EP2049150A2 - Recombinant ou transgenic factor vii compound having a majority of glycan, biantennary, bisialylated and non-fucosylated forms - Google Patents

Abstract

The present invention concerns a recombinant or transgenic factor VII composition, each factor VII molecule of the composition having glycan forms linked to N-glycosylation sites, wherein among all the factor VII molecules in said composition, glycan, biantennary, bisialylated, and non-fucosylated forms are in the majority. The invention also concerns such a composition for use as a medication, and a method for preparing said composition, among others.

Description

 Composition of recombinant or transgenic factor VII, predominantly containing biantennary, bisialylated and non-fucosylated glycannic forms

Factor VII (FVII) is a vitamin K-dependent glycoprotein that, in its form. activated

(FVIIa), participates in the coagulation process by activating factor X and factor IX in the presence of calcium and tissue factor. FVII is secreted as a single peptide chain of 406 residues, which has a molecular weight of about 50 kDa. FVII has four distinct structural domains: the N-terminal γ-carboxylic (GI) domain, two epidermal growth factor (EGF) -like domains, and a serine protease domain. The activation of FVII in FVIIa is characterized by the cleavage of the Argi ₅₂ -Ilei53 (Arginine 152-Isoleucine 153) linkage. FVIIa is therefore composed of a light chain of 152 amino acids with a molecular weight of approximately 20 kDa and a heavy chain of 254 amino acids with a molecular mass of approximately 30 kDa linked together by a single disulfide bridge (Cysi ₃₅ -Cys ₂ 62) •

Plasma FVIIa (FVIIa, p) has several post-translational modifications: the first ten glutamic acids are γ-carboxylated, Asp ₆₃ (aspartic acid) is partially hydroxylated, Sers ₂ (Serine 52) and Serεo (Serine 60 ) are O-glycosylated and bear respectively the units Glucose (Xylose) 0-2 and Fucose, Asni4 ₅ (Asparagine 145) and Asn ₃ 22 (Asparagine 322) are predominantly N-glycosylated by bisantiylated biantennary complexed glycan forms .

FVII is used in the treatment of hemophilia patients with factor VIII (haemophilia A) or factor IX deficiency

(Type B hemophilia), as well as patients with other disability factors. coagulation, for example an inherited deficiency in FVII. FVII is also recommended for the treatment of stroke. It is therefore necessary to have injectable FVIIa concentrates.

The oldest method of obtaining FVIIa concentrates was the purification of FVIIa from plasma proteins from fractionation. For this purpose, the document EP 0 346 241 describes the preparation of a fraction enriched in FVIIa, obtained after adsorption then eluting a by-product of the fractionation of plasma proteins containing FVII and FVIIa and other proteins such as the factors IX (FIX), X (FX) and II (FII), including the pre-eluate of PPSB (P = prothrombin or FII, P = proconvertin or FVII, S = Stuart factor or FX and B = antihemophilic factor B or FIX). The disadvantage of this method is that the FVII obtained still contains some traces of other coagulation factors.

Similarly, EP 0 547 932 describes a method of manufacturing a high purity FVIIa concentrate essentially free of vitamin K dependent factors and FVIII. The FVII obtained by this method, despite its purity, has residual thrombogenic activity.

One of the major drawbacks of these methods is that they only make it possible to obtain small quantities of products. In addition, the volume of plasma collected from blood donors remains limited.

As a result, as early as the 1980s, DNA encoding human factor VII was isolated (Hagen et al.

(1986); Proc. Natl. Acad. Sci. USA; Apr 83 (8): 2412-6) and the corresponding protein was expressed in BHK (Baby Hamster Kidney) mammalian cells (EP 0 200 421). The patent application FR 06 04872 filed by the Applicant also describes the production of FVIIa in a transgenic animal.

These production methods make it possible to obtain secure proteins in terms of contamination by viruses or other pathogens. In addition, such methods make it possible to obtain proteins whose primary sequence, that is to say an identical sequence between the different amino acids, is identical to the human primary sequence. However, the human plasma FVII has complex post- translational modifications: the first ten glutamic acids are γ-carboxylated, Asp ₆ b 3 is partially-ydroxylé (aspartic acid 63) Ser ₅₂ (Serine 52) and the Serβo (Serine 60) are O-glycosylated and carry respectively the units Glucose (Xylose) 0-2 and Fucose, 1 Asni ₄₅ (Asparagine 145) and Asn ₃₂ 2 (Asparagine 322) are predominantly N-glycosylated by forms of the type complex, biantennées and bisialylées. In particular, the addition of N-glycans (glycans linked to 1-asparagine) is particularly important for correct protein folding, in vitro and in vivo stability, bioactivity and pharmacokinetic properties (bioavailability). for example) of the heterologous protein produced. Thus, variations of all or part of the post-translational modifications expose, on the one hand, to the risk that the protein is not active, and, on the other hand, to the risk that it is immunogenic.

However, existing recombinant or transgenic FVIIs can exhibit, by their expression in systems different from human systems, a glycosylation which differs from the glycosylation of human plasma FVII, which can lead to the appearance of antibodies directed against the recombinant protein, and thereby less effective than human FVII purified from human plasma. There is therefore a need for therapeutic or prophylactic compositions of FVIIa, the functional characteristics of which are similar to human FVII purified from human plasma, and whose mode of production is compatible with the need for large quantities of this protein.

Thus, the invention relates to a recombinant or transgenic factor VII composition, each factor VII molecule of the composition comprising glycan forms linked to the N-glycosylation sites, characterized in that, among all the factor VII molecules of said composition, the biantennary, bisialylated and non-fucosylated glycan forms are in the majority with respect to all the glycan forms linked to the N-glycosylation sites of the factor VII of the composition.

The Applicant has surprisingly discovered that a recombinant or transgenic FVII composition, whose biantennary forms, bisialylated and non-fucosylated are the majority, has an increased bioavailability, a decreased clearance and an increased stability compared to a recombinant FVII composition. or transgenic whose rate of bisialylated forms is less, that is to say with respect to a recombinant or transgenic FVII composition whose majority of biantennary, monosialylated and nonfucosylated forms. The FVII of the invention therefore makes it possible to envisage less frequent administrations to the patient, as well as lower doses compared to a recombinant or transgenic FVII composition whose rate of bisialylated forms is less, that is to say whose rate of monosilalylated forms is the majority. By bioavailability is meant the percentage of administered FVII diffusing into the bloodstream and therefore particularly likely to reach the bleeding site. By clearance, denotes the fraction of a theoretical volume totally purified, that is to say no longer containing the FVII per unit of time. In other words, this corresponds to the hypothetical quantity of fluid that would be completely free of the substance in a unit time interval.

Stability refers to the ability of FVII to maintain its chemical, physical, microbiological and biopharmaceutical properties within specific limits throughout its lifetime. By "biantennary, bisialylated and non-fucosylated glycan forms" means the forms below:

Form A2 (biantennate, bisialylated and not fucosylated)

A: Sialic acid

éεgà: Galactose

: N-acetylglucosamine (GIcNAc) φ -.Mannose

These glycannic forms are linked to the N-glycosylation sites consisting of asparagine 145 (Asn145) and asparagine 322 (Asn322). Indeed, the FVII of the invention comprises, like the human FVII, two N-glycosylation sites, at position 145 and 322, and 2 O-glycosylation sites, at position 52 and 60. At a N-glycosylation site Oligosaccharide chains are linked to asparagine (N-linked). On a 0-glycosylation site, the oligosaccharide chains are linked to a serine. Each FVII molecule of the invention therefore comprises two N-linked oligosaccharide chains. However, the FVII molecules of the composition do not exhibit homogeneous glycosylation, i.e. all N-linked oligosaccharide chains are not identical to each other. It is a mixture of different forms of glycan.

Indeed, any FVII, be it plasmatic, recombinant or transgenic, is in the form of a mixture of several FVII proteins, these proteins differentiating among themselves in particular by their glycosylation and otherwise called glycoforms. This glycosylation is due to post-translational processing carried out by the cell organelles during the transfer of the FVII protein between the different cell compartments. This biochemical modification deeply modifies the protein, so that the final protein is perfectly structured and therefore both active and well tolerated by the body. This chemical modification contributes to the regulation of the activity of the protein as well as to its location. It is therefore possible to quantify, for the entire composition, and therefore for all the N-linked oligosaccharide chains. of the composition of FVII, the level of each glycan form or of each sugar present in said composition.

The percentages of the different glycans given in the present application do not take into account the O-glycosylation.

By "FVII composition" is meant a composition whose only molecular entity is FVII, preferably activated. Each FVII molecule of the composition has the same primary sequence, but glycosylation varies from one molecule to another. The term "FVII composition" therefore designates a mixture of molecules with the same primary sequence, characterized by its contents in glycan forms. For purposes of the invention, the terms "FVII" and "composition of FVII" are equivalent. Therefore, within the scope of the invention, the term "FVII" refers to either the FVII molecule as such, or a mixture of such molecules, having the stated characteristics.

The FVII composition of the invention is a composition of FVII whose biantennary, bisialylated and nonfucosylated glycan forms are the majority. This means that among the set of N-linked oligosaccharides of the composition, that is to say all glycan forms linked to N-glycosylation sites of factor VII, the biantennary, bisialylated and non-fucosylated forms are the most represented. .

Advantageously, the level of biantennary, bisialylated and non-fucosylated glycan forms is greater than or equal to 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. Particularly advantageously, the level of biantennary, bisialylated and non-fucosylated glycan forms is greater than or equal to 45%. In particular Advantageously, the level of biantennary, bisialylated and non-fucosylated glycan forms is between 45% and 65%, and preferably between 50% and 60%. The rates of sialylated species can be determined empirically by HPCE-LIF (High Performance Capillary-Laser Fluorescence Electrophoresis) and / or NP-HPLC (High Performance Liquid Chromatography) analyzes with quantification by measurement of the area of the peaks corresponding to the different glycans, or by any other method known to those skilled in the art.

The FVII composition of the invention may also comprise monosialylated and triantennary biantennary minority forms, as well as neutral forms, which do not have sialic acids.

By "recombinant or transgenic FVII" is meant any FVII derived from genetic engineering, that is to say produced by cells whose DNA has been modified by genetic recombination so as to express a molecule of FVII, and presenting the glycosylation characteristics stated.

Thus, the FVII of the invention is derived from the transcription and then the translation of a DNA molecule encoding FVII in a cellular host or by a transgenic animal. The recombinant or transgenic FVII of the invention can be obtained using standard techniques, well known to those skilled in the art, allowing the expression of a protein in a biological system.

More particularly, the term "recombinant FVII" means any FVII obtained by genetic recombination and expressed by a cultured cell line. By way of example, mention may be made of the following lines: BHK (Baby Hamster Kidney) and in particular BHK tk ^" tsl3 (CRL 10314, Waechter and Baserga, Proc Natl Acad Sci USA 79: 1106-1110, 1982 ), CHO (ATCC CCL 61), COS-I (ATCC CRL 1650), HEK293 (ATCC CRL 1573, Graham et al., J. Gen. Virol 36: 59-72, 1977), Rat Hep I (Rat hepatoma, ATCC CRL 1600), Rat Hep II ( Rat hepatoma, ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (CHO cell line) (Urlaub and Chasin, Proc Natl Acad Sci USA 77: 4216-4220, 1980), 3T3 cells, Namalwa cells, or BHK cells adapted for serum-free culture (US 6,903,069). In addition, "transgenic FVII" is understood to mean any FVII obtained by genetic recombination and expressed by a living tissue, animal or plant.

The rate of bisialylated forms of FVII of the invention can be achieved in different ways.

In a particular embodiment, the FVII of the invention is expressed in a microorganism, cell, plant or animal conferring the stated glycosylation characteristics, that is to say a majority of biantennal, bisialylated and nonfucosylated forms. .

In another embodiment, the FVII of the invention is expressed in a microorganism, plant or animal that does not make it possible to obtain an FVII composition exhibiting a majority of bisialylated and nonfucosylated biantennary forms, the sialylation being carried out by subsequently in vitro, using one or more enzymes to confer the desired sialylation, that is to say that biantennées and bisialylées forms become the majority and triantennées forms become trisialylées.

By way of example, a sialyltransferase, in vitro, can be made to act on an FVII composition chosen for its advantageous properties under appropriate conditions to allow the desired sialylation. Thus, the FVII composition of the invention is capable of being obtained by sialyltransferase on a partially sialylated FVII composition (starting FVII composition).

Advantageously, the starting FVII composition has predominantly monosialylated biantennary glycan forms. Advantageously, the starting FVII composition has biantennary, monosialylated and nonfucosylated glycan forms predominant. The action of the sialyltransferase makes it possible to graft an additional sialic acid onto the monosialylated forms, converting them to a bisialylated form. Advantageously, these biantennary, monosialylated forms are present in the starting FVII composition at a level greater than 40%, particularly advantageously at a level greater than 50%, or even at 60%. Advantageously, the starting composition has a level of biantennane, monosialylated and non-fucosylated glycan forms greater than 20%, or particularly preferably greater than 30%, 40% or even 50%.

Advantageously, at least some of the sialic acids of the starting FVII composition involve α2-6 linkages. Particularly advantageously, the level of sialic acids involving α2-β bonds is greater than 60%, or greater than 70%, 80%, or 90%. In particular, this rate is between 60% and 90%.

Preferentially, all the sialic acids of the starting FVII composition involve α2-6 bonds.

In a particular embodiment, if the starting FVII composition comprises fucosylated forms in too large a proportion, for example greater than 50%, or even 60%, it is possible to obtain biantennary, bisialylated and nonfucosylated forms. using one or more enzymes to defucosylate the composition. As for example, mention may be made of the use of a fucosidase, for a time necessary for obtaining predominantly biantennary, bisialylated and nonfucosylated glycannic forms. In a particularly advantageous manner, the starting FVII composition is chosen for its low immunogenicity.

Advantageously, the starting composition is the composition of FVII described in document FR 06 04872, the content of which is considered to be included in the present document.

Advantageously, the FVII of the invention is a polypeptide whose peptide sequence may be that of natural human FVII, that is to say the sequence present in humans not exhibiting FVII-related disorders. Such a sequence can be coded for example by the sequence Ib described in the document EP

0, 200,421.

Advantageously, the sequence of the FVII of the invention is the sequence of SEQ ID NO: 1.

In another embodiment, the FVII of the invention may be a variant of natural human FVII, since this variant is not more immunogenic than natural FVII. Thus, the peptide sequence of this variant may have at least 70% identity, and advantageously at least 80% or 90%, and even more advantageously at least 99% identity with the sequence of natural human FVII. such a variant having essentially the same biological activity as natural FVII.

Furthermore, the FVII of the invention also refers to any modified FVII sequence so that the biological activity of the protein is reduced compared to the natural human FVII. By way of example, mention may be made of recombinant human FVII inactivated FFR-FVIIa, used for the treatment or prevention of thromboses (Holst et al, Eur J.Vasc.Endovasc.Surg., 1998 Jun, 15 (6): 515-520). Such FVIIs are polypeptides having an amino acid sequence which differs from the natural FVII sequence by insertion, deletion or substitution of one or more amino acids.

The biological activity of the FVII of the invention can be quantified by measuring the ability of an FVII composition to induce blood coagulation by means of FVII deficient plasma and thromboplastin, as for example described in US Pat. 5997864. In the test described in US Pat. No. 5,997,864, the biological activity is expressed by a reduction of the coagulation time relative to the control sample, and is converted into "units of FVII" by comparison with a human serum standard (pool ) containing 1 unit (1 U of FVII activity) / ml of serum. The FVII composition of the invention has glycosylation characteristics approximating that of plasma FVII. Indeed, the major N-glycans form of plasma FVII (or plasma FVII composition) is also the biantennary, bisialylated form.

Advantageously, the level of biantennary, bisialylated (fucosylated and non-fucosylated) forms of the FVII of the composition of the invention is greater than 30%, or 40% or 50%. Particularly advantageously, the rate of biantennal, bisialylated forms is greater than 60%, or 70%, or 80% or even 90%. Particularly advantageously, the level of bisialylated forms (fucosylated and non-fucosylated) is between 50% and 80%, or between 60% and 90%, or, preferably, between 70% and 85%. Advantageously, the fucose content of the FVII composition of the invention is greater than 20%, and is advantageously between 20% and 50%. This rate corresponds to the fucose rate measured among all the glycan forms of the FVII of the composition.

This feature is one of the advantages of the FVII of the invention. In fact, the commercially available recombinant FVII has a fucosylation level of 100%, whereas the plasmatic FVII has a fucosylation level of about 16%. Thus, the fucosylation of the FVII of the invention is close to that of the plasma FVII, which confers an advantage on the FVII of the invention in terms of safety.

Advantageously, at least some of the sialic acids of the factor VII composition of the invention involve α2-6 linkages. Particularly advantageously, the level of sialic acids involving α2-6 bonds is greater than 60%, or greater than 70%, 80%, or 90%. In particular, this rate is between 60% and 90%.

The FVII composition of the invention therefore comprises a non-zero level of sialic acid involving α2-6 bonds. This is an advantage over commercial recombinant FVII, which contains only sialic acids involving α2-3 bonds, whereas plasma FVII has them.

In a particular embodiment of the invention, all the sialic acids of the FVII composition of the invention involve ^'α2-6 bonds.

Particularly preferably, all sialic acids involve α2,6 bonds, i.e., all sialic acids are linked to galactose via a? 2,6 bond, and in particular at least 90%? sialic acids of FVII involve α2,6 bonds. The composition of FVII according to the invention may further comprise sialigic acids of α2-3 bonds.

The fact that sialic acids of the FVII of the composition involve α 2, 6 branches is one of the advantages of the FVII of the invention. Indeed, the sialic acids of the commercially available recombinant FVII involve only α2, 3 bonds. However, the plasma FVII is a mixture of these two isomers. Such plasma FVII contains, for example, 40% oc2,3 isomers and 60% 2,6-isomers. However, the latter has more α2,6 bonds, which brings the FVII even closer to the invention of plasma FVII.

In another embodiment, certain sialic acids of the FVII composition of the invention involve α2-3 linkages.

Thus, in a particular embodiment of the invention, the recombinant or transgenic FVII of the composition has biantennary, bisilalylated and nonfucosylated glycan forms predominant with respect to all the glycan forms linked to the factor VII N-glycosylation sites. and a sialic acid level involving cx2-6 bonds greater than 90%. In a particularly preferred embodiment of the invention, the recombinant or transgenic FVII of the composition has biantennary, bisilalylated and nonfucosylated glycan forms predominant with respect to all the glycan forms linked to the factor VII N-glycosylation sites, and a level of sialic acids involving α2-6 bonds equal to 100%.

In a particular embodiment of the invention, the recombinant or transgenic FVII of the composition has biantennary, bisilalylated and nonfucosylated glycan forms predominant in relation to all forms. glycans linked to the N-glycosylation sites of Factor VII, the fucose content of the FVII composition being between 20% and 50%.

In a particular embodiment of the invention, the recombinant or transgenic FVII of the composition has biantennary, bisilalylated and nonfucosylated glycan forms predominant with respect to all the glycan forms linked to the N-glycosylation sites of factor VII, all sialic acids involving α2-6 linkages, and the fucose content of the FVII composition being between 20% and 50%.

Advantageously, the FVII composition of the invention is capable of being produced by a transgenic non-human mammal.

In this embodiment, the FVII composition of the invention will therefore be described as "transgenic". By transgenic mammal means any mammal with the exception of the human being, genetically manipulated to express an exogenous protein, for example the rabbit, the goat, the mouse, the rat, the cattle, the horse, the pig, the insects, sheep, this list is not limiting. The exogenous protein is FVII, preferably human FVII. The transgenic non-human mammal may, in addition to FVII, express an exogenous enzyme so as to impart the desired sialylation to the transgenic FVII composition. As such, the transgenic non-human animal can co-express the gene encoding FVII and the gene encoding a sialyltransferase.

In a particular embodiment of the invention, the transgenic FVII of the invention is expressed in the mammary glands of the transgenic mammal and produced in its milk. As such, expression of the transgene is tissue-dependent, by means of a promoter ensuring the production of the transgene in the mammary glands. the animal. There may be mentioned the WAP (whey acidic protein) promoter, the casein promoter, in particular the β-casein promoter or α-casein, the β-lactoglobulin promoter, the α-lactalbumin promoter. , this list not being limiting.

Advantageously, the FVII composition of the invention is capable of being produced by a transgenic rabbit, said composition then being subjected to in vitro sialylation so that the biantennary, bisialylated forms become majoritarian.

The rabbit is a particularly advantageous species for the production of therapeutic proteins because the rabbit does not appear to be susceptible to prions, especially to transmissible spongiform encephalopathy, which constitutes a major public health problem.

In addition, the species barrier between the rabbit and the man is important. In contrast, the species barrier between man and hamster, which is the biological system in which commercially available recombinant FVII is produced, is less important.

Thus, the production of FVII in the rabbit is advantageous in terms of safety as regards the transmission of pathogens, including unconventional pathogens of the prion type.

In a preferred embodiment of the invention, the FVII of the invention is produced in the mammary glands of transgenic rabbits.

The secretion by the mammary glands of the protein of interest, allowing its secretion in the milk of the transgenic mammal is a technique well known to those skilled in the art, which involves the control of the expression of the recombinant protein in tissues. dependent. Tissue control άe expression is performed through sequences allowing expression of the protein to a particular tissue of the animal. These sequences are in particular the promoter sequences, as well as the signal peptide sequences.

Examples of promoters allowing the expression of a protein of interest in the mammary glands are the WAP (whey acidic protein) promoter, the casein promoter, in particular the β-casein promoter, α-casein, the promoter of β-lactoglobulin, α-lactalbumin, this list not being limiting. Particularly advantageously, the expression in the mammary glands of the rabbit is carried out under the control of the β-casein promoter.

A method for producing a recombinant protein in the milk of a transgenic animal may comprise the following steps: a synthetic DNA molecule comprising a gene coding for human FVII, this gene being under the control of a promoter of a a protein secreted naturally in milk, is integrated into an embryo of a non-human mammal. The embryo is then placed in a female mammal of the same species. Once the mammal from the embryo has grown sufficiently, the lactation of the mammal is induced, then the milk collected. The milk then contains the transgenic FVII of interest.

An example of a process for preparing a transgenic protein in the milk of a mammalian female other than a human is given in EP 0 264 166, the teaching of which may be repeated for the production of the FVII of the invention. .

Another example of a process for preparing protein in the milk of a mammalian female other than humans is given in EP 0 527 063, whose teaching can be repeated for the production of the FVII of the invention.

The composition of FVII produced in the mammary glands of rabbit is characterized in that at least some of the factor VII sialic acids involve a.2-6 linkages.

In a particularly preferred manner, all the sialic acids involve α 2, 6 bonds, and in particular at least 90% of the sialic acids of the FVII involve α 2, 6 bonds. The FVII composition according to the invention can moreover include sialic acids of α2-3 bonds.

Particularly advantageously, the level of sialic acids involving 2-6 bonds is greater than 60%, or greater than 70%, 80%, or 90%. In particular, this rate is between 60% and 90%.

Of the biantennary, monosialylated glycan forms of the FVII composition expressed by the rabbit, the majority glycan forms are non-fucosylated. Advantageously, these biantennary glycan forms, monosialylated and non-fucosylated, are present in the FVII of this composition at a rate greater than 20%. Advantageously, this level is greater than 25%, or even greater than 40%.

In one embodiment of the invention, the fucosylation rate of the FVII of this composition of the invention is between 20% and 50%. In another embodiment of the invention, this rate may be less than 15%.

Transgenic rabbit FVII has several post-translational modifications: the first nine or ten N-terminal glutamic acids are γ carboxylated, Asp63 (Asparagine63) is partially hydroxylated, Sers2 (Serine 52) and Ser ₆₀ (Serine 60) are O-glycosylated and carry respectively the units Glucose (Xylose) 0-2 and Fucose, Asni ₄₅ and Asn ₃₂ 2 are N- predominantly glycosylated by monosialylated biantennary complex glycan forms.

Such a composition of FVII produced by the mammary glands of rabbit is described in the document FR 06 04872, the content of which is incorporated in the present teaching.

FVII produced in milk by transgenic mammals can be purified from milk by techniques known to those skilled in the art.

For example, a method of purifying a protein of interest from milk as described in US Pat. No. 6,268,487 may include the following steps: a) subjecting the milk to tangential filtration on a porosity membrane sufficient to form a retentate and a permeate, the permeate containing the exogenous protein, b) subject the permeate to a chromatographic capture apparatus so as to displace the exogenous protein and obtain an effluent, c) combine the effluent and the retentate, d) Repeat steps a) through c) until FVII is separated from lipids, casein micelles, and the FVII is at least 75% recovered.

Another technique for purifying FVII produced in the milk of a transgenic mammal is described in the patent application FR 06 04864 filed by the Applicant, the content of which is incorporated by reference. This process for extracting and purifying FVII (Method A), contained in milk of a transgenic animal, comprises the following steps: a) extracting FVII from milk, Factor VII being salt-bound and and / or organic and / or inorganic complexes of calcium of said milk, by the precipitation of calcium compounds obtained by adding a soluble salt to the milk, of which the anion is chosen for its ability to form said insoluble calcium compounds to thereby release factor VII from said salts and / or complexes, factor VII being present in a liquid phase, b) separating the enriched liquid phase into the protein of the precipitate of calcium compounds, said liquid phase being further separated into a lipid phase and a non-lipidic aqueous phase comprising the protein, c) subjecting the non-lipidic aqueous phase to an affinity chromotography step, using an elution buffer based on a phosphate salt at a predetermined concentration, and d) subjecting the factor VII eluate, obtained according to step c), to two or three chromatography steps on low base type anion exchange columns. using appropriate buffers for successive elutions of the factor VII retained on said columns. Indeed, the Applicant has surprisingly found that FVII, even under the control of a promoter of a protein naturally produced in whey, such as the WAP promoter or the β-casein promoter, is nevertheless likely to be associated with the calcium ions of milk, and thus with casein micelles.

Another technique for purifying FVII produced in the milk of a transgenic mammal is described in patent application FR 06 11536 filed by the Applicant, the content of which is incorporated by reference. This extraction and purification process FVII contained in milk of a transgenic animal (Method B), comprising the following steps of: a) Creaming and delipidation of said milk, b) Passage of the delipidated and skimmed fraction containing said protein on a chromatographic medium on which is grafted a ligand having both a hydrophobic character and an ionic character, under pH conditions allowing said protein to be retained on said support, c) Elution of the protein, d) Purification of the eluted fraction by elimination of the proteins of the milk of said fraction eluted, and e) recovering said protein.

When the FVII composition is produced by a transgenic rabbit, it is subjected to in vitro sialylation so that the biantennal, bisialylated forms become the majority.

In a particular embodiment of the invention, the sialylation is carried out by means of a sialyltransferase, for example α2,6- (N) -sialyltransferase (or β-D-Galactosyl-β1,4-N-acetyl). - β-D-glucosamine-α2, 6-sialyltransferase), or the GaI beta 1,3GaINAc alpha 2,3-sialyltransferase, or the GaI beta 1,3 (4) GIcNAc alpha 2,3 sialyltransferase, or GaINAc alpha-2 , 6-sialyltransferase I, these enzymes being commercially available. Preferably, the sialyltransferase used is a sialyltransferase for transferring sialic acids via an α2,6 bond. Indeed, it is advantageous that the FVII composition of the invention has sialic acids involving "2-6" linkages, as this isomer is more represented in the plasma FVII. Sialylation can be carried out with a sialic acid donor substrate, such as sialic acid itself, or any molecule that has sialic acid and is capable of releasing sialic acid.

According to one embodiment of the invention, if the enzyme is α2, 6- (N) -sialyltransferase, the substrate is cytidine-5'-monophospho-N-acetylneuraminic acid, in a reaction medium suitable for allowing the transfer of the sialic acid from the sialic acid donor group to the FVII, the biantennary, bisialylated forms becoming the majority. This reaction medium may, for example, be based on a buffer composed of 3-morpholino propanesulfone acid, and a buffer, for example, based on Tween.

According to another embodiment of the invention, the substrate can be synthesized in the reaction medium by including in this medium a cytidine monophosphate (CMP) -sialic acid synthetase, sialic acid, CTP (cytidine triphosphate) and a sufficient level of a divalent metal cation to allow the reaction to occur. By way of example, the divalent metal cation may be calcium ion, zinc ion, magnesium ion, chromium ion, copper ion, iron ion or cobalt ion.

Whatever the embodiment of the sialylation of the FVII composition, the reaction is always carried out for a sufficient time and suitable conditions to allow the increase of the bisialylated forms sufficiently to become the majority. As an indication, the reaction may be carried out for at least 0.5 hours, particularly advantageously for at least 5 hours, or for 7 hours, or for 8 hours, 9 hours or 10 hours. Preferably, the incubation takes place during any a night. In particular, this reaction is carried out for periods of between 5 and 12 hours.

Advantageously, the FVII of the composition of the invention is activated (FVIIa). As such, the FVIIa can exhibit a coagulant activity 25 to 100 times greater than that of the FVII

(not activated) when the latter interacts in place of the first with the tissue factor (FT).

Activation of FVII results in vivo cleavage of the zymogen by different proteases (FIXa, FXa, FVIIa) into two chains joined by a disulfide bridge. FVIIa alone has very little enzymatic activity, but complexed with its cofactor, tissue factor (FT), it triggers the coagulation process by activating FX and FIX. FVIIa is the coagulation factor responsible for hemostasis in hemophiliacs with circulating antibodies, for example. In a particularly advantageous manner, the FVII of the invention is fully activated. Advantageously, the FVIIa of the invention comprises several post-translational modifications: the first nine or ten N-terminal glutamic acids are γ -carboxylated, the Asp63 is partially hydroxylated, the Ser ₅ 2 and the Sergo are 0-glycosylated and carry respectively Glucose (Xylose) 0-2 and Fucose, the Asni45 and Asn ₃₂₂ are N-glycosylated mainly by complex biantennary, bisialylated and non fucosylated.

The activation of FVII may also result from a process carried out in vitro, for example during the purification of the FVII of the invention (see Example 2).

The FVIIa of the invention is therefore composed of a light chain of 152 amino acids with a molecular weight of approximately 20 kDa and a heavy chain of 254 amino acids with a molecular weight of approximately 30 kDa linked together by a single molecule. disulfide bridge (Cysi35-Cys ₂ 62) - Thus, the FVII of the invention is an activated FVII having an activity and a structure close to the plasma FVII.

FVIIa has a coagulant activity 25 to 100 times greater than that of FVII when interacting with tissue factor (FT).

In one embodiment of the invention, FVII can be activated in vitro by factors Xa, VIIa, Ha, IXa and XIIa. The FVII of the invention may also be activated during its purification process.

Another object of the invention is an FVII composition of the invention for use as a medicament.

Another object of the invention is the use of a factor VII composition according to the invention, for the preparation of a medicament for the treatment of patients with hemophilia.

Another object of the invention is the use of a factor VII composition according to the invention for the preparation of a medicament for the treatment of multiple hemorrhagic traumas.

Another subject of the invention is the use of a factor VII composition according to the invention for the preparation of a medicament for the treatment of hemorrhages due to an overdose of anticoagulants.

Another subject of the invention is a pharmaceutical composition comprising factor VII according to the invention and a pharmaceutically acceptable excipient and / or vehicle. Another subject of the invention is a method for preparing a recombinant or transgenic factor VII composition, each factor VII molecule of which comprises glycan forms linked to N-glycosylation sites and in which, among all the molecules, of factor VII of said composition, the biantennary, bisialylated glycan forms are in the majority, comprising a sialylation step by contacting a partially sialylated transgenic or recombinant factor VII composition as defined above with a sialic acid donor substrate and a sialyltrasferase, in a reaction medium suitable to allow the activity of the sialyltransferase, for a sufficient time and under suitable conditions to allow a transfer of the sialic acid from the sialic acid donor substrate to FVII and an increase of the bisialylated forms of sufficiently, said bisialylated forms thus becoming majo ritaires. The conditions for carrying out the reaction are illustrated above, as well as in the examples.

The term "partially sialylated" means a composition of FVII whose N-bonded glycan forms are not all bisialylated, that is to say of which some forms are monosialylated. Advantageously, these biantenned, monosialylated forms are present at a level greater than 40%, particularly advantageously at a level greater than 50%, or even at 60%. Advantageously, the level of biantenned, monosialylated and non-fucosylated glycan forms is greater than 20%, or particularly preferably greater than 30%, 40% or even 50%.

Advantageously, the sialyltransferase is α2,6- (N) -sialyltransferase (or β-D-Galactosyl-β1,4-N-acetyl-β-D-glucosamine-α2,6-sialyltransferase), or GaI beta 1 , 3GaINAc alpha 2, 3-sialyltransferase, or the GaI beta 1,3 (4) GIcNAc alpha 2,3 sialyltransferase, or GaINAc alpha-2,6-sialyltransferase I.

Preferably, the sialyltransferase used is a sialyltransferase which makes it possible to transfer sialic acids via an oc2, 6 bond. In fact, it is advantageous for the FVII of the composition of the invention to have sialic acids involving α2-6 bonds, because this isomer is more represented in the plasma FVII. Sialylation can be performed with any sialic acid donor substrate.

According to one embodiment, if the enzyme is α2, 6- (N) -sialyltransferase, the substrate is cytidine-5'-monophospho-N-acetylneuraminic acid, in a suitable reaction medium to allow the transfer of the sialic acid of the sialic acid donor group to FVII, the biantennary, bisialylated forms becoming the majority.

The reaction medium may be based on a biologically compatible surfactant, such as

Tween®80 or Triton®X-100 or a mixture of both at a concentration of 0.01% to 0.2%, or a divalent metal cation, such as Ca ²⁺ , Mn ²⁺ , Mg cations ²⁺ or Co ²⁺ , Ca ²⁺ being preferred, at a concentration of between 5mM and 10mM. This reaction medium may further comprise agents for adjusting the ionic strength and / or contributing to the maintenance of the pH of the medium, such as sodium cacodylate, 3-morpholinopropanesulphonic acid, Tris and NaCl at varying concentrations. from 40 mM to 60 mM. PH values are typically between 6 and 7.5. Finally, the reaction medium may also comprise bovine serum albumin (BSA) at a concentration of between 0.05 and 0.15 mg / ml. According to another embodiment, the substrate can be synthesized in the reaction medium by placing in this medium an acidic CMP-sialic synthetase, sialic acid, CTP (cytidine triphosphate) and a sufficient level of a divalent metal cation, examples of which are given above. Whatever the embodiment of the sialylation of the FVII composition, the reaction is always carried out for a sufficient time and appropriate conditions to allow the increase of the bisialylated forms sufficiently so that they become the majority, as defined more high .

When the process uses an immobilized enzyme, the duration of the reaction is preferably between 0.5 and 3 hours at a temperature advantageously between 4 and 37 ^° C., preferably between ^{40 °} C. and 20 ^° C.

When the process involves a batch reaction, the duration of the reaction is preferably between 1 hour and 9 hours, preferably between 1 hour and 6 hours, at a temperature advantageously between 4 and 37 ⁰ C, preferably between ^{40 °} C. and 20 ^° C.

Preferably, the method of the invention is a method for improving the bioavailability of the partially sialylated transgenic or recombinant factor VII composition. This improvement in biodisposibility is achieved by contacting said composition with a sialic acid donor substrate and a sialyltrasferase, as mentioned above.

By "bioavailability enhancement" is meant an increase of at least 5%, or at least 10%, or preferably at least 30% or 50%, and preferably at least 80% or more. 90% of the bioavailability of the FVII composition compared to the same composition of FVII whose sialylation was not modified. In another particular embodiment, a galactosylation step is carried out before the sialylation step. The purpose of this step is to graft galactose onto galactose deficient forms, i.e. the agalactosylated and monogalactosylated forms of FVII. Galactose binds to the GIcNAc, and may be able to bind a sialic acid residue in the subsequent sialylation step. This galactosylation step can be carried out using a galactosyltransferase, in a reaction medium including UDP-galuridine (5'-diphosphogalactose), known to those skilled in the art.

Advantageously, the major glycan forms of the partially sialylated FVII composition are of the complex, biantenned, monosialylated type.

Such glycan forms are represented below:

To: Sialic acid

galactose

: N-acetylglucosamine (GIcNAc) ^• .Mannose

fucose

In a particular embodiment of the invention, the partially sialylated FVII composition also comprises non-sialylated (fucosylated or non-fucosylated), non-sialylated triantenes, and bisialylated (fucosylated or non-fucosylated) non-sialylated complex forms.

Advantageously, among the monosialylated biantennary glycannic forms of said partially sialylated FVII composition, the predominant glycannic forms are non-fucosylated.

Advantageously, the partially sialylated FVII composition has at least some of the sialic acids involving α2-6 bonds, as indicated above. Preferably, the method further comprises, prior to the sialylation step, a step of producing the transgenic FVII composition partially sialylated by transgenic rabbits. This step is implemented as indicated above. This step can also be carried out before the galactosylation step.

Advantageously, the FVII of the partially sialylated FVII composition is activated.

The method of the invention makes it possible to obtain, among all the factor VII molecules of said composition, a rate of biantennary, bisialylated major forms.

Advantageously, the sialic acid donor group is cytidine-5'-monophospho-N-acetylneuraminic acid and the sialyltransferase is α 2, 6- (N) -sialyltransferase. Such a partially sialylated FVII composition may be a transgenic FVII composition produced in the mammary glands of a transgenic rabbit. In a particularly advantageous manner, the partially sialylated FVII composition is the composition described in document FR 06 04872, the content of which is considered to be included in the present document.

Other aspects and advantages of the invention will be described in the examples which follow, which should be considered as illustrative and do not limit the scope of the invention.

abbreviations

FVII-Tg = FVIIa-Tg: Activated transgenic FVII according to

The invention

FVII-r = FVIIa-r: commercially available recombinant activated FVII

FVII-p = FVIIa-p: FVII of activated plasma origin, i.e., purified from human plasma.

BIALDI-TOF: Matrix Assisted Laser Desorption Ionization - Time of Flight

HPCE-LIF: High Performance Capillary Electrophoresis-

Laser Induced Fluorescence (High Performance Capillary Electrophoresis-Laser Induced Fluorescence)

MS-ESI: Electrospray mass ionization spectrometry.

LC-ESIMS: Liquid Chromatography - Electrospray Mass Ionization Spectrometry

NP-HPLC: High Performance Liquid Chromatography

PNGase F Normal Phase: Peptide: N-glycosidase F

LC-MS: Liquid Chromatography-Mass Spectrometry Description, figures

Figure 1: Extraction and purification of the FVII composition obtained in Example 1.

Figure 2: Deconvolved ESI mass spectra of peptides carrying N-glycosylation sites.

Figure 3: HPCE-LIF electropherograms after deglycoεylation of FVII by PNGase F;

Legend: top electropherogram: FVIIa, p; the two electropherograms of the medium: FVII-Tg; lower electropherogram: FVIIa, r.

Figure 4: Characterization of FVII by NP-HPLC;

Legend: top chromatogram: FVIIa, p; middle chromatogram: FVII-Tg; bottom chromatogram: FVIIa, r.

Figure 5: Identification of the major glycan forms of FVII-Tg by MALDI-TOFMS.

Figure 6: Identification of the major glycan forms of FVIIa, r by MALDI-TOFMS.

Figure 7: HPCE-LIF analyzes of in vitro resialylation: (low) oligosaccharide map of native FVII-Tg; (top) oligosaccharide map of FVII-Tg after resialylation.

Figure 8: Sialylation kinetics of FVII-Tg according to the percentage of biantennary, bisialylated, non-fucosylated (A2) and fucosylated (A2F) forms over time. Figure 9: Results of the comparative PK (PK: pharmacokinetics) comparative study in rabbits, non-resialylated transgenic FVII (FVIITgNRS) compared with Resialylated transgenic FVII (FVIITgRS): semi-log elimination curves.

Examples

Example 1 Production of transgenic rabbits producing a human FVII protein in their milk

A pi plasmid is first prepared by introducing the WAP gene sequence (described in Devinoy et al, Nucleic Acids Research, Vol 16, No. 16, Aug. 25, 1988, p 8180) into the vector polylinker. p-poly III-I (described in Lathe et al., Gene (1987) 57, 193-201).

Plasmid p2, obtained from plasmid p1, contains the rabbit WAP gene promoter and the human FVII gene.

Transgenic rabbits were obtained by the conventional microinjection technique (Brinster et al, Proc Natl Acad Sci USA (1985) 82, 4438-4442). 1-2 μl containing 500 copies of the gene were injected into the male pronucleus of rabbit embryos. Fragments of this vector containing the recombinant genes were microinjected. The embryos were then transferred to the oviduct of hormonally prepared adoptive females. About 10% of the embryos handled gave birth to rabbits and 2-5% of the embryos handled to transgenic rabbits. The presence of the transgenes was revealed by the Southern transfer technique from I ¹ DNA extracted from tails of the rabbits. FVII concentrations in the blood and milk of animals were evaluated using specific radioimmunoassays.

The biological activity of FVII was evaluated by adding milk to the culture medium of rabbit breast cells or explants.

EXAMPLE 2 Extraction and Purification of the FVII Obtained a) Extraction of FVII

500 ml of raw non skimmed milk are considered which are diluted with 9 volumes of 0.25 M sodium phosphate buffer, pH 8.2. After stirring for 30 minutes at room temperature, the aqueous phase enriched with FVII is centrifuged at 10,000 g for 1 hour at 15 ^° C. (Sorvall Evolution RC centrifuge - 6700 rpm - SLC-6000 rotor). 6 pots of about 835 ml are needed.

After centrifugation, three phases are present: a surface lipid phase (cream), a clear non-lipidic aqueous phase enriched in FVII (majority phase) and a solid white phase in pellet (precipitates of insoluble caseins and calcium compounds).

The non-lipidic aqueous phase FVII is collected at the peristaltic pump until the creamy phase. The creamy phase is collected separately. The solid phase (precipitate) is removed.

The non-lipidic aqueous phase, however still comprising very small amounts of lipids, is filtered through a sequence of filters (PaIl SLK7002U010ZP - 1 μm pore size glass fiber pre-filter - then SLK7002NXP - pore size nylon 66). 0.45 μm). At the end of the filtration, the lipid phase is passed on this filtration sequence which completely retains the lipid globules of the milk, and the filtrate is clear. The filtered non-lipidic aqueous phase is then dialyzed on an ultrafiltration membrane (Millipore Biomax 50 kDa - 0.1 m ² ) to make it compatible with the chromatography phase. The molecular weight FVII of about 50 kDa does not filter through the membrane, unlike the milk salts, sugars and peptides. At first, the solution

(about 5,000 ml) is concentrated to 500 ml and then Ultrafiltration dialysis, while maintaining the constant volume, eliminates the electrolytes and conditions the biological material for the chromatography step. The dialysis buffer is a 0.025M sodium phosphate buffer, pH 8.2.

This non-lipidic aqueous phase comprising FVII can be assimilated to whey enriched with FVII-Tg. This preparation is stored at -30 ^° C. before continuing the process. The overall recovery yield of FVII by this step is very satisfactory: 90% (91% phosphate extraction + 99% Dialysis / concentration).

The non-lipidic aqueous phase comprising FVII at the end of this step is perfectly clear and is compatible with the chromatographic steps which follow.

About 93,000 IU of FVII-Tg are extracted at this stage. The purity in FVII of this preparation is of the order of 0.2%.

b) Purification of the FVII

1. Chromatography on hydroxyapatite gel

(affinity chromatography)

An Amicon 90 column (9 cm diameter - 64 cm ² section) is filled with BioRad Ceramic Hydroxyapatite Gel Type I (CHT-I).

The gel is equilibrated in aqueous buffer A consisting of a mixture of 0.025 M sodium phosphate and 0.04 M sodium chloride, pH 8.0. The entire preparation stored at -30 ^° C. is thawed on a water bath at 37 ^° C. until the ice cube is completely dissolved and is then injected onto the gel (linear flow rate 100 cm / h, ie 105 ml / min). The unbound fraction is removed by passing a buffer of 0.025 M sodium and 0.04 M sodium chloride, pH 8.2, up to baseline (RLB).

Elution of the FVII-Tg containing fraction is via buffer B consisting of 0.25 M sodium phosphate and 0.4 M sodium chloride, pH 8.0. The eluted fraction is collected until the return to baseline. The detection of the compounds is ensured by measuring the absorbency at λ = 280 nm.

This chromatography makes it possible to recover more than 90% of the FVII-Tg, while eliminating more than 95% of the lactic proteins. The specific activity (A.S.) is multiplied by 25. About 85,000 IU of FVII-Tg of 4% purity are available at this stage.

2. Tangential filtration ICK) kDa and concentration / dialysis 50 kDa

The entire eluate from the previous step is filtered in tangential mode on a 100 kDa ultrafiltration membrane (PaIl OMEGA SC 100K - 0.1 m ² ). The FVII is filtered through the 100 kDa membrane, whereas the proteins of. Molecular weight greater than 100 kDa are not filterable.

The filtered fraction is then concentrated to about 500 ml and then dialyzed on the 50 kDa ultrafilter already described above. The dialysis buffer is 0.15M sodium chloride.

At this stage of the process, the product is stored at -30 ^° C. before passing through ion exchange chromatography.

This step made it possible to reduce the protein load of molecular weight greater than 100 kDa and in particular proenzymes. The 100 kDa membrane treatment retains about 50% of proteins including high molecular weight proteins, while filtering 95% of FVII-Tg, or 82,000 IU of FVII-Tg. This treatment makes it possible to reduce the risks of proteolytic hydrolysis during the downstream stages.

3. Q-Sepharose® FF Gel Chromatography (step d) - Method A)

These three successive chromatographies on Q-Sepharose® Fast Flow (QSFF) ion exchange gel are carried out to purify the active principle, to allow the activation of FVII in activated FVII (FVIIa) and finally to concentrate and formulate the FVII composition. The detection of the compounds is ensured by measuring the absorbance at λ = 280 nm.

3.1 Step Q-Sepharose® FF 1 - Elution "High Calcium"

A column 2.6 cm in diameter (5.3 cm ² section) is filled with 100 ml of Q-Sepharose® FF gel (GE Healthcare).

The gel is equilibrated in 0.05 M Tris buffer, pH 7.5.

The entire fraction stored at -30 ^° C. is thawed in a water bath at 37 ^° C. until the ice cube is completely dissolved. The fraction is diluted to ¹ A [v / v] with the equilibration buffer before injection on the gel (flow rate 13 ml / min, ie linear flow of 150 cm / h), then the non-retained fraction is eliminated by passage of the buffer until RLB.

A first low FVII protein fraction is eluted at 9 ml / min (ie 100 cm / h) with a buffer of 0.05 M Tris and 0.15 M sodium chloride, pH 7.5, and is then eliminated.

A second protein fraction rich in FVII is eluted at 9 ml / min (ie 100 cm / h) with a buffer of 0.05 M Tris, 0.05 M sodium chloride and 0.05 M calcium chloride, pH -7, 5. This second fraction is dialyzed on

The 50 kDa ultrafilter already described above. The dialysis buffer is sodium chloride 0.15 M. This fraction is stored at +4 ⁰ C overnight before 2 ^th passage chromatography anion exchange.

This step makes it possible to recover 73% of the FVII (ie 60000 IU of FVII-Tg), while eliminating 80% of the accompanying proteins. It also allows the activation of FVII in FVIIa.

3.2 Step Q-Sepharose® FF 2 - elution "Low Calcium"

A 2.5 cm diameter column (4.9 cm ² section) is filled with 30 ml of Q-Sepharose® FF gel (GE Healthcare). The gel is equilibrated in 0.05 M Tris buffer, pH 7.5.

The preceding eluted fraction (second fraction), stored at + ^{40 °} C., is diluted before injection on the gel (flow rate 9 ml / min, ie linear flow rate of 100 cm / h). After injection of the fraction, the gel is washed with the equilibration buffer for removal of the non-retained fraction, up to RLB.

A fraction containing very high purity FVII is eluted at 4.5 ml / min (ie 50 cm / h) in buffer of 0.05 M Tris, 0.05 M sodium chloride and 0.005 M calcium chloride, pH 7.5.

About 23,000 IU of FVII-Tg were purified, or 12 mg of FVII-Tg.

This step eliminates more than 95% of the accompanying proteins (rabbit milk proteins).

This eluate, of greater than 90% purity, has structural and functional characteristics close to the natural molecules of human FVII. It is concentrated and formulated by the third pass in ion exchange chromatography. 3.3 Step Q-Sepharose® FF 3 - elution "Sodium"

A column 2.5 cm in diameter (4.9 cm ² section) is filled with 10 ml of Q-Sepharose® FF gel (GE Healthcare).

The gel is equilibrated in 0.05 M Tris buffer, pH 7.5.

After injection of the fraction, the gel is washed with the equilibration buffer for removal of the non-retained fraction, up to RLB.

The purified eluted fraction of the preceding step is diluted five times with purified water for injection (PPI) before injection on the gel (flow rate 4.5 ml / min, ie linear flow of 50 cm / h).

The FVII-Tg is then eluted at a flow rate of 3 ml / min (ie 36 cm / h) with the buffer of 0.02 M Tris and 0.28 M sodium chloride, pH 7.0. A composition of FVII-Tg in concentrate form was prepared with purity greater than 95%. The product is compatible with intravenous injection. The process has a cumulative yield of 22%, which makes it possible to purify at least 20 mg of FVII per liter of milk used.

Table A summarizes the steps of the method according to a preferred embodiment of the invention to provide the purified FVII composition, and provides the different yields, purity and specific activities obtained at each step.

The FVII-Tg of the composition is then subjected to various structural analyzes, as developed in the examples which follow. Example 3 Characterization of glycosylation sites and glycopeptides by MS-ESI

The N-glycosylation sites of FVII-Tg, FVIIa, p (FVII plasma) and FVIIa, r were identified by

LC-ESIMS (/ MS), confirmed by MALDI-TOFMS, and the relative proportions of the different glycans present at each site were determined by LC-ESIMS.

Figure 2 shows the deconvoluted ESI spectra of the glycopeptides containing the two glycosylated Asn residues. The location of the glycosylation sites was confirmed by MALDI-TOF (/ TOF) as well as by Edman sequencing.

The analysis of the mass spectra of the glycopeptides [D ₁₂₃ -R ₁₅₂ ] and [K _31-6 -R ₃₅₃ ] of FVIIa, p, respectively presenting the Asni ₄₅ and Asn322 / N-glycosylation sites, reveals the presence of a biantennate form, non-fucosylated tdsialylated (A2) (observed mass of glycopeptide containing ASn ₁₄₅ : 5563.8 Da) and fucosylated (A2F) (mass observed for glycopeptide with Asni ₄₅ : 5709.8 Da). ASn _I45 also _shows the presence of trisialylated trifunctional oligosaccharides, non-fucosylated (A3) (obstruction 6220.0 Da) and fucosylated (A3F) (obstruction mass 6366.1 Da).

For the FVIIa, r, the ASn _I45 is modified by A2F, AlF and "AlF" type glycans, which corresponds to a monosialylated form with a GaINAc in the terminal position on the other antenna. The presence of A3F glycols (triantennate, trisialylated, fucosylated forms) is noted.

The FVII-Tg, the analysis of mass spectra of glycopeptides [D123-R152] and [K ₃ L ₆ -R _353] FVII-Tg, respectively having the N-glycosylation sites Asn ₃₂₂ Asni4 ₅ and / reveals the presence of biantennary, non-fucosylated bisialylated forms (A2) (observed mass of glycopeptide containing ASn ₁₄₅ : 5563.8 Da) and fucosylated (A2F) (observed mass: 5709.7 Da). We also note the majority presence of oligosaccharides, located on Asni ₄₅ , biantennés, monosialylés non fucosylés (Al) (observed mass: 5272,3 Da) and fucosylés (AlF) (observed mass: 5418,7 Da). Triantennial forms are poorly represented. It should be noted that no monosialylated form with a GaINAc in the terminal position on the other antenna is present. Concerning the majority glycoforms of Asn ₃₂ 2, the same glycan structures are observed in different proportions. Figure 1 reveals the presence of less mature forms (less antennae and sialylated) than on the Asni ₄₅ . For example, the triantennial forms are less represented on Asn ₃₂ 2 than on ASn _I45 for the plasma product and absent on FVIIa, r and FVII-Tg. It should also be noted that Asn 145 and 322 are 100% glycosylated. Although only semi-quantitative, these results are in agreement with the quantitative data obtained by HPCE-LIF and NP-HPLC.

Example 4 Quantification of N-glycans by HPCE-LIF

The identification and quantification of the N-linked oligosaccharides is carried out by HPCE-LIF after deglycosylation with PNGase F. The FVII samples are treated with exoglycosidases (sialidase)

(ENZYME / SUBSTRATE 1 mIU / 10 μg ratio), galactosidase, hexnacase (Prozyme kit), fucosidase (I / O ratio: 1 mIU / 10 μg) to ensure the identification and quantification of each structure isolated. The glycols obtained are labeled with a fluorochrome and separated according to their mass and charge. Two standards (homopolymers of glucose, oligosaccharides) make it possible to identify the structures. The quantification is done by the integration of each peak reduced, in percentage, to the set of quantized oligosaccharides. ProteomeLab PA800 capillary electrophoresis apparatus (Beckman Coulter) is used, the capillary of which is "coated" N-CHO (Beckman-Coulter) 50 cm × 50 μm internal diameter. A "buffer-N gel" separation buffer (Beckman-Coulter) is used. The migration is carried out by applying a voltage of 25 kV, for 20 min, at 20 ^° C. The detection takes place by laser at λ _ex quotation 488nm and λ _em ission 520 πm.

The fucosylation rate is calculated, after simultaneous deglycosylation by sialidase, galactosidase and hexacase, by the ratio between the peak areas corresponding to the "core" and "core" fucosylated.

The glycols of FVIIa, p are mainly of the biantennés type, bisialylés non fucosylés (A2), and biantennés bisialylés fucosylés (A2F). The glycan profiles of FVII-Tg reveal the presence of biantennary, monosialylated, fucosylated or non-fucosylated (AlF, Al), and biantennal, bisialylated, fucosylated or non-fucosylated forms (A2F, A2). The distribution between these different forms varies between the two lots.

FVJIa, r shows biantennial sialylated fucosylated forms with predominant A2F forms, and fucosylated monosialylated biantennates (AlF). Atypical migration times for the A2F and AlF forms are noted with respect to the migration times usually encountered for these structures.

The glycan profiles of two lots (A and B) of FVII-Tg (cf figure 3-the two electropherograms of the medium) reveal the presence of biantennous, monosialylated, fucosylated or non-fucosylated (AlF, Al), and biantenned, bisialylated, fucosylated forms. or not (A2F, A2).

Table 1. Summary of the percentage of sialylated forms from native samples of different batches of FVII.

The quantitative analysis of the different glycan forms (Table 1) shows, for FVIIa, p, the predominance of sialylated forms with 51% of bisialylated glycans (A2 and A2F), and 30% of non-fucosylated and fucosylated sialylated triantennial forms (respectively G3 and G3F) (results not shown). FVII-Tg (lots A and B) is less sialylated than FVIIa, p with 35% bisialylated biantennal forms, and only 6% sialylated triantennial forms (results not shown). The majority forms are monosialylated with 50% Al and AlF structures. FVIIa, r is also less sialylated than FVIIa, p with 45% A2F structures, and only 6% sialylated tri-annealed glycans (results not shown). For the FVIIa, we note the absence of non-fucosylated forms.

Table 2. Fucosylation rate of different FVII

FVIIa, p FVII-Tg FVII-Tg FVIIa, r Lot A f * Lot B

Rate of 16. 23. 6 41. 8 100 fucosylation (%) The results show the low fucosylation rate of FVIIa, p (16%), fucosylation rate 24 to 42% for FVII-Tg and 100% fucosylation for FVIIa, r.

EXAMPLE 5 Quantification of N-glycans by NP-HPLC The qualitative and quantitative analysis of the N-glycosylation of FVIIa, p, FVIIa, r and FVII-Tg was studied by NP-HPLC (see FIG. After desalting and drying of the protein, it is denatured and reduced according to implementations known to those skilled in the art. The glycans are then released enzymatically (endoglycosidase PNGase F), and purified by ethanol precipitation. The glycans thus obtained are labeled with a fluorophore, 2-aminobenzamide (2-AB). The labeled glycans are separated according to their hydrophilicity by HPLC chromatography in normal phase on an Amide-80 column, 4.6 x 250 mm (Tosohaas) thermostated at 30 ^° C. Before injection of the sample, the column is equilibrated in buffer at 80% acetonitrile. The oligosaccharides are eluted by a growing gradient of 50 mM ammonium formate, pH 4.45, for times greater than or equal to 140 minutes. Detection is performed by fluorometry λ _exc i _tat i _on to 330 nm and λemission SL 420nitl. The chromatographic profile of FVIIa, p shows that the majority of molecules are biantennés, bisialylés (A2) with a rate of 39%. Bainenic, bisialylated, fucosylated (A2F), monosialylated (Al) and trisialylated fucosylated and nonfucosylated (A3F and A3) forms are also observed in smaller amounts.

The analysis by NP-HPLC carried out on FVII-Tg confirms the presence of oligosaccharides predominantly Al type, at a rate of 27%. The AlF, A2 and A2F forms are less represented and the triantenned forms are present in the trace state. This highlights a sialylation difference between FVIIa, p and factor FVII-Tg (lot B), less sialylated.

The same analysis carried out on a factor FVIIa, r reveals majority forms of type A2F present at a rate of 30%. AlF forms are less represented and triantennial forms are present as traces. The analysis of FVIIa, r also shows a significant shift in the retention time of AlF and A2F forms suggesting different forms from those present in FVIIa, p and in FVII-Tg.

All these results are consistent with those obtained in HPCE-LIF.

Example 6 Identification with MALDI-TQFMS MALDI-TOF MS (Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) mass spectrometry is a technique for measuring the molecular mass of peptides, proteins, glycans, oligonucleotides, and the majority of ionizable polymers with high accuracy.

The peptides, proteins and glycans to be analyzed are mixed with a matrix that absorbs at the wavelength of the laser used. The main matrices are α-cyano-4-hydroxycinnamic acid (HCCA) for peptide analysis, sinapinic acid (SA) for proteins and 2,5-dihydroxybenzoic acid (DHB) for oligosaccharides.

The method consists in irradiating the matrix / analyte co-crystals with the aid of a pulsed laser, this entails the joint desorption of the matrix and analyte molecules. After ionization in the gas phase, the analyte molecules pass through a time-of-flight detector. As the masses and flight times are directly related, the measurement of the latter allows the determination of the mass of the target analyte. The identification is carried out by measuring the mass observed, compared with the theoretical mass. The Sequencing can be performed in the MS / MS mode on the basis of the fragment ions obtained. The instrument used is a Bruker Autoflex 2 operating in the TOF and TOF / TOF modes. In order to identify the glycan forms present in FVII-Tg and FVIIa, r, MALDI-TOF MS analyzes were performed from elution fractions derived from preparative NP-HPLC.

The MALDI-TOF analysis of FVII-Tg confirmed the identification of NP-HPLC-separated glycans, namely predominantly monosialyl Al forms and AlF, A2F and A2 minority forms.

This study also made it possible to identify the triantennial bisialylated and trisialylated forms, hybrid forms and oligomannoses of the Man5 and Man6-P-HexNAc type (see Figure 5).

MALDI-TOF MS analysis performed on FVIIa, r revealed the presence of the glycan forms shown in Figure 6. FVIIa factor, r is almost completely fucosylated, unlike FVII-Tg which is only partially. We note that the. The predominant glycan form is A2F with a quantified level in NP-HPLC at 30%. Biantenned, monosialylated, fucosylated (AlF) forms and having a GaINAc in the terminal position on the other antenna are identified as well as fucosylated biantennary neutral forms with Hex-NAc-HexNAc motifs on one and / or two antennas. There is also the presence of triantennate, trisialylated and fucosylated type glycans. Non-fucosylated forms are present in the trace state.

Example 7 HPCE-LIF Analysis of the Binding of Sialic Acids - Galactose

As regards the study of the sialic-galactose acid bond ("branching"), the experimental procedure is similar to that developed in Example 4. After deglycosylation with PNGaseF, the oligosaccharides are treated with specific exosialidases so as to ensure the identification of the binding and the quantification of each isolated structure. The sialidases used are recombinant enzymes derived from S. pneumoniae (specific for the "2-3, 0.02 IU, E / S = 0.4 m / m) binding, C. perfringens (specific for α2-3 and α2-6 bonds, 0.04 IU, I / O = 0.1 m / m) and A. urefaciens (hydrolyzing the α2-3, α2-6, α2-8 and "2-9, 0.01 IU, E / S = 0.05 m / m linkages). Assays have shown that FVIIa, r has biantennary, sialylated, fucosylated forms with predominant A2F forms, and biantenned, monosialylated, fucosylated (AlF) forms. Atypical migration times for these A2F and AlF structures are noted with respect to the migration times usually encountered for these forms. In particular, these sialylated oligosaccharide forms have atypical migration times in HPCE-LIF and NP-HPLC compared to those of FVII-Tg. On the other hand, the analysis of the monosaccharide composition did not reveal any particular sialic acid other than Neu5Ac and the mass spectrometry tools reveal mass molecules in accordance with bisialylated types. Finally, the desialylation of the FVIIa, r glycan allows to recover chromatographic and electrophoretic behaviors equivalent to those of the oligosaccharides of FVII-Tg.

These differences in electrophoretic and chromatographic behavior can therefore be explained by a different branching of sialic acids. This hypothesis has been evaluated by the different approaches of HPCE-LIF and MS.

The results are summarized in Table 3 below. Table 3: Sialic acid connections on the different lots of FVII.

The results show distinct isomers at the sialic acid level between the two FVIIs. Indeed, the sialic acids of FVIIa, r involve α2-3 bonds while FVII-Tg has α2-6 connections.

The differences in HPCE-LIF and NP-HPLC behaviors observed for FVIIa, r compared to FVII-Tg glycols are related to these differences in sialic acid isomerism.

Example 8 Resialylation in vitro of FVII-Tg

The literature (Zhang X. et al, Biochim Biophys Acta 1998, 1425, 441-52) mentions that more complete sialylation of a glycoprotein contributes to better stabilities in vitro and in vivo. The objective of this study is therefore to demonstrate the feasibility of in vitro sialylation.

Resialylation was performed using an α2, 6- (N) -sialyltransferase (rat, Spodotera frugiperda,

AS> 1 unit / mg, 41 kDa, Calbiochem) and cytidine-5'-monophospho-N-acetylneuraminic acid substrate

(Calbiochem). These two reagents are stored at -80 ^° C. because of their instability. The sialylation substrate

(cytidine-5'-monophospho-N-acetylneuraminic acid) and the enzyme (α2, 6- (N) -sialyltransferase) are mixed in the reaction buffer overnight at 37 ^° C. The reaction buffer used contains 50 mM acid morpholino-3 propanesulfonic acid, 0.1% Tween®80, 0.1 mg / ml BSA, adjusted to pH 7.4 (Sigma reagents).

Table 4 below summarizes the experimental conditions.

Table 4: Summary of experimental conditions

The electropherogram of native FVII-Tg, as obtained after the purification of Example 2 (FIG. 7, bottom profile), shows the predominantly Al monosialylated biantenate form (42%) and the A2, A2F and AlF minus structures. represented. After resialylation (FIG. 7, top profile), the monosialylated form represents only 6% in favor of the bisialylated form, in particular not fucosylated, which has become a majority (52%).

Quantification of the glycans before and after resialylation is shown in Table 5 below. Table 5: Quantification of oligosaccharide structures before and after sialylation

The kinetics of the sialylation of transgenic FVII is shown in Figure 8.

This study shows the effectiveness of in vitro resialylation with a rate of bisialylated forms increased by more than 100%.

Example 9 Comparative pharmacokinetic study in the rabbit of a non-resialylated transgenic FVII (FVII Tg NRS) relative to the resialylated transgenic FVII (FVII Tg RS), obtained at the end of Example 8)

The purpose of this study is to compare the pharmacokinetic profiles of FVII-TgRS versus FVII-TgNRS in the New Zealand Vigilant Male Rabbit.

The dose tested is 200 μg / kg per animal, which corresponds to twice the therapeutic dose in humans described for recombinant FVII.

The blood samples are taken on D-4 (4 days before the injection of the product) and on D1 (day of the injection of the product) at T0.17h (the day of the injection of the product, 10 minutes after the injection). injection), T0.33h (the day of injection of the product, 20 minutes after the injection), TIh (the day of the injection of the product, 1 hour after the injection), T3h (the day of the injection) injection of the product, 3 hours after the injection), T6h (the day of the injection of the product, 6 hours after the injection), T8h (the day of the injection of the product, 8 hours after the injection).

FVII: Ag (FVII antigen) assays are performed by ELISA (Asserachrom kit). The results of the FVII: Ag assays in rabbit plasma make it possible to determine, on the one hand, the elimination profiles and, on the other hand, the pharmacokinetic parameters. The dosages and experimental groups are shown in Table 6.

The elimination curves are shown in Figure 9.

The results are retranscribed in Table 7.

At the doses administered, the elimination half-life, the mean time of presence (MRT), the maximum concentration (Cmax) and the recovery rate ("recovery") are comparable in the 2 experimental groups.

FVII-TgRS has a different kinetic profile than FVII-TgNRS. Resialylation of FVII-Tg markedly improves half-life, mean residence time (MRT), Cmax, and recovery.

A difference is observed in AUC (peak area), Cl (clearance) and volume of distribution (Vd) parameters (This volume is obtained by dividing the administered or absorbed dose by plasma concentration) suggesting elimination of FVII-TgRS less important blood circulation.

Resialylation of FVII-Tg induces an increase in product bioavailability of approximately 30%. Table A

'

Yield: Yield

Claims

claims

1. Composition of recombinant or transgenic factor VII, each factor VII molecule of the composition comprising glycan forms linked to the N-glycosylation sites, characterized in that, among all the factor VII molecules of said composition, the glycan forms biantennées , Bisialylated and non-fucosylated are the majority with respect to all glycan forms linked to the N-glycosylation sites of factor VII of the composition.

2. Composition according to claim 1, characterized in that the rate of biantennal, bisialylated fucosylated and non-fucosylated forms is greater than 50%.

3. Composition according to claim 1 or 2, characterized in that, among all the factor VII molecules of said composition, the fucose content is between 20% and 50%.

4. Composition according to any one of claims 1 to 3, characterized in that at least some of the sialic acid factor VII of said composition involve α2-β bonds.

5. Composition according to claim 4, characterized in that all the sialic acids of the factor VII of said composition involve α2-6 bonds.

6. Composition according to claim 4, characterized in that further sialic acids of FVII of said composition involve bonds "2-3.

7. Composition according to any one of claims 1 to 6, characterized in that said FVII is activated.

8. Composition according to any one of claims 1 to 7 for its use as a medicament.

9. Use of a factor VII composition according to any one of claims 1 to 7 for the preparation of a medicament for the treatment of patients with hemophilia.

10. Use of a factor VII composition according to any one of claims 1 to 7 for the preparation of a medicament for the treatment of multiple bleeding disorders.

11. Use of an FVII composition according to any one of claims 1 to 7, for the preparation of a medicament for the treatment of hemorrhages due to an anticoagulant overdose.

A pharmaceutical composition comprising an FVII as defined in any one of claims 1 to 7, and a pharmaceutically acceptable excipient and / or carrier.

A process for the preparation of a recombinant or transgene factor VII composition, wherein each factor VII molecule of the composition comprises glycan forms linked to the N-glycosylation sites and in which out of all the factor VII molecules of said composition, the biantennary, bisialylated glycan forms are predominant, comprising a step of sialylation by bringing into contact a transgenic or recombinant factor VII composition partially sialylated with a sialic acid-donor substrate and a sialyltransferase, in a reaction medium suitable for allowing the activity of the sialyltransferase, for a sufficient time and under suitable conditions to allow transfer of the sialic acid from the donor substrate. FVII sialic acid and an increase of bisialylated forms sufficiently so that said bisialylated forms become the majority.

The method of claim 13, wherein prior to the sialylation step, a galactosylation step is performed to graft galactose onto galactose deficient forms representing the agalactosylated and monogalactosylated forms of FVII.

15. Method according to one of claims 13 and 14, characterized in that said partially sialylated FVII composition has predominantly monosialylated biantennary glycan forms.

16. The method according to claim 15, characterized in that, among the monosialylated biantennary glycan forms of said partially sialylated FVII composition, the majority glycan forms are non-fucosylated.

17. A method according to any one of claims 13 to 16, characterized in that said partially sialylated FVII composition has at least some of the sialic acids involving α2-6 bonds.

18. A method according to any one of claims 13 to 17, characterized in that it further comprises, prior to the sialylation step, a step of producing the transgenic FVII composition partially sialylated by transgenic rabbits.

19. The method according to any one of claims 13 to 18, characterized in that the FVII of said partially sialylated FVII composition is activated.

20. Process according to any one of claims 13 to 19, characterized in that said sialyltransferase is 1'a2, 6- (N) -sialyltransférase, and in that said sialic acid donor group is cytidine- 5'-monophospho-N-acetylneuraminic.