WO2017025566A1

WO2017025566A1 - Improved recombinant factor vii

Info

Publication number: WO2017025566A1
Application number: PCT/EP2016/069044
Authority: WO
Inventors: Steffen Goletz
Original assignee: Glycotope Gmbh
Priority date: 2015-08-10
Filing date: 2016-08-10
Publication date: 2017-02-16
Also published as: AR105669A1

Abstract

The present invention pertains to improved factor VII preparations which have a superior glycosylation pattern. These improved factor VII preparations can be used in the treatment of bleeding disorders.

Description

..Improved recombinant factor VII"

FIELD OF THE INVENTION

The present invention pertains to the field of blood coagulation factors. In particular, improved recombinant factor VII is provided. This improved factor VII is useful in the treatment of bleeding disorders, in particular in human patients.

BACKGROUND OF THE INVENTION

Blood coagulation is an important natural process for sealing wounds in the human or animal body. It consists of a complex interaction of various blood components which eventually result in a fibrin clot. The blood components, coagulation factors, which are involved in the coagulation cascade are proenzymes, enzymatically inactive proteins which are converted to protease enzymes by proteolytic cleavage. Thereby a coagulation cascade is formed wherein one coagulation factor cleaves and activates the next coagulation factor. Coagulation factors which have undergone such a conversion are generally referred to as "activated factors" and are designated by the addition of a lowercase postscript "a" (e.g., factor Vila). The coagulation factors are generally serine proteases which act by cleaving downstream proteins. Examples of these serine protease factors are thrombin and factors VII, IX, X, XI and XII.

Factor VII is a single-chain glycoprotein with a molecular weight of about 50 kDa Activation of factor VII to Vila may be catalyzed by several different plasma proteases, such as factor Xlla. Activation of factor VII results in the formation of two polypeptide chains, a heavy chain of 28 kDa and a light chain of 17 kDa held together by at least one disulfide bond. Factor IX is also a single-chain precursor of 57 kDa and is converted to an active serine protease (factor IXa) upon cleavage by factor XIa. Factor IXa consists of a 16 kDa light chain and a 29 kDa heavy chain.

Various coagulation factors such as in particular factors VII are used in medicine in the treatment of hemophilia or to prevent uncontrollable hemorrhage. Many properties of glycosylated therapeutic protein, including their circulation half-life, enzymatic activity, immunogenicity and others, are influenced by their specific glycosylation pattern. This is in particular important for therapeutic glycoproteins used in the treatment of congenital disorders, since here the therapeutic agent is administered frequently over a long period of time. In view of this, it is one object of the present invention to provide improved factor VII preparations. Furthermore, it is an object of the present invention to provide factor VII preparations which have an improved glycosylation pattern.

SUMMARY OF THE INVENTION

The present inventors have found that improved factor VII preparations obtained from human cells which preferably have been selected for an optimized glycosylation exhibit advantageous properties compared to conventional factor VII preparations. Especially they are more stable and less prone to fragmentation, they have a higher biologic and therapeutic activity, including a stronger platelet binding and a higher thrombogenic activity, they have an elongated circulation half-life in patients and have a lower immunogenicity in humans. Therefore, the factor VII preparations according to the present invention have surprising characteristics which are useful in therapy.

The present invention provides, in a first aspect, a recombinant factor VII preparation, wherein the recombinant factor VII in the preparation has a glycosylation pattern at its N-glycosylation sites comprising the following characteristics:

(i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of at least 10 % of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation;

In a second aspect, the present invention provides a recombinant factor VII preparation obtainable by production in human cells or a human cell line, preferably in the cell line GT-5s (DSM ACC3078, deposited on July 28, 2010). It was found that factor VII produced in a respective cell line results in an improved glycosylation profile as is described above and below. Also provided is a pharmaceutical composition, comprising the recombinant factor VII preparation according to the present invention. Furthermore, the present invention pertains to the recombinant factor VII preparation or the pharmaceutical composition according to the present invention for use in medicine, in particular in the treatment of bleeding disorders.

Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, which indicate preferred embodiments of the application, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

DEFINITIONS As used herein, the following expressions are generally intended to preferably have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

The expression "comprise", as used herein, besides its regular meaning also includes and specifically refers to the expressions "consist essentially of" and "consist of". Thus, the expression "comprise" refers to embodiments wherein the subject-matter which

"comprises" specifically listed elements does not comprise further elements as well as embodiments wherein the subject-matter which "comprises" specifically listed elements may and/or indeed does encompass further elements.

The term "factor VII" refers to the blood coagulation factor VII. The term "factor VI I" as used herein includes the preproprotein comprising the signal sequence, the proprotein consisting of one polypeptide chain, and the activated protein comprising a light chain and a heavy chain. Thus, the term "factor VII" also refers to factor Vila except it is explicitly stated otherwise. Preferably, the factor VII is human factor VII, in particular human factor VII comprising the amino acid sequence of SEQ ID NO: 1 , or a sequence derived therefrom, wherein this sequence is present in one polypeptide chain if factor VII is the proprotein or preproprotein, or the light chain comprises the amino acid sequence of amino acid position 1 to 152 of SEQ ID NO: 1 , or a sequence derived therefrom, and the heavy chain comprises the amino acid sequence of amino acid position 153-406 of SEQ ID NO: 1 , or a sequence derived therefrom, if factor VII is the activated protein. In certain embodiments, one or more, such as 1 , 1 or 2, up to 3, up to 5, up to 10 or up to 20, amino acid substitution, addition and/or deletions may be present in one or both amino acid sequences. Preferably, the amino acid sequence of the light chain or the part of the (pre)proprotein corresponding thereto shares an overall homology or identity of at least 80%, more preferably at least 85%, at least 90%, at least 95% or at least 98% with the amino acid sequence according to amino acid position 1 to 152 of SEQ ID NO: 1. Furthermore, the amino acid sequence of the heavy chain or the part of the (pre)proprotein corresponding thereto preferably shares an overall homology or identity of at least 80%, more preferably at least 85%, at least 90%, at least 95% or at least 98% with the amino acid sequence according to amino acid position 153-406 of SEQ ID NO: 1 . Preferably, the factor VII according to the invention is capable of activating, in particular cleaving by limited proteolysis, factor X to form factor Xa, preferably human factor X. The term "factor VII" as used herein in particular refers to all factor VII proteins in a preparation. Thus, the term "factor VII" in particular refers to the entirety of all factor VII proteins in a factor VII preparation or composition. Preferably, the factor VII protein comprises one or more carbohydrate structures attached to the polypeptide chain. Especially, one carbohydrate structure is attached to an asparagine residue in the light chain, in particular asparagine 145 of SEQ ID NO: 1 or an asparagine residue corresponding thereto, and another carbohydrate structure is attached to an asparagine residue in the heavy chain, in particular asparagine 322 of SEQ ID NO: 1 or an asparagine residue corresponding thereto. Furthermore, the factor

VII protein may comprise one or more, in particular 2, carbohydrate structures which are attached to a serine or threonine residue, in particular to a serine residue. These carbohydrate structures are in particular attached to serine 52 and serine 60 of SEQ ID NO: 1 or a serine residue corresponding thereto. Factor VII as used according to the present invention preferably is recombinant factor

VII, more preferably recombinant human factor VII. The term "recombinant factor VII" refers to factor VII which is not naturally produced by a living human or animal body and then obtained from a sample derived therefrom, such as blood or other body liquid or tissue of the human or animal body. Preferably, recombinant factor VII is obtained from cells which have been biotechnologically engineered, in particular cells which have been transformed or transfected with a nucleic acid encoding factor VII or the light chain and heavy chain of factor Vila. According to preferred embodiments, recombinant factor VII is obtained from human cells comprising an exogenous nucleic acid encoding factor VII. Respective exogenous nucleic acids can be introduced e.g. by using one or more expression vectors, which can be introduced into the host cell e.g. via transfection. Respective methods for recombinantly producing proteins such as factor VII are well known in the prior art and thus, need no further description. The factor VII according to the invention preferably is factor VII, more preferably human factor VII, obtainable by production in a human cell, preferably a human cell line. The human cell line preferably is derived from human blood cells, in particular it is a myeloid cell line, preferably a myeloid leukemia cell line. The cell line preferably is immortalized. In a preferred embodiment, the cell line for the production of the factor VII according to the invention is the cell line GT-5s, deposited on July 28, 2010 under the accession number DSM ACC3078 according to the requirements of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), ΙηϊιοίίβηβίΓΒββ 7B, 38124 Braunschweig (DE) by the Glycotope GmbH, Robert-Rossle-Str. 10, 13125 Berlin (DE), or a cell line derived therefrom, or a homologous cell line. GT-5s is an immortalized human myeloid leukemia cell line which is capable of providing a specific glycosylation pattern as described herein. According to the present invention, the terms "GT-5s" and "GT-5s cell line" also include cells or cell lines derived from GT-5s. A cell line which is derived from GT-5s can be for example obtained by randomly or specifically selecting a single clone or a group of cells from a GT-5s culture, optionally after treating the GT-5s cells in order to enhance their mutation rate, or by genetically altering a GT-5s cell line. The selected clone or group of cells may further be treated as described above and/or further rounds of selection may be performed. A cell line which is homologous to GT-5s in particular is an immortalized human myeloid cell line. Preferably, a cell line derived from or homologous to GT-5s is capable of providing factor VII having a glycosylation pattern similar to that obtained from GT-5s. Preferably, factor VII that is produced by a cell line derived from or homologous to GT-5s has one or more of the glycosylation characteristics as described herein, in particular a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of at least 15% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or a relative amount of tetraantennary glycans of at least 8% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or a relative amount of glycans carrying four galactose residues of at least 3% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation. According to one embodiment, the cell line derived from or homologous to GT-5s is capable of expressing factor VII having a glycosylation pattern as is described as preferred herein, in particular a glycosylation pattern selected from Table 1 . The similar glycosylation pattern of factor VII that is produced by the cell line derived from or homologous to GT-5s preferably differs from the glycosylation pattern of factor VII obtained from GT-5s by 20% or less, more preferably 15% or less, 10% or less or 5% or less, in particular in one or more, preferably all of the glycosylation properties selected from the group consisting of the relative amount of bisGlcNAc, the relative amount of tetraantennary glycans, the relative amount of glycans carrying four galactose residues, the relative amount of sialylated glycans, the relative amount of fucosylated glycans, the relative amount of glycans carrying at least one terminal galactose residue, the relative amount of glycans carrying galactose, and the relative amount of glycans carrying at least one terminal GalNAc residue. Furthermore, the factor VII according to the invention preferably is factor VII, more preferably human factor VII, having one or more specific glycosylation characteristics as disclosed herein, preferably a glycosylation pattern selected from Table 1 . The cell line GT-5s as well as cell lines derived therefrom and cell lines homologous thereto are in particular advantageous since they provide a very stable and homogeneous protein production, in particular with respect to factor VII protein. They have a very good batch-to-batch consistency, i.e. the produced proteins and their glycosylation pattern are similar when obtained from different production runs or when produced at different scales and/or with different culturing procedures. In particular, the diverse glycosylation pattern as described herein is highly reproducible in different production runs using these cell lines for expressing factor VII.

The factor VII according to the present invention is glycosylated. In particular, it is modified by one or more, preferably four, oligosaccharides attached to the polypeptides chains. These oligosaccharides, also named glycans or carbohydrates, may be linear or branched saccharide chains. In particular, One or two, especially two, of the oligosaccharides are complex-type N-linked oligosaccharide chains. Depending on the number of branches the oligosaccharide is termed mono-, bi-, tri- or tetraantennary (or even pentaantennary). A monoantennary oligosaccharide is unbranched while a bi-, tri- or tetraantennary oligosaccharide has one, two or three branches, respectively. A glycoprotein with a higher antennarity thus has more oligosaccharide endpoints and can carry more functional terminal saccharide units such as, for example, sialic acids. "At least triantennary" as used herein refers to oligosaccharides having an antennarity of at least 3, including triantennary, tetraantennary and pentaantennary oligosaccharides. "At least tetraantennary" as used herein refers to oligosaccharides having an antennarity of at least 4, including tetraantennary and pentaantennary oligosaccharides. With respect to complex-type N-glycans, a bisecting GlcNAc residue and/or a fucose residue, preferably is not considered as a branch or antenna and thus, does not add to the antennarity of factor VII. The glycosylation pattern of factor VII as referred to herein in particular refers to the overall glycosylation pattern of all factor VII proteins in a factor VII preparation according to the present invention. In particular, any glycan structures comprised in the factor VII protein and thus, attached to the factor VII polypeptide chains in the factor VII preparation are considered and reflected in the glycosylation pattern. In specific embodiments, only the glycan structures attached to asparagine residues, i.e. the N- glycosylation, are considered.

The term "sialic acid" in particular refers to any N- or O-substituted derivatives of neuraminic acid. It may refer to both 5-N-acetylneuraminic acid and 5-N- glycolylneuraminic acid, but preferably only refers to 5-N-acetylneuraminic acid. The sialic acid, in particular the 5-N-acetylneuraminic acid preferably is attached to a carbohydrate chain via a 2,3- or 2,6-linkage. Preferably, in the factor VII preparations described herein both 2,3- as well as 2,6-coupled sialic acids are present. A "relative amount of glycans" according to the invention refers to a specific percentage or percentage range of the glycans attached to the factor VII glycoproteins of a factor VII preparation or in a composition comprising factor VII, respectively. In particular, the relative amount of glycans refers to a specific percentage or percentage range of all glycans comprised in the factor VII proteins and thus, attached to the factor VII polypeptide chains in a factor VII preparation or in a composition comprising factor VII.

100% of the glycans refers to all glycans attached to the factor VII glycoproteins of the factor VII preparation or in a composition comprising factor VII respectively. In specific embodiments, only the glycans attached to the N-glycosylation sites of factor VII, i.e. the glycans attached to asparagine residues of factor VII, are considered. For example, a relative amount of glycans carrying bisecting GlcNAc of 20% refers to a factor VII preparation wherein 20% of all glycans attached to the N-glycosylation sites of the factor VII proteins and thus, attached to asparagine residues of the factor VII polypeptide chains in said factor VII preparation comprise a bisecting GlcNAc residue while 80% of all glycans attached to the N-glycosylation sites of the factor VII proteins and thus, attached to asparagine residues of the factor VII polypeptide chains in said factor VII preparation do not comprise a bisecting GlcNAc residue.

The numbers given herein, in particular the relative amounts of a specific glycosylation property, are preferably to be understood as approximate numbers. In particular, the numbers preferably may be up to 10% higher and/or lower, in particular up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1 % higher and/or lower.

A "factor VII preparation" may be any composition or substance comprising or consisting of factor VII. It may be in solid or fluid form and may comprise further ingredients in addition to factor VII. In particular, a factor VII preparation may be a solution comprising factor VII and a suitable solvent such as water and/or alcohol, or a powder obtained, for example, after lyophilization of a solution containing factor VII.

Suitable examples of a factor VII preparation are composition obtained after expression of factor VII in cells, in particular after purification of the factor VII, or pharmaceutical compositions comprising factor VII. A factor VII preparation may contain, in addition to factor VII, for example solvents, diluents, excipients, stabilizers, preservatives, salts, adjuvants and/or surfactants. The terms "factor VII preparation" is used herein in particular in the meaning of a "composition comprising factor VII". These terms are preferably used synonymously herein. The term "nucleic acid" includes single-stranded and double-stranded nucleic acids and ribonucleic acids as well as deoxyribonucleic acids. It may comprise naturally occurring as well as synthetic nucleotides and can be naturally or synthetically modified, for example by methylation, 5'- and/or 3'-capping. The term "vector" is used herein in its most general meaning and comprises any intermediary vehicle for a nucleic acid which enables said nucleic acid, for example, to be introduced into prokaryotic and/or eukaryotic host cells and, where appropriate, to be integrated into a genome of the host cell. Vectors of this kind are preferably replicated and/or expressed in the host cells. A vector preferably comprises one or more selection markers for selecting host cells comprising the vector. Suitable selection markers are resistance genes which provide the host cell with a resistance e.g. against a specific antibiotic. Further suitable selection markers are, for example, genes for enzymes such as DHFR or GS. Vectors enabling the expression of recombinant proteins including factor VII as well as suitable expression cassettes and expression elements which enable the expression of a recombinant protein with high yield in a host cell are well known in the prior art and are also commercially available, and thus, need no detailed description here.

The terms "cell" and "cells" and "cell line" used interchangeably, preferably refer to one or more mammalian cells, in particular human cells. The term includes progeny of a cell or cell population. Those skilled in the art will recognize that "cells" include progeny of a single cell, and the progeny can not necessarily be completely identical (in morphology or of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. "Cell" preferably refers to isolated cells and/or cultivated cells which are not incorporated in a living human or animal body. The term "patient" means according to the invention a human being, a nonhuman primate or another animal, in particular a mammal such as a cow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouse and rat. In a particularly preferred embodiment, the patient is a human being. In case of a human patient, the factor VII preferably is human factor VII. The term "pharmaceutical composition" particularly refers to a composition suitable for administering to a human or animal, i.e., a composition containing components which are pharmaceutically acceptable. Preferably, a pharmaceutical composition comprises an active compound or a salt or prodrug thereof together with a carrier, diluent or pharmaceutical excipient such as buffer, preservative and tonicity modifier. The term "bleeding disorders" according to the invention means the treatment of a dysfunction or disease related to blood coagulation. In particular, a diminished blood coagulation ability can lead to uncontrolled bleeding with a slow formation of blood clots which can stop the bleeding. Furthermore, also treatments wherein the normal blood coagulation is enhanced are included, such as during or after surgery to accelerate blood clotting and wound healing.

The term "about" as used herein, when referring to a value, means the value as such and a range around the value with a lower border at the value minus 10% and an upper border at the value plus 10%, in particular plus/minus 5%. Concerning percentage values, "about" in particular refers to the given percentage value and a range of 3 percentage point, in particular 2 percentage points, 1 percentage point or 0.5 percentage points, above and below the given percentage value.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is according to one aspect based on the finding that an improved recombinant factor VII preparation having an optimal glycosylation pattern has superior properties compared with conventional factor VII. Especially, the improved factor VII is more stable and less prone to fragmentation, has a higher biologic and therapeutic activity, including a stronger platelet binding and a higher thrombogenic activity, has an elongated circulation half-life in patients and a lower immunogenicity in humans. Therefore, the factor VII preparations according to the present invention have surprising characteristics which are useful in therapy. The analysis of the glycosylation patterns of different factor VII constructs according to the invention revealed that the main difference to commonly used factor VII preparations which do not show the advantageous properties provided by the invention is the presence of a significant amount of bisecting GlcNAc residues. Indeed, neither plasma-derived factor VII nor commonly used recombinant factor VII show any bisecting GlcNAc residues in their glycosylation pattern.

In view of these findings, the present invention provides, in a first aspect, a factor VII preparation, wherein the recombinant factor VII in the preparation has a glycosylation pattern at its N-glycosylation sites comprising the following characteristics:

(i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of at least 10% of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation.

In particularly preferred embodiments, the glycosylation pattern at its N-glycosylation sites comprises the following characteristics: (i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of at least 15% of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation.

In specific embodiments, the present invention provides a factor VII preparation, wherein the recombinant factor VII in the preparation has a glycosylation pattern at its

N-glycosylation sites comprising one or more of the following characteristics:

(i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of at least 15% of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation; (ii) a relative amount of tetraantennary glycans of at least 8% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or

(iii) a relative amount of glycans carrying four galactose residues of at least 3% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation.

In certain embodiments, the present invention provides a factor VII preparation, wherein the factor VII in the preparation has a glycosylation pattern comprising one or more of the following characteristics:

(i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of at least 20% of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation;

(ii) a relative amount of tetraantennary glycans of at least 10% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or (iii) a relative amount of glycans carrying four galactose residues of at least 4% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation.

Preferably, said factor VII is a recombinant factor VII and thus, is obtained by recombinant production in a host cell, which preferably is a human host cell. Suitable human host cells which provide a respective glycosylation pattern are described subsequently. Preferably, the glycosylation pattern comprises at least two of the features (i), (ii) and (iii) (in particular features (i) and (ii), (i) and (iii), or (ii) and (iii)), and more preferably all of the features (i), (ii) and (iii).

Furthermore, the glycosylation pattern may further comprise a relative amount of glycans carrying at least one sialic acid residue of at least 80%, in particular at least

85% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or a relative amount of glycans carrying at least one galactose residue of at least 87%, in particular at least 90% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or a relative amount of glycans carrying at least one terminal galactose residue of at least 10%, in particular at least 15% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or a relative amount of glycans carrying fucose of at least 30%, in particular at least 40% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or a relative amount of glycans carrying at least four sialic acid residue of at least 0.5%, in particular at least 1 % of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or a relative amount of glycans carrying at least one terminal GalNAc residue of not more than 15%, in particular not more than 10% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or no detectable NeuGc and/or Gala1 ,3-Gal structures. In certain embodiments, the factor VII in the preparation has a human glycosylation pattern.

The relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation is preferably at least 18% or at least 20%. It may in particular also be at least 25%, at least 27.5%, at least 30%, at least 32.5%, at least 34% or at least 35%.

More preferably, it is in the range of from about 15% to about 60% or from about 18% to about 60%, or it may be in the range of from about 25% to about 60%, in particular in the range of from about 30% to about 50% or in the range of from about 32.5% to about 45% or in the range of from about 34% to about 42.5%. According to one embodiment, it is about 37%.

The relative amount of tetraantennary glycans of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation is preferably at least 2%, at least 5% or at least 8%, in particular at least 12.5%, at least 15%, at least 17.5% or at least 20%. More preferably, it is in the range of from about 13% to about 50%, in particular in the range of from about 15% to about 40% or in the range of from about

17% to about 35% or in the range of from about 18% to about 30%. According to one embodiment, it is about 24%. The relative amount of glycans carrying four galactose residues of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation is preferably at least 0.5%, at least 1 % or at least 2%, in particular at least 5%, at least 6%, at least 7% or at least 8%. More preferably, it is in the range of from about 5% to about 30%, in particular in the range of from about 6% to about 25% or in the range of from about 7% to about 20% or in the range of from about 8% to about 17.5%. According to one embodiment, it is about 1 1 %.

The relative amount of glycans carrying at least one sialic acid residue of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation is preferably at least 40%, at least 45% or at least 50%, in particular at least 87.5%, at least 90%, at least 91 % or at least 92%. More preferably, it is in the range of from about 85% to about 100%, in particular in the range of from about 88% to about 99.5% or in the range of from about 90% to about 99%. According to one embodiment, it is about 94%. The relative amount of glycans carrying at least one galactose residue of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation is preferably at least 86%, at least 88%, at least 89% or at least 90%, in particular at least 92%, at least 93%, at least 94% or at least 95%. More preferably, it is in the range of from about 91 % to about 100%, in particular in the range of from about 92% to about 99.5% or in the range of from about 93% to about 99%. According to one embodiment, it is about 97%.

The relative amount of glycans carrying at least one terminal galactose residue of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation is preferably at least 20%, at least 25%, at least 30% or at least 35%. More preferably, it is in the range of from about 20% to about 60%, in particular in the range of from about 25% to about 55% or in the range of from about 30% to about 50%. According to one embodiment, it is about 45%.

The relative amount of glycans carrying fucose of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation is preferably at least 45%, at least 50%, at least 55% or at least 57%. More preferably, it is in the range of from about 45% to about 90%, in particular in the range of from about 47.5% to about 80% or in the range of from about 50% to about 75% or in the range of from about 52.5% to about 70%. According to one embodiment, it is about 60%. In certain embodiments, the elative amount of glycans carrying fucose of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation is at least 85%, in particular at least

90%. The relative amount of glycans carrying at least four sialic acid residues of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation is preferably at least 1.5%, at least 2% or at least 2.5%. More preferably, it is in the range of from about 0.5% to about 15%, in particular in the range of from about 1 % to about 10% or in the range of from about 1.5% to about 7.5%. According to one embodiment, it is about 3%.

The relative amount of glycans carrying at least one terminal GalNAc residue of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation is preferably not more than 9%, not more than 8%, not more than 7% or not more than 6%. More preferably, it is in the range of from about 0% to about 8%, in particular in the range of from about 0.5% to about 7.5% or in the range of from about 1 % to about 7%.

In preferred embodiments, the recombinant factor VII preparation according to the invention does not comprise N-glycolyl neuraminic acids (NeuGc) or detectable amounts of NeuGc. Furthermore, the recombinant factor VII preparation according to the invention preferably also does not comprise Galili epitopes (Gala1 ,3-Gal structures) or detectable amounts of the Galili epitope. In particular, the relative amount of glycans carrying NeuGc and/or Gala1 ,3-Gal structures is less than 0.1 % or even less than 0.02% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation.

The present invention in particular provides a factor VII with a human glycosylation pattern. Due to these glycosylation properties, foreign immunogenic non-human structures which induce side effects are absent which means that unwanted side effects or disadvantages known to be caused by certain foreign sugar structures such as the immunogenic non-human sialic acids (NeuGc) or the Galili epitope (Gal-Gal structures), both known for rodent production systems, or other structures like immunogenic high-mannose structures as known from e.g. yeast systems are avoided.

In certain embodiments the glycosylation pattern of the recombinant factor VII in the preparation according to the present invention comprises one or more, preferably all of the following characteristics:

(i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) in the range of from 15% to 45% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation;

(ii) a relative amount of tetraantennary glycans in the range of from 1 % to 35% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; (iii) a relative amount of glycans carrying at least one galactose residue in the range of from 88% to 100% of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation;

(i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) in the range of from 25% to 45% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation;

(ii) a relative amount of tetraantennary glycans in the range of from 13% to 35% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation;

(iii) a relative amount of glycans carrying four galactose residues in the range of from 5% to 18% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation.

In certain preferred embodiments, the recombinant factor VII preparation according to the invention has one of the glycosylation patterns listed in the following Table 1 :

Table 1 : Specific glycosylation parameters

Embodiment B A4 G4 S>0 tG F

1 ≥20 ≥ 10 ≥4

2 ≥20 ≥ 10 ≥4 ≥85 ≥ 15 ≥40

3 ≥20 ≥ 10 ≥4 ≥90

4 ≥20 ≥ 10 ≥4 ≥20

5 ≥20 ≥ 10 ≥4 ≥50

6 ≥20 ≥ 10 ≥4 ≥90 ≥20 ≥50

7 20-50 ≥ 10 ≥4

8 ≥20 10-40 ≥4

9 ≥20 ≥ 10 4-20

10 20-50 10-40 4-20

1 1 ≥20 ≥ 10 ≥4 85-100 ≥ 15 ≥40

12 ≥20 ≥ 10 ≥4 ≥85 15-60 ≥40

13 ≥20 ≥ 10 ≥4 ≥85 ≥ 15 40-80

14 20-50 10-40 4-20 85-100 15-60 40-80

15 ≥25 ≥ 15 ≥5 16 ≥30 ≥ 15 ≥5

17 ≥25 ≥ 18 ≥5

18 ≥25 ≥ 15 ≥6

19 ≥30 ≥ 18 ≥6

20 ≥25 ≥ 15 ≥5 ≥85 ≥ 15 ≥40

21 ≥25 ≥ 15 ≥5 ≥90 ≥20 ≥50

22 ≥30 ≥ 18 ≥6 ≥90 ≥20 ≥50 shown are the relative amounts of glycans at the N-glycosylation sites of factor VII having the following property:

B: bisecting GlcNAc; A4: tetraantennary glycans; G4: at least four galactose residues; S>0: at least one sialic acid; tG: at least one terminal galactose; F: fucose

The glycosylation patterns listed in table 1 preferably are human glycosylation patterns and/or do not comprise NeuGc and the Galili epitope.

In specific embodiments, the factor VII in the preparation has a higher amount of sialylation. In particular, the factor VII preparation may be produced in a cell line with higher sialylation activity and/or the factor VII preparation may be subjected to in vitro sialylation using respective sialyltransferases. In these embodiments, the relative amount of glycans carrying at least one sialic acid residue of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation may be higher than described herein. Furthermore, the relative amount of glycans carrying at least four sialic acid residues of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation may be higher than described herein. In addition, the relative amount of glycans carrying at least one terminal galactose residues of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation may be lower than described herein, in particular it may be 35% or less, especially 30% or less, and it may be in the range of from about 5% to about 35% or from about 10% to about 30%.

In certain embodiments, the recombinant factor VII in the preparation comprises one or two, in particular two O-glycosylation sites. These O-glycosylation sites are present at serines corresponding to Ser52 and Ser60 of SEQ ID NO: 1 . In specific embodiments, at least 90% of the factor VII proteins in the preparation are glycosylated at the O- glycosylation site corresponding to Ser60 of SEQ ID NO: 1 with a fucose residue. In particular, at least 95%, especially at least 97%, at least 98% or at least 99% of the factor VII proteins in the preparation are glycosylated at the O-glycosylation site corresponding to Ser60 of SEQ ID NO: 1 with a fucose residue. In further embodiments, at least 90% of the factor VII proteins in the preparation are glycosylated at the O-glycosylation site corresponding to Ser52 of SEQ ID NO: 1 with a glucose residue optionally carrying one or two xylose residues. In particular, at least 95%, especially at least 97%, at least 98% or at least 99% of the factor VII proteins in the preparation are glycosylated at the O-glycosylation site corresponding to Ser52 of SEQ ID NO: 1 with a glucose residue optionally carrying one or two xylose residues.

In specific embodiments, about 25% to about 55% of the factor VII proteins in the preparation are glycosylated at the O-glycosylation site corresponding to Ser52 of SEQ

ID NO: 1 with a glucose residue. In particular, about 30% to about 50%, especially about 35% to about 45% or about 37% to about 43%, specifically about 39% to about 40% of the factor VII proteins in the preparation are glycosylated at the O-glycosylation site corresponding to Ser52 of SEQ ID NO: 1 with a glucose residue. In further embodiments, about 45% to about 75% of the factor VII proteins in the preparation are glycosylated at the O-glycosylation site corresponding to Ser52 of SEQ ID NO: 1 with the structure glucose-xylose-xylose. In particular, about 50% to about 70%, especially about 55% to about 65% or about 57% to about 63%, specifically about 59% to about 60% of the factor VII proteins in the preparation are glycosylated at the O-glycosylation site corresponding to Ser52 of SEQ ID NO: 1 with the structure glucose-xylose-xylose.

In specific embodiments, the recombinant factor VII in the preparation is v- carboxylated. The γ-carboxylated glutamate residues (Gla residues) in particular are present in the Gla domain in the N-terminal region of factor VII, especially at one or more glutamate residues corresponding to the amino acid positions 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 of SEQ ID NO: 1. In certain embodiments, the recombinant factor VII in the preparation has an average amount of at least 9 γ-carboxylated glutamic acid residues. In further embodiments, at least 10%, in particular at least 15%, at least 17%, at least 18%, at least 19% or at least 20% of the recombinant factor VII in the preparation comprise 10 γ-carboxylated glutamic acid residues. The factor VII in particular is activated factor VII (factor Vila) comprising a light chain and a heavy chain. In certain embodiments, factor VI I is produced by the host cells as inactive proprotein and is then activated by proteolytic digestion. In alternative embodiments, the light chain and the heavy chain of factor VII are produced by the host cells as separate polypeptide chains. In certain embodiments, factor VII is a fusion protein comprising one or more fusion partners. The fusion partners may be fused to the proprotein or to one or both of the light chain and the heavy chain of factor VII. The light chain and the heavy chain of factor VII may be fused to different fusion partners. In one embodiment, the fusion partner elongates the half-life of factor VII in the human circulation, such as albumin, in particular human serum albumin or a part thereof, of an antibody or a part thereof, in particular the Fc part. In further embodiments, the fusion partner comprises a dimerization domain. In these embodiments, in particular the light chain of factor VII is fused to one dimerization domain and the heavy chain of factor VII is fused to another dimerization domain and the two dimerization domains bind to each other. Especially, the glycosylation pattern of the fusion partner is not considered when calculating, analyzing and/or determining the glycosylation pattern of the factor VII. The glycosylation characteristics described herein for factor VII in particular do not include any glycosylation characteristics of any fusion partner.

In specific embodiments, one or more further agents are conjugated to factor VII, forming a factor VII conjugate. These agents in particular are conjugated to the polypeptide chain of factor VII, either directly or via a suitable linker. In certain embodiments, the agents are conjugated to the polypeptide chain of factor VII via its glycan structures, optionally further comprising a linker. The further agent may be useful in therapy of a disease, in particular bleeding disorders. The further agent may be any compound, including proteins such as blood coagulation factors, polymers such as PEG, HES and saccharides, and detectable labels such as fluorescent dyes, chelators for radioactive labeling and radioisotopes.

Furthermore, the present invention provides a recombinant factor VII preparation that is obtainable by production in a human host cell or a human cell line. Preferably, the recombinant factor VII is obtainable from a human myeloid cell line, preferably an immortalized human myeloid leukemia cell line, in particular the cell line GT-5s or a cell line derived therefrom or a cell line homologous to GT-5s. It was found that an factor VII produced in said cell line exhibits a glycosylation pattern as described above and in particular exhibits the advantageous therapeutic and pharmacological effects described herein. Thus, the present invention also pertains to a method for producing a recombinant factor VII preparation by recombinantly expressing the factor VII in a suitable cell line, in particular a cell line as described above, preferably the cell line GT- 5s, a cell line derived from GT-5s or a cell line homologous to GT-5s. The recombinant factor VII respectively produced can be isolated and optionally purified.

Thus, the recombinant factor VII preparation preferably is obtainable by a process comprising the steps of:

(i) cultivating a human host cell, preferably derived from the cell line GT-5s or a homologous cell line, comprising nucleic acids coding for factor VII under conditions suitable for expression of the factor VII; and

(ii) isolating factor VII.

The human host cells used for expression preferably are myeloid cells, in particular immortalized myeloid leukemia cells, and preferably are or are derived from the cell line GT-5s or is a cell line homologous thereto. The human host cells are cultured so that they express factor VI I. In certain embodiments, factor VI I is secreted by the host cell. Suitable culture conditions are known to the skilled person.

The isolation of factor VII preferably comprises the further steps of:

(a) obtaining the culture supernatant where the factor VI I is secreted by the human cells, or lysing the human cells where the factor VII is not secreted;

(b) isolating the factor VI I from the culture supernatant or cell lysate using chromatographic steps such as affinity chromatography and multimodal anion exchange chromatography; and

(c) optionally activating non-activated factor VI I by incubation in a solution comprising calcium ions at a pH of 7.5 or higher for at least 1 day at 10°C or less.

Preferably, the nucleic acid coding for factor VI I is comprised in one or more expression cassettes comprised in a suitable expression vector that allows the expression in a human host cell. If factor Vi la is expressed, the nucleic acid coding for the light chain of factor VI I and the nucleic acid coding for the heavy chain of factor VI I may be comprised in the same vector or in separate vectors. Furthermore, they may also be expressed from one expression cassette using appropriate elements such as an I RES element. Preferably, the factor VI I is secreted by the human cells. In preferred embodiments, cultivation of the human cells is performed in a fermenter and/or under serum-free conditions.

A suitable purification process for the recombinant factor VI I is described, for example, in the Luxembourg patent application no. LU 92751 .

The recombinant factor VI I preparation obtainable by production in human host cells or a human cell line preferably exhibits the features described herein with respect to the recombinant factor VI I preparation according to the present invention. In particular, its glycosylation pattern comprises one or more of the characteristics described above, preferably at least one glycosylation pattern as described in Table 1 and/or in claims 1 to 1 1 .

In preferred embodiments of the aspects of the present invention, the recombinant factor VI I according to the present invention is recombinant human factor VI I, preferably obtainable by production in a human cell line, such as the cell line GT-5s, which comprises one or more nucleic acids encoding the human factor VI I and elements for expressing said one or more nucleic acids in the host cell. Preferably, the light chain of the factor VI I has the amino acid sequence according to amino acid positions 1 to 152 of SEQ ID NO: 1 or an amino acid sequence having a homology or preferably identity to amino acid positions 1 to 152 of SEQ ID NO: 1 over its entire length of at least 80 %, preferably at least 85%, at least 90%, at least 95% or at least 98%. In preferred embodiments, the light chain of factor VII is glycosylated at an asparagine residue corresponding to Asn145 of SEQ ID NO: 1. The heavy chain of factor VII preferably has the amino acid sequence according to amino acid positions

153 to 406 of SEQ ID NO: 1 or an amino acid sequence having a homology or preferably identity to amino acid positions 153 to 406 of SEQ ID NO: 1 over its entire length of at least 80 %, preferably at least 85%, at least 90%, at least 95% or at least 98%. In preferred embodiments, the heavy chain of factor VII comprises is glycosylated at an asparagine residue corresponding to Asn322 of SEQ ID NO: 1 .

The recombinant factor VII preparation according to the present invention preferably is present in a pharmaceutical composition. Thus, another aspect of the present invention is a pharmaceutical composition comprising the recombinant factor VII preparation according to the present invention for use in medicine, in particular for use in the treatment of bleeding disorders. The bleeding disorder may be selected from the group consisting of hemophilia A and B; acquired hemophilia; congenital factor VI l-deficiency; hemophilia prophylaxis for patients with inhibitors, trauma, bleeding in emergencies or surgical intervention such as spinal and cardiac surgery; Glanzmann's thrombasthenia; blast lung injury; and hemorrhages such as intracerebral hemorrhage, unspecific hemorrhage, or diffuse alveolar hemorrhage. Especially, the bleeding disorder is hemophilia A or B, in particular in patients with inhibitors, factor VII deficiency or Glanzmann's thrombasthenia.

The pharmaceutical composition may include further pharmaceutically active agents, in particular further agents useful in the treatment of bleeding disorders such as other blood coagulation factors. Alternatively, the pharmaceutical composition comprising the recombinant factor VII may be designed for use in combination with such further pharmaceutically active agents.

Furthermore, the present invention provides the recombinant factor VII preparation according to the present invention or the pharmaceutical composition according to the present invention for use in the treatment of bleeding disorders as well as a method for the treatment of bleeding disorders comprising the administration of the recombinant factor VII preparation according to the present invention or the pharmaceutical composition according to the present invention to the patient, in particular in a therapeutically active amount. Preferably, the recombinant factor VII preparation according to the present invention is for parenteral administration to the patient. In particular, the recombinant factor VII is to be administered by injection or infusion, for example intravenously, intramuscularly or subcutaneously. In certain embodiments of the present invention, the recombinant factor VII is present in a pharmaceutical composition. Suitable dosage regiments can be determined by the skilled artisan and can be derived from the general knowledge in the field.

The pharmaceutical composition according to the invention may be in the form of a single unit dose or a multiple unit dose. Preferably, the pharmaceutical composition is a sterile solution comprising the recombinant factor VII according to the present invention, further comprising one or more ingredients selected from the group consisting of solvents such as water, buffer substances, stabilizers, preservatives, excipients, surfactants and salts. A multiple unit dose comprises enough factor VII to provide for multiple single doses, in particular at least 5, at least 10, at least 20 or at least 50 single doses.

Numeric ranges described herein are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects or embodiments of this invention which can be read by reference to the specification as a whole. According to one embodiment, subject matter described herein as comprising certain steps in the case of methods or as comprising certain ingredients in the case of compositions refers to subject-matter consisting of the respective steps or ingredients. It is preferred to select and combine specific aspects and embodiments described herein and the specific subject-matter arising from a respective combination of specific embodiments also belongs to the present disclosure.

FIGURES

Figure 1 shows a gel picture of a denaturating non-reducing SDS-PAGE separation of reconstituted factor VII. The intact monomer is visible as a band of about 50 kDa and degradation products can be observed as bands of lower molecular weight. Lane 1 : NovoSeven™; lane 2: Factor Vila (invention); M: molecular weight marker.

Figure 2 shows a chromatogram of an analytical size exclusion chromatography of reconstituted NovoSeven™ and Factor Vila (invention). Degradation products of lower molecular weight are visible as peaks at higher elution times.

Figure 3 shows the activities of NovoSeven™ and Factor Vila (invention) determined by Quick^'s prothrombin time. Prothrombin time was determined by Ceveron® alpha TGA (Technoclone). Factor Vila (invention) was measured in three different test series each representing six independent determinations measured in duplicates. NovoSeven™ was included in each test series as a single sample measured in duplicates. Figure 4 shows the activities of NovoSeven and Factor Vila (invention) determined by chromogenic COASET® FVII assay. Factor Vila (invention) was measured in duplicates several times in 4 different assays. NovoSeven™ was included in each analysis as a single sample measured in duplicates. Figure 5 shows the tissue factor binding of Factor Vila (invention) and NovoSeven™ by ELISA. Mean and SD from duplicate measurements are given.

Figure 6 shows stability of the factor Vila construct after storage for 14 days at different temperature as analyzed by denaturating reducing SDS-PAGE using 4-20% Mini-PROTEAN® TGX™ Precast Gels followed by Coomassie staining, lane 1 : 4°C; lane 2: room temperature; lane 3: 37°C.

EXAMPLES

Example 1 : Preparation of factor VII (invention)

Factor VII is produced by cultivation of GT-5s cells stably transfected with an expression construct harbouring the proprotein of human factor VII (UniProt accession number P08709) for secretory production.

After fermentation factor VII is isolated from the supernatant using affinity chromatography as capture step followed by multimodal anion exchange chromatography. Then, factor VII is activated by incubation in a solution comprising 25 mM Tris, 30 mM CaCI₂ and 100 μΜ ZnCI₂, pH 8.0, at 2 to 8°C for several days. Factor Vila is gained at high purity.

In a similar manner, also a fusion protein of factor VII fused to albumin and a dimer construct wherein the light chain of activated factor VII (factor Vila) is fused to a first dimerization domain and the heavy chain of factor Vila is fused to a second dimerization domain were produced.

Example 2: Glycoprofiling - N-glycosylation

The glycoprofiles of the different preparations of the factor VII constructs were determined by structural analysis of the glycosylation. Glycoprofiling generates information on the complex glycan structure of the glycosylation sites. For glycoprofiling of the N-glycosylation, the intact N-glycans were released from the protein core employing PNGase F. The digestion was performed in a gel or gel block for unambiguous workup. Free N-glycans were labeled with a fluorescence marker and the purified sample of labeled N-glycans was separated by means of hydrophilic interaction chromatography (HILIC) with fluorometric detection. This analysis gave the following results ("factor VII inv.": factor VII according to the present invention; "albumin fusion": factor VII - albumin fusion protein according to the invention; "factor Vila inv." dimer construct of the activated factor VII according to the invention; "plasma": plasma- derived factor VII obtained from human donors; "Novo7": factor VII recombinantly expressed in BHK cells, commercial product NovoSeven™ of Novo Nordisk):

Table 2: Relative amounts of different glycosylation properties

shown are the relative amounts of glycans having the following property:

F: fucose; B: bisecting GlcNAc; SO - S4: zero to four sialic acids; S>0: at least one sialic acid; GO - G4: zero to four galactoses; G>0: at least one galactose; A1 - A4: mono- to tetraantennary; tG: at least one terminal galactose; tG1 - tG4: one to four terminal galactoses; M: mannose-rich glycans; NeuGc: glycans with NeuGc; Galili: glycans with Galili epitope

In conclusion, the factor VII according to the present invention (Factor VII (invention)) has a high degree of bisecting N-acetlyglucosamine, a high antennarity and a high degree of galactosylation. It is assumed that because of one or more of these three glycosylation parameters, the factor VII (invention) has superior properties compared to the common recombinant or plasma-derived factor VII preparations. Furthermore, the factor VII (invention) is also highly sialylated and highly fucosylated.

Furthermore, the glycan structures of the factor VII preparation was also analyzed by a much less accurate analysis method, wherein the following results were obtained: Table 3: Relative amounts of different glycosylation properties

These data are only shown as reference and are less reliable than the data shown in Table 2.

Example 3: Glycoprofiling - O-glycosylation

For glycoprofiling of the O-glycosylation, factor VII was proteolytically digested with Trypsin and the peptide comprising the O-glycosylation sites at amino acid positions 52 and 60 of factor VII was analyzed by LC-MS/MS. This analysis gave the following results:

Table 4: Relative molar amounts of peptides with different glvcosylation properties

shown are the relative amounts of peptides having the following glycosylation:

F: fucose; Glc: glucose; Glc-Xyl: glucose-xylose; Glc-Xyl-Xyl: glucose-xylose-xylose; w/o: without glycosylation

In conclusion, nearly in every factor VII according to the present invention the O- glycosylation site at serine 60 is occupied by a fucose residue and the O-glycosylation site at serine 52 is occupied by a glucose residue carrying 0 to 2, in particular either 0 (-40%) or 2 (-60%), xylose residues. Example 4: γ-Carboxylation

The N-terminal sequence of human FVII comprises 10 glutamic acid residues which can be γ-carboxylated (GLA domain). It was shown that the extent of γ-carboxylation for recombinant factor Vila is variable. However, it was found that the ability of factor Vila to reduce prolonged aPTT in hemophilia A and B plasma was comparable for recombinant factor Vila with 9 γ-carboxylated glutaminic acid moieties and one partially γ-carboxylated glutaminic acid moiety versus plasma derived factor Vila which is fully γ-carboxylated.

To evaluate the γ-carboxylated glutamic acid residues, factor Vila according to the invention was digested with Lys-C and subsequently deglycosylated employing

PNGase F treatment. The resulting mixture of peptides is separated by use of a modified reversed phase chromatography column (Charge Surface Hybrid C18) with mass detection (High resolution ESI-QTOF).

Digestion of factor VII with Lys-C results in one peptide comprising all 10 v- carboxylated glutamic acid residues, which has the sequence

ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTK (amino acid positions 1 to 38 of SEQ ID NO: 1 ). The high density of negative charge in the N-terminal region is probably responsible for two missed cleavage sites.

The peptide was separated from all other peptides resulting from Lys-C digestion. Identification of peptide species with different content of γ-carboxylated glutamic acid residues was performed on base of exact mass using high resolution ESI-QTOF mass spectrometry and additionally CID-MS/MS fragmentation for sequence analysis. Peptide species comprising 0-10 γ-carboxylated glutamic acid residues were screened, only highly γ-carboxylated species with 9 or 10 γ-carboxylated glutamic acid residues were detected.

Relative quantification of γ-carboxylation is based on peak areas of the extracted ion chromatograms generated for both detected carboxylation isoforms of the peptide. Mass spectrometric quantification resulted in 79% of N-terminal peptide comprising 9 v- carboxylated glutamic acid residues and 21 % of peptide species with complete γ- carboxylation.

Factor VII according to the invention is highly γ-carboxylated in the GLA domain even though ca. 80% of factor VII lack one γ-carboxylation at one of the glutamic acid moieties of the GLA domain. However, it was found that recombinant factor Vila with only partial γ-carboxylation at one glutamic acid residue provided essentially the same bioactivity with respect to blood clotting compared to plasma derived factor VII with complete γ-carboxylation. The exact position of partially γ-carboxylated glutamic acid in the GLA domain of factor Vi la (invention) could not be determined exactly although CID-MS/MS indicate most probably position 35 to be partially γ-carboxylated which is in concert with data reported for recombinant factor Vila.

Example 5: Stability in solution

The stability of factor VI I (invention) in solution was tested. The lyophylized preparation of factor VI I (invention) was reconstituted and analyzed for degradation fragments using non-reducing SDS-PAGE and size exclusion chromatography (SEC). Reconstituted recombinant factor VI I produced in BH K cells (NovoSeven™ of Novo Nordisk) was used as comparative example.

SDS-PAGE: Degradation was analyzed by denaturating non-reducing SDS-PAGE using 4-20% Mini-PROTEAN® TGX™ Precast Gels followed by Coomassie staining. Degradation bands were quantified by densitometric evaluation (see Figure 1 ).

SEC: Analytical size exclusion chromatography was used to analyze degradation under native conditions. Samples were analyzed by HPLC on a BEH200 column at 0.3 mL/min in 1 x TBS pH 7.5. Results are given as percent of total peak area (see Figure 2).

Table 5: Degradation of reconstituted factor VI I

Assay factor VII (inv.) NovoSeven™

SDS-PAGE <1 % fragments -10% fragments

SEC -1 .2% fragments -3.8% fragments In conclusion, the factor VI I having the superior glycosylation pattern according to the invention is significantly more stable than the commercially available recombinant factor VI I obtained from BH K cells which do not provide a comparable glycosylation pattern.

Example 6: Prothrombin time The prothrombin time (PT, also Quick's prothrombin time or Quick assay) was developed to measure prothrombin (Factor II) and is sensitive to abnormalities of factors VI I, X, V, I I and fibrinogen. It is a one-stage test based upon on the time required to form a fibrin clot after the addition of tissue factor (TF) (thromboplastin), phospholipid and calcium to decalcified, platelet poor plasma. In principle, plasma is mixed with TF and phospholipid at 37°C. Coagulation is initiated by addition of calcium chloride. The time taken from the addition of calcium to the formation of the fibrin clot is known as the prothrombin time.

Factor Vila activity was measured using the Quick assay, performed on the fully automated coagulation analyzer Ceveron® alpha TGA (Technoclone, Austria).

Samples of factor Vila according to the invention and NovoSeven™ were diluted in PBS/10% factor VII deficient plasma and the PT-assay was run according to the manufacturer's protocol. Factor Vila activity was calculated using the internal calibration of the coagulation analyzer. Factor Vila (invention) shows comparable activity to NovoSeven™ in Quick's prothrombin time indicating comparable coagulation activity in vitro (see Figure 3). Since both proteins mediate blood coagulation in an identical manner, comparable coagulation activity of factor Vila (invention) and NovoSeven™ can be expected in vivo. Example 7: Chromogenic Assay

To determine factor VII / factor Vila activity in plasma a chromogenic assay (COASET® FVII, Chromogenix) was used. In this assay, factor X is activated to factor Xa and factor VII, if not yet, is completely activated to factor Vila. Subsequently, factor Xa separates the chromophoric group pNA from a substrate (S-2765) that can be measured photometrically at 405 nm (see also following reaction scheme).

TF, Ca2 +, FVU→FVIla

FX > FXa

FXa

S— 2765— Peptide + pNA (yellow color)

Activation of factor X by factor Vila (invention) is similar to activation of factor X by NovoSeven™ (see Figure 4). Activities of both proteins are identical. Initiation of coagulation and following thrombin generation would have proceeded in the same time as it is detectable in the Quick assay (example 6).

Example 8: Binding to tissue factor

Tissue factor (TF) is a membrane protein that plays a role in initiation of thrombin generation by forming a complex with circulating factor VII or factor Vila and subsequent activation of factor X and factor IX. Optimal binding of factor VII to TF is therefore essential for an optimal activity of factor VI I / factor Vila. To evaluate the binding properties of factor Vila (invention) to TF, recombinant human TF (American Diagnostica) was bound on a 96-well microtiter plate (Nunc™ MaxiSorp™), blocked with Blocker Casein (Thermo Scientific) and incubated with different concentrations of factor Vila (invention). Bound factor Vila (invention) as well as NovoSeven™ was detected by an anti-factor VII antibody (abeam). The secondary antibody (dianova) was conjugated with peroxidase which reacts with its substrate TMB (TMB One Component HRP Microwell Substrate, tebu-bio) under color development. After stopping the reaction with sulfuric acid, soluble yellow reaction product was quantified at 450 nm in a plate reader (Tecan).

Factor Vila (invention) shows concentration-dependent binding to tissue factor comparable to NovoSeven™ (see Figure 5). Interaction of factor Vila with TF enhances its activity and is therefore a key feature of the protein.

Example 9: Stability in solution of the factor Vila construct

The stability of the factor Vila construct (invention) in solution was tested at different storage temperatures for 14d and analyzed for degradation using reducing and non- reducing SDS-PAGE (see Figure 6) and size exclusion chromatography (SEC).

SEC: Analytical size exclusion chromatography was used to analyze degradation under native conditions. Samples were analyzed by HPLC on a BEH200 column at 0.3 mL/min in 1 x TBS pH 7.5. Results are given as percent of total peak area (see Table 6).

Table 6: Stability of fusion factor VII in solution relative area [%]

Sample dimerization /

monomer fragment

aggregation storage 14d, 4°C 97,57 0,1 1 2,32

storage 14d, RT 97,83 0,59 1 ,57

storage 14d, 37°C 97,58 0,86 1 ,56

In conclusion, SDS-PAGE reveals no detectable changes in band appearance of the factor Vila construct according to the invention after storage at 4 °C, RT or 37 °C for 14d in solution. Furthermore, fragmentation analyzed by SEC-HPLC shows only minor increase by elevated storage temperatures whereas dimerization and aggregation content decreases slightly.

Claims

A recombinant factor VII preparation, wherein the recombinant factor VII in the preparation has a glycosylation pattern at its N-glycosylation sites comprising the following characteristics:

The recombinant factor VII preparation according to claim 1 , wherein the glycosylation pattern comprises the following characteristics:

(i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of at least 15% of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation.

The recombinant factor VII preparation according to claim 1 or 2, wherein the glycosylation pattern comprises the following characteristics:

(ii) a relative amount of tetraantennary glycans of at least 10% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or

(iii) a relative amount of glycans carrying four galactose residues of at least 4% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation.

The recombinant factor VII preparation according to claim 3, wherein the glycosylation pattern comprises at least two of the features (i), (ii) and (iii), and preferably all of the features (i), (ii) and (iii).

A recombinant factor VII preparation obtainable by production in the human cell line GT-5s or a cell line derived therefrom or a cell line homologous thereto.

The recombinant factor VII preparation according to any one of claim 1 to 5, wherein the recombinant factor VII in the preparation has a glycosylation pattern at its N-glycosylation sites comprising one or more of the following further characteristics:

(a) a relative amount of glycans carrying at least one galactose residue of at least 90% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or

(b) a relative amount of glycans carrying at least one terminal galactose residue of at least 15% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or

(c) a relative amount of glycans carrying fucose of at least 40% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or

(d) a relative amount of glycans carrying at least one terminal GalNAc residue of not more than 10% of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation; and/or

(e) no detectable NeuGc and/or Galal ,3-Gal structures; and/or

(f) it is a human glycosylation pattern.

The recombinant factor VII preparation according to any one of claim 1 to 6, wherein the recombinant factor VII in the preparation has a glycosylation pattern at its N-glycosylation sites comprising one or more of the following characteristics:

(ii) a relative amount of tetraantennary glycans in the range of from 1 % to 35% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or

(iii) a relative amount of glycans carrying at least one galactose residues in the range of from 88% to 100% of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation.

The recombinant factor VII preparation according to any one of claim 1 to 7, wherein the recombinant factor VII in the preparation has a glycosylation pattern at its N-glycosylation sites comprising one or more of the following characteristics: (i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of at least 15% of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation; and/or

(ii) a relative amount of glycans carrying at least one galactose residues of at least 90% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation.

9. The recombinant factor VII preparation according to any one of claim 1 to 8, wherein the recombinant factor VII in the preparation has a glycosylation pattern at its N-glycosylation sites comprising one or more of the following further characteristics:

(a) a relative amount of glycans carrying at least one galactose residue in the range of from 90% to 100% of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation; and/or

(b) a relative amount of glycans carrying at least one terminal galactose residue in the range of from 20% to 60% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or

(c) a relative amount of glycans carrying fucose in the range of from 50% to 100% of the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation; and/or

(d) a relative amount of glycans carrying at least one terminal GalNAc residue of not more than 7% of the total amount of glycans attached to the N- glycosylation sites of factor VII in the preparation; and/or

(e) a relative amount of glycans carrying NeuGc and/or Gala1 ,3-Gal structures of less than 0.1 % the total amount of glycans attached to the N-glycosylation sites of factor VII in the preparation.

10. The recombinant factor VII preparation according to any one of claim 1 to 9, wherein the recombinant factor VII in the preparation has an average amount of at least 9 γ-carboxylated glutamic acid residues.

1 1 . The recombinant factor VII preparation according to any one of claim 1 to 10, wherein at least 15% of the recombinant factor VII in the preparation comprise 10 γ-carboxylated glutamic acid residues.

12. The recombinant factor VII preparation according to any one of claim 1 to 1 1 , wherein the recombinant factor VII in the preparation has one or more of the following characteristics

(a) it is human recombinant factor VII; and/or (b) it is activated factor Vila; and/or

(c) it is a fusion protein, wherein the non-activated single chain factor VII is fused to a fusion partner, or wherein one or both of the light chain and the heavy chain of the activated factor Vila are fused to separate fusion partners; and/or

(d) it comprises one or more further agents conjugated to its polypeptide chain(s); and/or

(b) it is produced by a human cell line or human cells.

13. The recombinant factor VII preparation according to any one of claim 1 to 12, wherein the recombinant factor VII in the preparation has a glycosylation pattern at its O-glycosylation sites comprising one or more of the following further characteristics:

(a) at least 90% of the factor VII proteins in the preparation are glycosylated at the O-glycosylation site corresponding to Ser60 of SEQ ID NO: 1 with a fucose residue; and/or

(b) about 30% to about 50% of the factor VII proteins in the preparation are glycosylated at the O-glycosylation site corresponding to Ser52 of SEQ ID NO:

1 with a glucose residue; and/or

(c) about 50% to about 70% of the factor VII proteins in the preparation are glycosylated at the O-glycosylation site corresponding to Ser52 of SEQ ID NO: 1 with the structure glucose-xylose-xylose.

14. A pharmaceutical composition comprising the recombinant factor VII preparation according to any one of claims 1 to 13.

15. The recombinant factor VII preparation according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 14 for use in medicine, in particular in the treatment of bleeding disorders.

16. The recombinant factor VII preparation or the pharmaceutical composition according to claim 15 for use in the treatment of bleeding disorders, wherein the bleeding disorder is selected from the group consisting of hemophilia A and B; acquired hemophilia; congenital factor Vll-deficiency; hemophilia prophylaxis for patients with inhibitors, trauma, bleeding in emergencies or surgical intervention such as spinal and cardiac surgery; Glanzmann's thrombasthenia; blast lung injury; and hemorrhages such as intracerebral hemorrhage, unspecific hemorrhage, or diffuse alveolar hemorrhage.