulting activated factor VII can then be prepared and administered as described below.

Functional Properties of Factor VII Preparations

Preparations of factor VII polypeptides and factor VII-related polypeptides having predetermined oligosaccharide configurations in accordance with the present invention exhibit improved functional properties compared to reference preparations. Improved functional properties may include, without limitation, a) physical properties, such as, for example, storage stability; b) pharmacokinetic properties, such as, for example, bioavailability and half-life; and c) immunogenicity for humans.

The term reference preparation refers to a preparation containing a polypeptide that is identical to that contained in the preparation of the present invention with which it is compared (such as, for example, wild-type factor VII or a specific variant, or a chemically modified form), except for demonstration excellent configuration of asparagine-linked glycosylation. For example, reference preparations typically have one or more of the following glycoform configurations: (i) less than about 93% (e.g., less than about 92% or less than about 90%) of the oligosaccharide chains contain at least one residue sialic acid; (ii) at least about 6% (for example, at least about 7.5% or at least about 10%) of the oligosaccharide chains do not contain any sialic acid (i.e., are neutral); (iii) at least about 10% (for example at least about 12.5% or at least about 15%) of the oligosaccharide chains contain at least one final galactose residue; (iv) at least about 15% (for example at least about 20% or at least about 25%) of the oligosaccharide chains contain at least one final N-acetylgalactosamine residue; (v) at least about 25% (for example at least about 30% or at least about 35%) of the oligosaccharide chains contain at least one non-blocked antenna (i.e., contain at least one final residue of galactose or N-acetylgalactosamine); or (vi) substantially undetectable levels (e.g., less than about 0.2%) of antennar fucose residues.

The storage stability of the factor VII preparation can be assessed by measuring (a) the time required for a 20% reduction in the biological activity of the drug when it is stored as a dry powder at 25 ° C, and / or (b) the time required to double the proportion aggregates of factor VIIa in the preparation.

In some embodiments, the preparations of the present invention show an increase of at least about 30%, preferably at least about 60%, and more preferably at least about 100% of the time required for a 20% reduction in biological activity, compared with the time required for the same phenomenon in the reference preparation, when both preparations are stored in the form of dry powders at 25 ° C. Biological activity measurements can be performed using any assay from: coagulation assay, proteolysis assay, TF-binding assay, or TF-independent thrombin generation assay.

In some embodiments, the preparations of the present invention exhibit an increase of at least about 30%, preferably at least about 60%, and more preferably at least about 100% of the time required to double the number of aggregates, compared to the reference preparation, when both preparations are stored as dry powders, at 25 ° C. The aggregate content is determined by gel permeation HPLC on a Protein Pak 300 SW column (7.5 × 300 mm) (Waters, 80013) as follows. The column is equilibrated with eluent A (0.2 M ammonium sulfate, 5% isopropanol, the pH is adjusted to 2.5 with phosphoric acid, and then the pH is adjusted to 7.0 with triethylamine), after which 25 μg is introduced into the column sample. Elution is performed using eluent A at a flow rate of 0.5 ml / min for 30 minutes and detection is carried out by measuring the absorption coefficient at 215 nm. The aggregate content is calculated as the peak area of factor VII aggregates / total peak area of factor VII (monomer and aggregates).

The term "bioavailability" refers to the proportion of the administered dose of a factor VII preparation or a factor VII related drug that can be detected in blood plasma at predetermined times after administration. Typically, bioavailability is measured in the test animals by administering a dose of the drug in the range of about 25-250 mcg / kg; obtaining blood plasma samples at predetermined times after administration; and determining the content of factor VII polypeptides or factor VII related polypeptides in the samples using one or more assays from a coagulation assay (or any biological assay), immunological assay, or any equivalent thereof. Data is typically presented graphically as [factor VII] as a function of time, and bioavailability is expressed as the area under the curve (AUC). The relative bioavailability of the study drug refers to the ratio of the AUC of the study drug to the same parameter for the reference drug.

In some embodiments, the preparations of the present invention exhibit a relative bioavailability of at least about 110%, preferably at least about 120%, more preferably at least about 130%, and most preferably at least about 140% of the bioavailability of a reference preparation. Bioavailability can be measured on any mammalian species, preferably dogs, and predetermined times used to calculate AUC can span various intervals from 10 minutes to 8 hours.

The term "half life" refers to the time required to lower the concentration of factor VII polypeptides and factor VII related polypeptides in blood plasma from a specific value to half this value. Half life can be determined using the same procedure as for bioavailability. In some embodiments, the preparations of the present invention demonstrate an increase in half life by at least about 0.25 hours, preferably at least about 0.5 hours, more preferably at least about 1 hour, and most preferably at least about 2 hours relative to half the life of the reference preparation.

The term “immunogenicity” of a drug refers to the ability of this drug, when administered to humans, to elicit a harmful immune response, either humoral or cellular, or both together. factor VIIa polypeptides and factor VIIa related polypeptides are not known to cause detectable immune responses in humans. However, in any human subpopulation, individuals may exist that are sensitive to the particular protein being introduced. Immunogenicity can be measured by quantifying the presence of antifactor VII antibodies and / or factor VII-sensitive T lymphocytes in a sensitive individual using conventional methods known in the art. In some embodiments, the preparations of the present invention exhibit a decrease in immunogenicity in a susceptible individual by at least about 10%, preferably at least about 25%, more preferably at least about 40%, and most preferably at least about 50% with respect to the immunogenicity of the reference preparation in the same individual.

Pharmaceutical compositions and methods for their use

The preparations of the present invention can be used to treat any factor VII sensitive syndrome, such as, for example, bleeding disorders, including, but not limited to, those caused by deficiency of a coagulation factor (for example, hemophilia A and B, or deficiency of coagulation factors XI or VII); thrombocytopenia or von Willebrand disease, or coagulation factor inhibitors, or excessive bleeding of any origin. Drugs can also be administered to patients due to surgery or other trauma, or to patients receiving therapeutic treatment with anticoagulants.

Preparations containing factor VII-related polypeptides in accordance with the present invention, which have significantly reduced biological activity compared to wild-type factor VII, can be used as anticoagulants, for example, in patients undergoing angioplastic surgery or other surgical procedures that may increase the risk of thrombosis or occlusion of blood vessels, as occurs, for example, with restenosis. Other medical indications for which anticoagulants are prescribed include, without limitation, deep vein thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIG), fibrin deposition in the lungs and kidneys associated with gram-negative endotoxemia, myocardial infarction; acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), hemolytic uremia syndrome (HUS), MOF and TTP.

Pharmaceutical compositions containing factor VII preparations and factor VII related products of the present invention are intended primarily for parenteral administration for prophylactic and / or therapeutic treatment. Preferably, the pharmaceutical compositions are administered parenterally, i.e. intravenously, subcutaneously or intramuscularly. They can be administered by continuous or pulsating infusion.

The pharmaceutical compositions or preparations comprise the preparation of the present invention in combination with a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent, preferably dissolved in it. many aqueous vehicles can be used, such as water, buffer water, 0.4% saline, 0.3% glycine and the like. The preparations of the present invention can also be prepared in the form of liposome preparations for delivery or targeted transport to the lesion sites. Liposome preparations are described generally, for example, in US Pat. Nos. 4,837,028, 4,501,728 and 4,975,282. The compositions can be sterilized using conventional, well-known sterilization techniques. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, while the lyophilized preparation is combined with a sterile aqueous solution immediately before administration.

The compositions may contain pharmaceutically acceptable excipients or adjuvants, including, but not limited to, pH adjusting agents and buffers, and / or tonicity agents, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and things like that.

The concentration of factor VII polypeptides or factor VII-related polypeptides in these preparations can vary widely, that is, from less than about 0.5 wt.%, Typically, or at least about 1 wt.%, Up to 15 or 20 wt.%, And should be selected, first of all, in accordance with the volume of liquid, viscosities and the like in accordance with the particular chosen route of administration.

Thus, a typical pharmaceutical composition for intravenous administration containing up to 250 ml of sterile Ringer's solution and 10 mg of the preparation can be made. Current methods for the preparation of parenterally administered compositions should be known or apparent to one skilled in the art and are described in more detail, for example, in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA (1990).

Compositions containing the preparations of the present invention can be administered for prophylactic and / or therapeutic treatment. In therapeutic applications, the compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure, alleviate or partially stop the disease and its complications. An amount adequate to achieve this is defined as a “therapeutically effective amount”. Effective amounts for each goal should depend on the severity of the disease or damage, as well as the weight and general condition of the patient. Typically, however, an effective amount should be in the range of about 0.05 mg to about 500 mg of the drug per day for 70 kg of the patient, with doses of about 1.0 mg to about 200 mg of the drug per day being used more often. It will be understood that the determination of the appropriate dosage can be carried out using routine experiments by constructing a matrix of values and examining various points in the matrix.

Local delivery of the preparations of the present invention, such as, for example, topical application, can be carried out, for example, by spray, perfusion, double balloon catheters, a stent, inserted into transplants or stents in vessels, hydrogels used for application to balloon catheters, or using other well-known methods. In any case, the pharmaceutical compositions should provide an amount of the drug sufficient to effectively treat the patient.

The pharmaceutical compositions of the present invention may additionally contain other biologically active agents, such as, for example, non-factor VII coagulants or anticoagulants.

The following examples are considered as non-limiting illustrations of the present invention.

Example 1: Preparation and analysis of a factor VII preparation showing an altered glycoform configuration

The following experiment is carried out to obtain a factor VII preparation having an altered glycoform configuration.

I. Production: The BHK cell line transformed with a plasmid encoding factor VII is adapted for growth in suspension culture in the absence of serum. Cells are grown continuously in a culture with stirring, and when the number of cells increases, the volume is gradually increased by adding a new medium.

Finally, 6 L of the seed culture was inoculated into a 100-liter industrial biological reactor containing macroporous Cytopore 1 carriers (Pharmacia), after which the suspended cells were immobilized on the carriers. The culture is maintained at 36 ° C, at a pH of 6.7-6.9 and at a DO value of 50%. The volume of an industrial biological reactor is gradually increasing by adding a new medium as the number of cells increases. When the cell density reaches approximately 2 × 10 ⁶ cells / ml, a production phase is initiated and a medium change is carried out every 24 hours. Stirring is stopped to allow sedimentation of the media containing cells, and then 80% of the culture supernatant is collected and replaced with a new medium. The collected culture supernatant is filtered to remove unreached cells and cell debris, and then transported for further processing.

During the production phase, cells reach a density of 3-6 × 10 ⁶ cells / ml and a titer of 2-7 mg of factor VII / liter.

II. Analysis of the configuration of glycoform recombinant factor VII

The oligosaccharide configurations of the following preparations are compared: (a) preparations of recombinant factor VII obtained as described in Part I (n = 7); and two reference drugs: (b) recombinant factor VII preparations obtained in BHK cells in the presence of calf serum

(n = 10); and (c) a factor VII preparation isolated from human blood plasma.

N-linked oligosaccharides are released from the polypeptides by chemical cleavage (hydrazinolysis on a GlycoPrep 1000 device, Oxford GlycoSciences) or by enzymatic cleavage (N-glycosidase F, for example, from Boehringer Mannheim). The released oligosaccharides are labeled with 2-aminobenzamide (use the K-404 signaling kit, Oxford GlycoSciences or Glyko). Labeled oligosaccharides were analyzed using anion exchange HPLC on a CarboPac PA100 column (4 × 250 mm, Dionex, P / N 43055) together with a Guard column (4 × 50 mm, Dionex, P / N 43054). The column is equilibrated with 150 mm sodium hydroxide and eluted with a gradient of 0-150 mm sodium acetate, 150 mm sodium hydroxide. Oligosaccharides are detected using fluorescence with excitation at 330 nm and emission at 420 nm.

The relative contents of the various oligosaccharide configurations of factor VII (Klausen et al., 1998) are calculated as the relative peak areas for carbohydrate peaks when analyzed by anion exchange HPLC. Based on the structural elements of each oligosaccharide, they are mapped to one of the following chains: (i) chains containing at least one sialic acid; (ii) chains lacking any sialic acid (i.e. neutral); (iii) chains containing at least one final galactose residue; (iv) chains containing at least one final N-acetylgalactosamine residue; and (v) chains containing at least one unblocked antenna (i.e., at least one final galactose or N-acetylgalactosamine residue). Finally, the sum of the relative contents of the oligosaccharide chains compared with each group is calculated as a percentage of all oligosaccharide chains. The standard deviation from this determination, as calculated, is 0.08% (variation throughout the day); 0.7% (spread between days); and 0.5% (range 1-100 μg).

The obtained glycoform configurations are illustrated in the following table:

  (i) (ii) (iii) (iv) (v) a 93.1-98.7 1.3-6.9 5.9-16.4 5.9-8.7 11.7-23.9 b 88.3-92.5 7.5-12.9 9.4-16.8 19.0-28.6 30.1-39.0 c 99.5% <0.5% 2-3% 0% 2-3%

Recombinant factor VII preparations obtained in accordance with this example (i.e., in the absence of serum) show a glycoform configuration that differs both from the glycoform of recombinant factor VII obtained in the presence of serum and from native factor VII isolated from human plasma . The oligosaccharides of recombinant factor VII obtained in the absence of serum are sialylated to a degree greater than those obtained in the presence of serum and contain fewer neutral chains and fewer chains that end with either galactose or N-acetylgalactosamine.

III. Bioavailability: The following experiment is performed to compare the bioavailability of two factor VII preparations, obtained as above (I and II), with the bioavailability of two factor VII reference preparations (i.e., obtained in the presence of serum) (A and B).

Groups of 8 rats are given either the study drug or the reference drug at a dose of 25 μg / kg (≈100 μg / rat) in glycylglycine buffer (pH 7.4) containing sodium chloride (7.87 mg / ml), calcium chloride dihydrate (1.48 mg / ml), mannitol (2.5 mg / ml) and polysorbate 80. Blood samples were taken at 10 min and 30 min after the initial injection. Plasma is recovered from the samples and factor VII is quantified by ELISA. The bioavailability of each sample is expressed as the dose-dependent area under the concentration curve for factor VII in plasma based on 10 and 30 minute samples (AUC _10-30 / dose). Relative bioavailability is expressed as the ratio of the average AUC of _10-30 / dose for the studied and reference samples × 100. 90% confidence intervals for relative bioavailability are calculated from 90% confidence intervals for differences between drugs.

The results are shown in the table below (% sialylation of each drug, which is measured as described above, is indicated in parentheses).

Researched Reference Relative bioavailability Lower limit of 90% confidence interval Upper limit of 90% confidence interval I (97.5%) A (93%) 128.6 116.1 141.1 I (97.5%) B (86%) 154.9 141.2 168.5 II (96.7%) A (93%) 117.3 104.8 129.8 II (96.7%) B (86%) 141.2 127.5 154.8

These results indicate that even relatively small differences in the proportion of oligosaccharide chains having at least one sialic acid residue, for example, between 93% and 96 or 97%, can have a noticeable effect on bioavailability (increase by 20-30%). Moreover, a 10% increase in% sialylation causes a 40-50% increase in bioavailability.

Example 2: Analysis of factor VII preparations showing an altered glycoform configuration

Factor VII is prepared as described above in Example 1, with the exception that factor VII is collected from 500 L of cultures. Glycoform analysis is carried out as described in Example 1. Three independent preparations (A, B and C) are analyzed and compared with the reference preparation (D).

Bioavailability is measured on a dog model as follows: The experiment is carried out as a cross-sectional study on four paws, on 12 hound dogs, divided into four groups. All animals receive each of the three studied preparations A, B, and C, and reference preparation D at a dose of ≈90 μg / kg in glycylglycine buffer (pH 5.5) containing sodium chloride (2.92 mg / ml), calcium chloride dihydrate (1.47 mg / ml), mannitol (30 mg / ml) and polysorbate 80. Blood samples were taken at 10, 30 and 60 minutes and at 2, 3, 4, 6 and 8 hours after the initial injection. Plasma is obtained from the samples and factor VII is quantified by ELISA.

The bioavailability of each sample is expressed as the area corresponding to the dose under the curve of the concentration of factor VII in the blood plasma (AUC / dose). Relative bioavailability is expressed as the ratio between the average AUC / dose for the test and reference preparation × 100 and 90% confidence intervals for relative bioavailability are calculated.

The results are shown in the table below. % sialylation of each drug, which is measured as described above in example 1, is indicated in parentheses.

Researched Reference Relative bioavailability Lower limit of 90% confidence interval Upper limit of 90% confidence interval A (98.7%) D (88.2%) 144 135 153 B (95.9%) D (88.2%) 127 119 136 C (93.1%) D (88.2%) 112 105 120

These results indicate that small differences in the proportion of oligosaccharide chains having at least one sialic acid residue have a significant effect on the bioavailability of factor VII. A 10% increase in% sialylation causes a 30-50% increase in bioavailability with a ratio close to linear for the three studied drugs and for the reference drug.

Example 3: factor VII preparations showing an altered glycoform configuration

The following experiment is carried out to obtain a factor VII preparation having an altered glycoform configuration.

I. Construction of cell lines and production of factor VII: The plasmid vector pLN174 for the expression of human FVII has been described (Persson and Nielsen. 1996, FEBS Lett. 385: 241-243). Briefly, it carries the nucleotide sequence of a cDNA encoding human FVII, including a propeptide, under the control of the mouse metallothionein promoter, for transcription of the cDNA insert, and mouse dihydrofoliate reductase cDNA, under the control of the SV40 early promoter, for use as a selectable marker.

To construct a plasmid vector encoding a gamma-carboxylation recognition sequence, a cloning vector (pBluescript II KS +, Stratagene) containing cDNA encoding FVII, including its propeptide (pLN171), is used. (Persson et al. 1997, J. Biol. Chem. 272: 19919-19924). The stop codon encoding nucleotide sequence is inserted into the cDNA encoding FVII after the FVII propeptide by reverse PCR-mediated mutagenesis using this cloning vector. The matrix plasmid was denatured by treatment with NaOH followed by PCR with Pwo polymerases (Boehringer-Mannheim) and Taq (Perkin-Elmer) with the following primers:

a) 5'-AGC GTT TTA GCG CCG GCG CCG GTG CAG GAC-3 '

b) 5'-CGC CGG CGC TAA AAC GCT TTC CTG GAG GAG CTG CGG CC-3 '

The resulting mixture was digested with Dpnl to digest the remainder of the template DNA, and Escherichia coli was transformed using the PCR product. Clones are scanned for the presence of the mutation by sequencing. cDNA from the correct clone is transferred as a BamHI-EcoRI fragment into the expressed plasmid pcDNA3 (Invitrogen). The resulting plasmid is defined as pLN329. CHO K1 cells (ATCC CCl61) are transfected with equal amounts of pLN174 and pLN329 using the Fugene6 method (Boehriner-Mannheim). Transfectants are selected by adding methotrexate up to 1 μM and G-418 up to 0.45 mg / ml. The transfectant pool is cloned by limiting dilution and the expression of FVII from the clones is measured.

The highly productive clone is further subcloned and clone E11 is selected with specific expression of FVII 2.4 pg / cell / day in Dulbecco's modified Eagle medium with 10% fetal calf serum. The clone is adapted to the culture of a suspension containing no serum in a commercially available CHO medium (JRH Bioscience) containing no animal derived components.

Adapted cells continuously multiply in cultures with stirring, and when the number of cells increases, the volume gradually increases by adding a new medium. After 25 days, 6 L of the culture with stirring are inoculated into a 50 liter biological reactor. Cells multiply in a biological reactor and when the number of cells increases, the volume gradually increases by adding a new medium.

Finally, 50 L of the seed culture was inoculated into a 500 liter industrial biological reactor containing macroporous Cytopore 1 carriers (Pharmacia), after which the cells from the suspension were immobilized on the carriers. The culture is maintained at 36 ° C, at a pH of 7.0-7.1 and at a dissolved oxygen pressure (DOT) of 50% of saturation. The volume in a biological reactor is gradually increased by adding a new medium as the number of cells increases. When the cell density reaches approximately 10-12 × 10 ⁵ cells / ml, the production phase is initiated and the medium is changed every 24 hours: stirring is stopped to allow sedimentation of carriers containing cells, and then 80% of the culture supernatant is collected and replaced with a new one Wednesday. The collected culture supernatant is filtered to remove unreached cells (i.e. cells that cannot be immobilized on carriers) and cell debris, and then transported for further processing.

During the production phase, cells reach a density of 2-3 × 10 ⁷ cells / ml and a titer of 8 mg of factor VII / liter.

II. Glycoform analysis:

A. the configuration of the oligosaccharides (a) of the factor VII preparation obtained as described above is compared with the configuration of (b) the preparations of recombinant factor VII produced in BHK cells in the presence of calf serum, and (c) the factor VII preparation isolated from blood plasma person. The methods used are essentially the same as described in example 1.

The results are shown in the table below. The classification of oligosaccharides is as follows: (i) chains containing at least one sialic acid; (ii) chains not containing any sialic acid (i.e. neutral); (iii) chains containing at least one final galactose residue; (iv) chains containing at least one final N-acetylgalactosamine residue; and (v) chains containing at least one unblocked antenna (i.e., at least one final residue of galactose or N-acetylgalactosamine).

(i) (ii) (iii) (iv) (v) a 95.2 4.8 22.9 0.1 23.0 b 88.3-92.5 7.5-12.9 9.4-16.8 19.0-28.6 30.1-39.0 c 99.5% <0.5% 2-3% 0% 2-3%

B. oligosaccharide configurations of five independent factor VII preparations prepared as described in this example, (a) are compared with structures (b) of recombinant factor VII preparations produced in BHK cells in the presence of calf serum, and (c) with factor VII preparation isolated from human blood plasma using the analytical methods described in example 1.

Based on the structural elements of each oligosaccharide, it is mapped to one of the following configurations: (i) chains containing at least one sialic acid; (ii) chains not containing any sialic acid (i.e. neutral); (iii) chains containing at least one fucose associated with the antenna. Finally, the sum of the relative contents of the oligosaccharide chains compared with each group is calculated as a percentage of the total number of oligosaccharide chains. The standard deviation from this definition is calculated as equal to 0.08% (spread throughout the day); 0.7% (spread between different days); and 0.5% (range 1-100 μg).

The resulting glycoform configurations are illustrated in the following table:

(i) (ii) (iii) a 89.0-97.9% 2.1-11.0% 6.3-21.3% b 88.3-92.5% 7.5-12.9% 0% c 99.5% <0.5% 0%

Recombinant factor VII preparations prepared in accordance with Example 1 (that is, obtained in the absence of serum using the CHO cell line) show a glycoform configuration that differs both from the glycoform configuration of recombinant factor VII obtained in the presence of serum and from native factor VII isolated from human blood plasma. The oligosaccharides of recombinant factor VII, obtained in the absence of serum using the CHO 282.4 cell line, include configurations with fucose associated with the antenna, which are absent in both reference preparations. Two of these configurations are isolated and characterized by laser desorption from an auxiliary matrix — ionization mass spectrometry, by treatment with fucosidases, specific for the binding, and by anion exchange HPLC, as described above. Both configurations, as shown, contain the Lewis x sialyl structure, i.e., fucose, linked α1-> 3 to the antennae N-acetylglucosamine in the sialylated oligosaccharide.

III. Biological Activity:

The five factor VII preparations obtained as described in this example are analyzed for (a) thrombin generation and (b) for binding to tissue factor (TF) and compared with recombinant factor VII produced in BHK cells in the presence of serum (reference) . The following table establishes correlations of glycoform configurations (% oligosaccharide chains containing sialic acid and% containing fucosylated antenna) and their biological activity.

Factor VII drug Oligosaccharide configuration Thrombin generation (% of reference) Binding to TF, Kd (nm) % sialyl % fucosyl one 98 6 125 2,8 2 94 13 123 2.0 3 93 fourteen 126 1.8 four 88 16 145 3.3 5 86 21 158 2,8 Reference 86-93 0 one hundred 2.2-6.6

These results show that factor VII preparations having fucosylated antennas exhibit higher TF-independent activity of factor VII (as demonstrated, for example, by thrombin generation) than factor VII preparations where fucosylated antennas are absent.

Example 4: in vitro hydrolysis assay

The following method can be used to analyze the biological activity of factor VIIa. The analysis is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden) at a final concentration of 1 mm was added to factor VIIa (final concentration of 100 nM) in 50 mm Hepes, pH 7.4, containing 0, 1 M NaCl, 5 mm CaCl ₂ and 1 mg / ml bovine serum albumin. The absorbance at 405 nm is continuously measured in a SpectraMax ™ 340 plate reader (Molecular Devices, USA). Absorption developed during a 20-minute incubation, after subtracting the absorption in an empty well containing no enzyme, is used to calculate the ratio of the activities of the studied and reference factor VIIa.

Example 5: analysis using proteolysis in vitro

The following method can be used to analyze the biological activity of factor VIIa. The analysis is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nm) and factor X (0.8 μM) in 100 μl of 50 mm Hepes, pH 7.4, containing 0.1 M NaCl, 5 mm CaCl ₂ and 1 mg / ml bovine serum albumin, incubated for 15 minutes. Then, the cleavage of factor X is stopped by adding 50 μl of 50 mm Hepes, pH 7.4, containing 0.1 M NaCl, 20 mm EDTA and 1 mg / ml bovine serum albumin. The amount of factor Xa generated is measured by adding the chromogenic substrate ZD-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). Absorption developed within 10 minutes after subtracting absorption in an empty well not containing FVIIa is used to calculate the ratio of proteolytic activities of the studied and reference factor VIIa.

All patents, patent applications, and references cited herein are hereby incorporated by reference in their entireties.

Specialists in this field in light of the above detailed description will offer many variations of the present invention. Such obvious variations are within the full intended scope of the appended claims.

Claims (53)

1. Factor VII polypeptides, which are a plurality of factor VII polypeptides or factor VII related polypeptides, where the polypeptides contain asparagine-linked oligosaccharide chains and where, in the range of about 94-99% oligosaccharide chains, contain at least one sialic acid residue for treatment syndromes associated with factor VII. 2. Factor VII polypeptides, which are a plurality of factor VII polypeptides or factor VII related polypeptides, where the polypeptides contain asparagine-linked oligosaccharide chains and where within about 1-7% of the oligosaccharide chains have a neutral charge for treating syndromes associated with factor VII. 3. Factor VII polypeptides, which are a plurality of factor VII polypeptides or factor VII related polypeptides, where the polypeptides contain asparagine-linked oligosaccharide chains and where in the range of about 6-16% of the oligosaccharide chains contain at least one final galactose residue for treatment syndromes associated with factor VII. 4. Factor VII polypeptides, which are a plurality of factor VII polypeptides or factor VII related polypeptides, where the polypeptides contain asparagine-linked oligosaccharide chains and where, within about 11-23% of the oligosaccharide chains, contain at least one terminal galactose or N-acetylgalactosamine residue intended for the treatment of syndromes associated with factor VII. 5. Factor VII polypeptides, which are a plurality of factor VII polypeptides or factor VII related polypeptides, where the polypeptides contain asparagine-linked oligosaccharide chains and where at least about 2% of the oligosaccharide chains contain at least one fucose residue bound by α1- > 3 with antennar N-acetylglucosamine, intended for the treatment of factor VII syndromes. 6. The polypeptides according to any one of claims 1 to 5, wherein the sialic residues in the oligosaccharide chains are linked to galactose via an α2-> 3 bond. 7. The polypeptides according to any one of claims 1 to 6, where the sialic acid residues contain N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). 8. The polypeptides according to any one of claims 1 to 7, where the oligosaccharides contain fucose bound α1-> 6 to the N-acetylglucosamine framework. 9. The polypeptides according to any one of claims 1 to 8, where in the range of about 95-98% of the oligosaccharide chains contain at least one sialic acid residue. 10. The polypeptides according to any one of claims 1 to 9, where in the range of about 96-97% of the oligosaccharide chains contain at least one sialic acid residue. 11. The polypeptides according to any one of claims 1 to 10, where within about 2-4% of the oligosaccharide chains have a neutral charge. 12. The polypeptides according to any one of claims 1 to 11, where in the range of about 8-12% of the oligosaccharide chains contain at least one final galactose residue. 13. The polypeptides according to any one of claims 1 to 12, where in the range of about 12-18% of the oligosaccharide chains contain at least one final residue of galactose or N-acetylgalactosamine. 14. The polypeptides according to any one of claims 1 to 13, where at least about 5% of the oligosaccharide chains contain at least one fucose residue associated with α1-> 3 with antennar N-acetylglucosamine. 15. The polypeptides according to any one of claims 1 to 14, where at least about 10% of the oligosaccharide chains contain at least one fucose residue associated with α1-> 3 with antennar N-acetylglucosamine. 16. The polypeptides according to any one of claims 1 to 15, where at least about 20% of the oligosaccharide chains contain at least one fucose residue associated with α1-> 3 with antennar N-acetylglucosamine. 17. The polypeptides according to any one of claims 1 to 16, where at least about 40% of the oligosaccharide chains contain at least one fucose residue associated with α1-> 3 with antennar N-acetylglucosamine. 18. The polypeptides according to any one of claims 1 to 17, where the polypeptides have a wild-type factor VII amino acid sequence. 19. The polypeptides according to any one of claims 1 to 18, where the polypeptides are wild-type factor VIIa. 20. The polypeptides according to any one of claims 1 to 17, where the factor VII polypeptides are selected from the group consisting of: S52A-factor VII, S60A-factor VII, factor VII, which is proteolytically cleaved between residues 290 and 291; factor VII, which is proteolytically cleaved between residues 315 and 316; and factor VII, which is oxidized. 21. The polypeptides according to any one of claims 1 to 17, where the polypeptides related to factor VII are selected from the group consisting of: R152E-factor VII, S344A-factor VII, FFR-factor VII and factor VIIa, where the Gla domain is missing. 22. Factor VII polypeptides, which are a plurality of factor VII polypeptides or factor VII related polypeptides, where the polypeptides contain asparagine-linked oligosaccharide chains and where (i) within about 94-100% of the oligosaccharide chains contain at least one sialic acid residue and (ii) within about 6-9% of the oligosaccharide chains contain at least one final N-acetylgalactosamine residue, intended for the treatment of factor VII-related syndromes. 23. The polypeptides of claim 22, wherein the factor VII polypeptides have a wild-type factor VII sequence. 24. Factor VII polypeptides, which are a plurality of factor Vila polypeptides having a wild-type factor VII sequence, where the polypeptides contain asparagine-linked oligosaccharide chains and where, within about 94-99% of the oligosaccharide chains, contain at least one sialic acid residue intended for treatment of syndromes associated with factor VII. 25. Factor VII polypeptides, which are a plurality of factor VIIa polypeptides having a wild-type factor VII sequence, where the polypeptides contain asparagine-linked oligosaccharide chains and where at least about 2% of the oligosaccharide chains contain at least one fucose residue bound α1 -> 3 with antennar N-acetylglucosamine, intended for the treatment of factor VII syndromes. 26. The polypeptides according to any one of claims 1 to 25, where the polypeptides are produced in a host cell selected from the group consisting of cells of fungi, insects and vertebrates. 27. The polypeptides of claim 26, wherein the host cell is a mammalian cell. 28. The polypeptides of claim 27, wherein the mammalian cell is obtained from a hamster. 29. The polypeptides of claim 28, wherein the hamster cell is selected from the group consisting of CHO cells and BHK cells. 30. The polypeptides of claim 27, wherein the mammalian cell is obtained from a human. 31. The polypeptides of claim 30, wherein the human cell is an HEK cell. 32. The polypeptides according to any one of claims 1 to 31, wherein the polypeptides exhibit bioavailability that is at least about 110% of the bioavailability of the reference preparation, and where about 93% or less of the oligosaccharide chains in the reference preparation contain at least at least one sialic acid residue. 33. The polypeptides of claim 32, wherein the polypeptides exhibit bioavailability that is at least about 120% of the bioavailability of a reference preparation. 34. The polypeptides of claim 33, wherein the polypeptides exhibit bioavailability that is at least about 130% of the bioavailability of a reference preparation. 35. A method for determining the glycoform configuration of factor VII polypeptides and factor VII related polypeptides, wherein the method includes: (a) culturing a culture of CHO-K1 cells expressing factor VII polypeptides or factor VII-related polypeptides in a first set of predetermined culturing conditions, where the cells are adapted for growth in the absence of serum and are cultured in the absence of serum both in the growth phase and in production phase; (b) recovering factor VII polypeptides or factor VII related polypeptides from the culture to produce polypeptides; and (c) analyzing the configuration of oligosaccharides associated with the polypeptides to determine the glycoform configuration of the polypeptides. 36. The method according to clause 35, further comprising: (d1) changing the cultivation conditions in step (a) to obtain a second set of predetermined culturing conditions; (e1) repeating steps (b) to (d1) until the desired glycoform configuration is obtained. 37. The method according to clause 35, further comprising: (d2) chemical or enzymatic treatment of polypeptides to alter the configuration of oligosaccharides; and (e2) repeating steps (b) to (d2) until the desired glycoform configuration is obtained. 38. The method of producing polypeptides according to any one of claims 1 to 34, wherein said method comprises culturing a culture of CHO-K1 cells expressing polypeptides, where the cells are adapted for growth in the absence of serum and are cultured in the absence of serum both in the growth phase and and in the production phase under conditions in which at least about 94% of the asparagine-linked oligosaccharides present in the polypeptides contain at least one sialic acid residue. 39. A pharmaceutical preparation containing the polypeptides according to any one of claims 1 to 34, and a pharmaceutically acceptable carrier or adjuvant for the treatment of syndromes associated with factor VII. 40. A method for treating a factor VII sensitive syndrome, wherein the method comprises administering the pharmaceutical preparation according to claim 39 to a patient in need of such treatment under conditions leading to a decrease in bleeding and / or to an increase in blood coagulation, wherein the preparation contains factor polypeptides VII. 41. The method of claim 40, wherein the syndrome is selected from the group consisting of hemophilia A, hemophilia B, factor XI deficiency, factor VII deficiency, thrombocytopenia, von Willebrand disease, the presence of a coagulation factor inhibitor, surgery, trauma and therapeutic treatment with anticoagulants. 42. A method for preventing unwanted bleeding, wherein the method comprises administering a pharmaceutical preparation according to claim 39 to a patient in need of such treatment under conditions leading to a decrease in bleeding and / or increased blood coagulation, wherein the preparation contains factor VII polypeptides. 43. A method for preventing unwanted blood coagulation, wherein the method comprises administering the pharmaceutical preparation of claim 39 to a patient in need of such treatment under conditions effective to inhibit coagulation, wherein the preparation contains polypeptides related to factor VII. 44. A method for preventing tissue factor mediated reactions, wherein the method comprises administering the pharmaceutical preparation of claim 39 to a patient in need of such treatment under conditions effective to inhibit coagulation, wherein the preparation contains polypeptides related to factor VII. 45. The method according to item 43, where the unwanted blood coagulation is associated with a condition selected from the group consisting of: angioplastic surgery, deep vein thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIG); deposits of fibrin in the lungs and kidneys associated with gram-negative endotoxemia, and myocardial infarction. 46. The method according to item 44, where the reaction mediated by tissue factor associated with a condition selected from the group consisting of SIRS, ARDS, MOF, HUS and TTP. 47. A drug intended for the treatment of a syndrome associated with factor VII, obtained using polypeptides according to any one of claims 1 to 34. 48. A drug for the treatment of a syndrome associated with factor VII according to clause 47, where the syndrome is selected from the group consisting of hemophilia A, hemophilia B, factor XI deficiency, factor VII deficiency, thrombocytopenia, von Willebrand disease, the presence of a factor inhibitor coagulation, surgery, trauma and therapeutic treatment with an anticoagulant. 49. A drug intended to prevent unwanted bleeding obtained using the polypeptides according to any one of claims 1 to 34. 50. A drug intended to prevent unwanted blood coagulation obtained using polypeptides according to any one of claims 1 to 34. 51. The drug is intended to prevent unwanted blood coagulation according to claim 50, wherein the unwanted blood coagulation is associated with a condition selected from the group consisting of: angioplasty, deep vein thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIG); deposits of fibrin in the lungs and kidneys associated with gram-negative endotoxemia, and myocardial infarction. 52. A drug intended to prevent tissue factor mediated reactions obtained using polypeptides according to any one of claims 1 to 34. 53. The drug is intended to prevent reactions mediated by the tissue factor according to paragraph 52, where the reactions mediated by the tissue factor are associated with a condition selected from the group consisting of SIRS, ARDS, MOF, HUS and TTP.