US20130011382A1 - Fibrinogen Preparations Enriched In Fibrinogen With An Extended Alpha Chain - Google Patents

Fibrinogen Preparations Enriched In Fibrinogen With An Extended Alpha Chain Download PDF

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US20130011382A1
US20130011382A1 US13/520,615 US201113520615A US2013011382A1 US 20130011382 A1 US20130011382 A1 US 20130011382A1 US 201113520615 A US201113520615 A US 201113520615A US 2013011382 A1 US2013011382 A1 US 2013011382A1
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fibrinogen
preparation
pharmaceutical composition
chain
plasma
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Joseph Grimbergen
Jacob Koopman
Abraham Bout
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Profibrix BV
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/363—Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4833—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins

Definitions

  • the present invention relates to fibrinogen preparations and the use of fibrinogen preparations in medical applications.
  • it relates to fibrinogen preparations which are enriched in fibrinogen with an extended alpha chain, to methods for producing it and to its applications.
  • Fibrinogen is a soluble plasma glycoprotein which is synthesized in the human body primarily by liver parenchymal cells. It is a dimeric molecule, consisting of two pairs of three polypeptide chains designated A ⁇ , B ⁇ and ⁇ , which are connected by disulfide bridges. The three polypeptide chains are encoded by three separate genes. The predominant form (HMW fib) harbors an A ⁇ chain which is synthesized as a 625 amino acid precursor and is present in fibrinogen found in blood plasma as a 610 amino acids polypeptide chain, the B ⁇ chain contains 461 and the ⁇ chain 411 amino acids. The three polypeptides are synthesized individually from 3 mRNAs. Assembly of the three component chains (A ⁇ , B ⁇ , and ⁇ ) into its final form as a six-chain dimer (A ⁇ , B ⁇ , ⁇ ) 2 occurs in the lumen of the endoplasmic reticulum (ER).
  • ER endoplasmic reticulum
  • Fibrinogen circulates in blood at high concentrations (1-2 g/L) and demonstrates a high degree of heterogeneity. It is estimated that in each individual about one million different fibrinogen molecules circulate. Most of these variants, which account for just a small portion of the total fibrinogen (in most cases not more than a few percents), differ in function and structure.
  • Fibrinogen is synthesized in the high-molecular weight form (HMW; molecular weight 340 kDa; the predominant form of A ⁇ chains in the circulation contains 610 amino acids).
  • HMW high-molecular weight form
  • LMW′ form 270 kDa
  • HMW high-molecular weight form
  • HMW and LMW′ variants show distinct differences in clotting time and fibrin polymer structure (Hasegawa N, Sasaki S. (1990) Thromb. Res. 57, 183).
  • the ⁇ -ext Fib or Fib420 variant which has a molecular weight of 420 kDa, accounts for 1-3% of the total circulating fibrinogen (de Maat and Verschuur (2005) Curr. Opin. Hematol. 12, 377).
  • the extended ⁇ -ext Fib isoform is distinguished from the conventional ⁇ -chain of fibrinogen by the presence of an additional 236 residue C-terminus globular domain due to alternative splicing. Contradictary data on the function and characteristics of Fib420 is given in literature.
  • the present invention relates to a fibrinogen preparation which contains at least 95% (w/w) fibrinogen and wherein at least 10% of the fibrinogen is in the form of Fib420.
  • Fib420 is also referred to as ‘ ⁇ -ext Fib’, ‘fibrinogen with an extended alpha chain’ or ‘fibrinogen 420 ’.
  • these terms are used interchangeably. All these terms refer to a symmetrical molecule of the structure (A ⁇ ext , B ⁇ , ⁇ ), wherein both conventional fibrinogen ⁇ -chains, as found in HMW fib, have been replaced by extended alpha chains.
  • fibrinogen preparation refers to fibrinogen in isolated form, for example to a plasma isolate or cell supernatant isolate of fibrinogen. It may also refer to a synthetic preparation of fibrinogen.
  • a fibrinogen preparation according to the invention preferably contains at least 65% w/w, at least 70% w/w, at least 75% w/w, at least 80% w/w or at least 85% fibrinogen, based on total protein.
  • a fibrinogen preparation according to the invention comprises at least 99% w/w or at least 99.5% w/w fibrinogen, based on total protein.
  • Such pure fibrinogen preparations are particularly suitable to formulate compositions which are used in medical applications, such as compositions for wound therapy and surgical repair.
  • At least 10% w/w of the fibrinogen in the preparation is in the form of Fib420.
  • at least 15% w/w, at least 20% w/w, at least 25% w/w or at least 30% w/w of the fibrinogen is in the form of Fib420.
  • at least 40% w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w or at least 90% w/w is in the form of Fib420.
  • at least 95% w/w, at least 99% w/w or all of the fibrinogen is in the form of Fib420.
  • fibrinogen preparation according to the invention is that plasmin-mediated digestion of the fibrinogen preparation is slower than for plasma derived fibrinogen. Its enhanced resistance to plasmin digestion is likely to be beneficial for treatment of patients who suffer from hyperfibrinolysis which is often seen in cases of acquired coagulopathy.
  • clots are formed faster and the clots which are formed have a higher clot firmness than clots formed with plasma derived fibrinogen or HMW fibrinogen.
  • fibrin clot stability is enhanced when using the fibrinogen preparation according to the invention and that the fibrinogen preparation according to the invention is more potent than state of the art preparations.
  • a more potent fibrinogen preparation allows for the reduction of both the amount of fluid and the amount of therapeutical protein to be administered. This is an advantage for intravenous use to treat dilutional coagulopathy.
  • fibrinogen for intravenous (i.v) injection of fibrinogen to compensate for low blood clotting activity, high dosages of fibrinogen are required ( ⁇ 5 gram of fibrinogen per treatment). This is administered by direct injection at a dose of 70 mg/kg. As fibrinogen in general can be dissolved at a maximum concentration of 20 mg/ml, this means that about 250 ml of fluid has to be administered intravenously to in an adult. Lowering this dose by providing a more potent fibrinogen would be beneficial.
  • fibrinogen preparation of the invention is less Factor XIII dependent than preparations of plasma derived fibrinogen or HMW fibrinogen.
  • the strength of a clot made in buffer by alpha-extended Fib is higher than for plasma-derived fibrinogen which will contain some Factor XIII. No Factor XIII is required to form firm clots. Therefore, in one embodiment, the fibrinogen preparation according to the invention is free of Factor XIII.
  • Clotting time (CT), clot formation time (CFT) and clot firmness of ⁇ -ext rhFib and plasma derived fibrinogen can be measured using ROTEM analysis.
  • ROTEM® Pultapharm GmbH, Kunststoff, Germany
  • the technique utilizes a rotating axis submerged in a (blood) sample in a disposable cuvette. Changes in elasticity under different clotting conditions result in a change in the rotation of the axis, which is visualized in a thromboelastogram, reflecting mechanical clot parameters (see e.g. Luddington R. J. (2005) Clin Lab Haematol. 2005 27(2):81).
  • a fibrinogen preparation according to the invention typically performs at least as good or better than a fibrinogen preparation or composition in which the fibrinogen variant distribution resembles the variant distribution in human plasma, viz. less than 5% w/w of the fibrinogen is of the Fib420 type.
  • a fibrinogen preparation or composition in which the fibrinogen variant distribution resembles the variant distribution in human plasma, viz. less than 5% w/w of the fibrinogen is of the Fib420 type.
  • fibrinogen concentrates which are based on plasma-derived fibrinogen.
  • Such concentrates are commercially available.
  • its clot formation time will be less than the clot formation time of a fibrinogen preparation in which less than 5% w/w of the fibrinogen is of the Fib420 type.
  • the clot formation time of a fibrinogen preparation according to the invention is maximally 80%, maximally 60%, maximally 50%, maximally 40%, maximally 30%, maximally 20%, maximally 10% of the clot formation time of a fibrinogen preparation in which less than 5% w/w of the fibrinogen is of the Fib420 type, such as plasma-derived fibrinogen preparations.
  • the ROTEM experiments with ⁇ -ext rhFib presented in the Examples indicate that a fibrinogen preparation according to the invention has a clot formation time (CFT) which is less than the CFT of purified plasma derived fibrinogen produced for intravenous applications.
  • CFT clot formation time
  • the invention in another aspect, relates to a composition comprising a fibrinogen preparation according to the invention.
  • the composition may comprise an activator, such as thrombin or a thrombin-like protein, such as reptilase. It may also comprise excipients which are suitable for use in an injectable preparation.
  • the preparation may be in dry form and subsequently reconstituted, with e.g. buffered saline, and the like, or it may be in liquid form, either as a suspension or solution.
  • Suitable excipient materials may include solvents and co-solvents, such as ethanol, glycerin, PEGs, oils, and the like; solubilizing, wetting, suspending, emulsifying or thickening agents, such as carboxymethylcellulose, hydrolyzed gelatin, pluronics, polysorbates, and the like; chelating agents, such as calcium EDTA, DTPA, and the like; antioxidants and reducing agents, such as BHT, ascorbic acid, sodium metabisulphite, and the like; antimicrobial preservatives, such as benzyl alcohol, phenol, parabens, and the like; buffers and pH adjusting agents, such as tromethamine, sodium phosphate, sodium acetate, sodium hydroxide, and the like; bulking agents, protectants, and tonicity adjustors, such as alanine, albumin, dextran, lactose, sorbitol, sodium chloride, histidine, and the like; special additives,
  • the composition may be used in any application in which plasma-derived or recombinant fibrinogen is used.
  • the main applications are hemostasis and to seal tissue.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition comprises a fibrinogen preparation according to the invention and a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” refers to a vehicle, auxiliary agent, adjuvant, diluent, excipient or carrier with which the fibrinogen preparation of the invention is administered.
  • Examples of pharmaceutically acceptable carriers include, without limitation, water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof.
  • Suitable pharmaceutically acceptable carriers and formulations are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995) and “Remington: The Science And Practice Of Pharmacy” by Alfonso R. Gennaro (Lippincott Williams & Wilkins, 2005).
  • the pharmaceutical composition may be applied topically and may include carriers which are water-soluble, water-absorbent, water-insoluble or water-swellable.
  • Suitable materials include saccharides such as mono-and di-saccharides, including lactose, mannitol and trehalose, or dextran and dextran polymers, like e.g. Sephadex, which are available in different particle sizes, starches, pullulan derivatives, hyaluronic acid esters, and the like.
  • Cellulose products such as microcrystalline cellulose (Avicel range), methylcellulose, carboxymethyl cellulose, microfine cellulose or hydroxy propyl cellulose, and other materials such as cross-linked polyvinyl pyrrolidone (PVP), may be used singly or in admixture.
  • suitable carriers include polyethylene glycol (PEG), preferably having a molecular weight of about 1000; polyvinylpyrrolidone (PVP), preferably having an average molecular weight of about 50,000; poly(acrylic acid), PVA, poly(methylvinylether co-maleic anhydride), poly(ethyleneoxide), and dextran, typically having an average molecular weight of about 40,000.
  • Tablet disintegrants may be included. These materials will absorb moisture from the wound, expand rapidly and thereby enhance the wettability of the hemostatic components of the powder blend. Suitable examples include sodium starch glycolate (Explotab® or Primojel®) which has an average particle size in the range of 35-55 ⁇ m. About 25% of the glucose units are carboxymethylated; cross-linked polyvinyl pyrrolidone (Polyplasdone®); alginates and alginic acid; cross-linked sodium carboxymethylcellulose (Ac-Di-Sol). Gums and gelling agents that can be used include, for example, tragacanth, karaya gum, soluble starch, gelatin, pectin, guar gum and gellan gum.
  • Emdex® i.e. a hydrated form of dextrates (spray crystallized dextrose containing small amounts of starch oligosaccharides). It is a highly refined product composed of white, free-flowing, spray-crystallized macroporous spheres with a median particle size of 190-220 ⁇ m.
  • NON-PAREIL SEEDS® (Sugar Spheres). These are used in multiple drug units for improved content uniformity, consistent and controlled drug release and high drug stability, size ranges from 200 to 2000 mm.
  • the pharmaceutically acceptable carrier may comprise an effervescent couple.
  • the gas produced following an effervescent reaction can expand the fibrin sealant into a ‘foam’ and/or increase wettability of the powders comprising the fibrin sealant.
  • the effervescent components dissolve, react and liberate, say, carbon dioxide, thereby increasing the wettability of the hemostatic components and thus enhancing time to clot formation.
  • the fibrin sealant will appear as a stable foam once fully reacted and the clot has formed.
  • the effervescent couple typically comprises citric acid or sodium hydrogen citrate and sodium bicarbonate, but other physiologically acceptable acid/alkaline or alkaline earth metal carbonate mixtures may be used, for example tartaric, adipic, fumaric or malic acids, and sodium, potassium or calcium (bi)carbonates or sodium glycine carbonate.
  • the pharmaceutical composition according to the invention is suitable for facilitating tissue adherence, improving wound healing or for intravenous administration.
  • the pharmaceutical composition according to the invention is a fibrin sealant.
  • the fibrin sealant according to the invention typically comprises thrombin.
  • the sealant may be in any convenient form, be it dry or liquid.
  • a suitable example of a dry sealant in which the fibrinogen preparation according to the invention may be used is Fibrocaps® powder sealant, which is described in WO97/44015 and which is based on micro-particles of fibrinogen and thrombin.
  • the separate components are prepared by spray-drying, fibrinogen with trehalose and thrombin with trehalose. Each powder component has a predominant particle size of approximately up to 50 ⁇ m in diameter.
  • the Fibrocaps® fibrin sealant which is a blend of these components, has been demonstrated to be an easy-to-use, stable and efficacious topical haemostat.
  • the product can be used immediately, without reconstitution and is useful in wound therapy, in surgical repair and as an extravascular stent.
  • aqueous fluid such as blood
  • the exposed active thrombin converts the exposed fibrinogen into insoluble fibrin polymers.
  • the fibrinogen preparation according to the invention would give further improved blood clotting properties.
  • the composition of the invention may be applied to wounds, sutures, incisions and other openings where bleeding may occur.
  • a wound includes damage to any tissue in a living organism.
  • the tissue may be an internal (e.g. organ) or external tissue (e.g. eye or skin), and may be a hard tissue (e.g. bone) or a soft tissue (e.g, liver or spleen).
  • the wound may have been caused by any agent, including infection, surgical intervention, burn or trauma.
  • the composition of the invention may be used for surgical interventions such as in the gastrointestinal system, e.g.
  • ENT ear, nose and throat area
  • ENT surgical interventions in the ear, nose and throat area
  • ENT dental surgery, cardiovascular surgery, aesthetic surgery, neurological surgery, lymphatic, biliary, and cerebrospinal (CSF) fistulae, air leakages during thoracic and pulmonary surgery
  • thoracic surgery including surgery of the trachea, bronchi and lungs, gynaecological, vascular, urological, bone (e.g. spongiosa resection), and emergency surgery.
  • the composition of the invention may be applied to the outside of a segment or the whole of a vein graft.
  • a suitable extravascular stent composition is a composition of a fibrinogen preparation according to the invention and thrombin.
  • the composition may be in any suitable for, be it liquid or dry.
  • a dry powder composition for instance one as described above, is used as an extravascular stent.
  • the dry powder composition polymerizes in the limited amounts of bodily fluids which are naturally present at the outside of the vessel wall, thus forming an extravascular stent.
  • a vein coated with an extravascular stent composition according to the present invention is also part of the invention.
  • the vein may be any kind of vein which needs to be protected from overextension or which needs support, for instance a varicose vein.
  • the vein is a venous graft.
  • the composition is applied before the venous graft is introduced in the human or animal body. In another embodiment, the composition is applied after the venous graft has been introduced in the human or animal body.
  • the present invention relates to the use of a fibrinogen preparation according to the invention as a medicament. It can be used for the preparation of a medicament for the treatment of acute bleeding episodes, hyperfibrinolysis, fibrinogen deficiency, be it acquired or congenital, or other bleeding disorders.
  • a composition according to the invention is used in a method for reducing bleeding at a hemorrhaging site.
  • a hemostatically effective amount of the composition according to the invention is used.
  • TTH time to hemostasis
  • a time to hemostasis (TTH) of about 10 minutes or less, about 5 minutes or less, or about 3 minutes or less may be achieved.
  • TTH is the time it takes to stop a bleeding. If a pressure sheet is used, measurement of TTH typically starts when the pressure sheet is applied to the bleeding site and runs until bleeding stops by visualization of the dressing and/or no bleeding through or around the dressing is observed.
  • the present invention relates to a method for preparing a fibrinogen preparation according to the invention.
  • Such preparation may be prepared in any suitable way, using techniques available in the art.
  • high expression levels of intact, functional fibrinogen are required and therefore recombinant production is preferred.
  • fibrinogen or a fibrinogen chain is ‘in intact form’ when the amino acid sequence contains all the amino acids which were encoded for by the nucleotide sequence, optionally without the amino acids which are removed during normal cell (secretion) processing. Therefore, alpha-ext chains having 866 or 847 amino acids are examples of an alpha chain in intact form.
  • Recombinant production of fibrinogen has many advantages over the use of plasma derived materials. These include its preferred safety profile, the possibility to make pure homogeneous preparations of variants free of any other blood born contaminants and the unlimited supply. In addition, for specific applications (e.g. use of fibrinogen as an intravenous hemostat) proper post-translational modifications (e.g. glycosylation) are required. Therefore, expression in eukaryotic, in particular mammalian systems, more particular in human systems, is preferred.
  • a fibrinogen preparation according to the invention is prepared by a method which comprises the steps of:
  • Expression vectors for eukaryotic hosts are known in the art and any of the vectors conventionally used for expression in eukaryotic cells may be used.
  • An expression vector typically contains a promoter operably linked to the nucleic acid sequence to be expressed and ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains and enhancers.
  • an expression vector for expression in mammalian cells such as for CHO or PER.C6® cells is used.
  • Such vectors are known in the art and suitable examples include pcDNA3.1 plasmids, GATEWAY (Invitrogen), pCMV/Bsd (Invitrogen), pFN vectors (Promega) and numerous other vector systems.
  • an alpha extended polypeptide chain of fibrinogen may refer to a fibrinogen alpha chain of 866 amino acids with a signal sequence or to one without a signal sequence and to any variants thereof which have arisen through genetic polymorphisms or differences in glycosylation and phosphorylation.
  • a suitable example of an alpha extended chain amino acid sequence is given in SEQ ID No. 1.
  • the terms ‘alpha chain’ and ‘A ⁇ chain’ are used interchangeably in the context of the present invention.
  • the eukaryotic cell must also contain nucleic acid sequences which encode the beta and gamma chain of fibrinogen to be able to produce a fibrinogen molecule. Recombinant production of fibrinogen from alpha, beta and gamma chains have been described before, see for instance PCT/EP2009/058754, U.S. Pat. No. 6,037,457 or WO 95/023868.
  • beta chain and ‘B ⁇ chain’ are used interchangeably. They may refer to both wild type and variants of the beta chain, with or without signal sequence.
  • a suitable example of a fibrinogen beta chain amino acid sequence is given in SEQ ID No. 2.
  • gamma chain and ‘ ⁇ chain’ are used interchangeably. They may refer to both wild type and variants of the gamma chain, with or without signal sequence. Suitable examples of a fibrinogen gamma chain amino acid sequence are given in SEQ ID No. 3 and 4.
  • the nucleic acid sequences encoding a fibrinogen chain are optimized.
  • An optimized nucleic acid sequence allows for the efficient expression of recombinant fibrinogen in intact form.
  • they are optimized for expression in a eukaryotic cell culture system, such as for expression in a COS cell, BHK cell, NS0 cell, Sp2/0 cell, CHO cell, a PER.C6 cell, a HEK293 cell or insect cell culture system. More preferably, they are optimized for expression in a mammalian cell culture system.
  • the nucleic acid sequences are optimized for expression in a human cell culture system, such as for a PER.C6 cell or a HEK293 cell culture system.
  • the nucleotide sequence which is optimized may be DNA or RNA. Preferably, it is cDNA.
  • An optimized nucleotide sequence encoding a fibrinogen alpha extended, beta or gamma chain shows at least 70% identity to its respective non-optimized counterpart.
  • an optimized nucleotide sequence encoding a fibrinogen ⁇ -ext Fib, B ⁇ and ⁇ chain shows 70-80% identity to its respective non-optimized sequences.
  • the optimized nucleotide sequences encoding a fibrinogen alpha extended, beta or gamma chain contain no cis-acting sites, such as splice sites and poly(A) signals.
  • An optimized nucleotide sequence which is used in the method according to the invention and which encodes a fibrinogen alpha extended chain contains no 39 basepair direct repeat sequences which are normally present in the gene encoding the alpha chain of human fibrinogen.
  • the repeating sequence must be changed without changing the encoded protein sequence.
  • an optimised nucleotide sequence according to SEQ ID NO. 5 or the part of this sequence without the signal is used for expressing the alpha extended chain.
  • an optimised nucleotide sequence according to SEQ ID NO. 6 or the part of this sequence without the signal sequence is used for expressing the beta chain.
  • an optimised nucleotide sequence according to SEQ ID NO. 7 or 8 or the part of these sequences without a signal sequence is used for expressing the gamma chain.
  • Nucleic acid sequences according to the invention may be encoding any type of fibrinogen chains. Preferably they are encoding mammalian fibrinogen chains, more preferably they are encoding primate fibrinogen chains, most preferably they are encoding human fibrinogen chains. Also combinations are possible, such as for example one or two mammalian fibrinogen chains combined with two or one rodent fibrinogen chains. Recombinant expression according to the method of the present invention allows for expression levels of Fib420 which are similar to those of recombinant HMW Fib.
  • FIG. 1 Western blot analysis.
  • Lane 1 is a control containing plasma derived wild-type fibrinogen (FIB3, Enzyme Research Laboratories).
  • Lane 2 contains culture supernatant of clone W115, a PER.C6 clones expressing ⁇ -ext rhFib.
  • the molecular weight marker (MW) is indicated at the left.
  • FIG. 2 ROTEM analysis. Clotting time, clot formation time and clot firmness were determined by ROTEM analysis.
  • the left panel displays the fibrinogen preparations in buffer, the right panel displays plasma mixed 1:1 with the fibrinogen preparations:
  • Plasma derived fibrinogen (Haemocomplettan, CSL Behring, Marburg, Germany) in buffer;
  • Plasma derived fibrinogen (Haemocomplettan, CSL Behring, Marburg, Germany) mixed with plasma;
  • FIG. 3 The picture shows a Coomassie stained protein gel loaded with material from plasmin degraded plasma derived material (ERL FIB3; upper panel, and ⁇ -ext Fib (lower panel). The conditions are indicated on top of the gel; the time of incubation (0, 1, 5, 30, 60 and 120 minutes and o/n (overnight)) is shown as well. At the right of the gel the MW marker is displayed.
  • cDNAs coding for human fibrinogen polypeptide chains of ⁇ -ext Fib (Fib420), B ⁇ and ⁇ were synthesized in codon optimized format by GeneArt (Regensburg, Germany): (i) cis-acting sites (splice sites, poly(A) signals) were removed; (ii) repeat sequence of A ⁇ chain was modified; (iii) GC content was increased for prolonged mRNA half life; (iv) Codon usage was adapted to CHO (codon adaption index—CAI ⁇ 0.95).
  • the codon optimized cDNAs for ⁇ -ext Fib (SEQ ID NO.5), B ⁇ (SEQ ID No. 6), and ⁇ (SEQ ID NO. 6) chain were subcloned in pcDNA3.1 deriviates. A ⁇ -extended (Fib420) in pcDNA3.1(+)neo, B ⁇ chains in pcDNA3.1(+)hygro and ⁇ chain in pcDNA3.1( ⁇ )hygro (Invitrogen, Carlsbad, USA).
  • PER.C6 cell lines producing recombinant human fibrinogen is similar as described before in PCT/EP2009/058754.
  • cells were cultured in suspension in MAb medium and transfected using the AMAXA nucleofection device (program A-27) and using Nucleofector kit T with three vectors encoding the three different chains of the human fibrinogen protein (A ⁇ - ext , B ⁇ , and ⁇ chain) and containing the optimized cDNA chains (SEQ ID no.5, SEQ ID no. 6, and SEQ ID no.7, respectively).
  • Recombinant human ⁇ -extended fibrinogen was purified from cell culture supernatant according to standard methods. Briefly, (NH 4 ) 2 SO 4 was added to the culture supernatant to 40% saturation and the precipitate was collected by centrifugation. Subsequently, the precipitate was dissolved in TMAE loading buffer (5 mM Tris-HCl pH 8.5, 0.01% Tween-20) followed by dialysis to the same buffer. The protein solution was then loaded on a Fractogel EMD TMAE (m) 40-90 ⁇ m (3 ml) (Merck KGaA, Darmstadt, Germany) Ion Exchange Column.
  • TMAE loading buffer 5 mM Tris-HCl pH 8.5, 0.01% Tween-20
  • Recombinant human ⁇ -extended fibrinogen was subsequently eluted using a continuous salt gradient of 0-1 M NaCl in 20 column volumes. Recombinant human fibrinogen in the peak fractions was precipitated again by adding (NH 4 ) 2 SO 4 to 40% saturation and collected by centrifugation. Finally the material was dissolved in TBS (50mM Tris-HCl, pH7.4, 100mM NaCl) and dialysed against TBS to remove any remaining (NH 4 ) 2 SO 4 .
  • TBS 50mM Tris-HCl, pH7.4, 100mM NaCl
  • Clotting time (CT), clot formation time (CFT) and clot firmness of ⁇ -ext rhFib and plasma derived fibrinogen were measured using ROTEM analysis.
  • ROTEM® Pultapharm GmbH, Kunststoff, Germany
  • the technique utilizes a rotating axis submerged in a (blood) sample in a disposable cuvette. Changes in elasticity under different clotting conditions result in a change in the rotation of the axis, which is visualized in a thromboelastogram, reflecting mechanical clot parameters (see e.g. Luddington R. J. (2005) Clin Lab Haematol. 2005 27(2):81).
  • ROTEM® analysis graphs for the fibrinogen preparations in buffer or mixed 1:1 with plasma are shown in FIG. 2 .
  • A10, A20, CFT and MCF values (mm) are shown in Table 1.
  • A10 and A20 reflect the firmness of the clot at 10 and 20 minutes post ⁇ -thrombin addition.
  • CFT reflects the time from initiaton of clotting until a clot firmness of 20 mm is detected.
  • MCF reflects maximum clot firmness.
  • Plasma derived fibrinogen has an A10 of 15 mm; this stronger clot formation in buffer can be explained by the activity of co-purified blood derived FXIII which will (partially) cross-link the fibrin monomers. In purified recombinant fibrinogen, no FXIII is present and hence no cross-linking can occur. This can be compensated for by running the experiment in diluted plasma (which contains FXIII). In this case, stronger clots are formed. Interestingly, in this situation, ⁇ -ext rhFib forms a stronger clot and forms it faster than HMW rhFib or plasma derived Fib, with A20 values of 21, 18 and 17 mm, resp.
  • Fibrinogenolysis of purified recombinant human ⁇ -ext rhFib was tested by incubation with plasmin. Briefly, fibrinogen was diluted in TBST (50 mM Tris-HCl, pH7.4, 100 mM NaCl, 0.01% Tween-20), CaCl2 or EDTA was added (5 mM final concentration) and plasmin was added (10 nM final concentration), followed by an incubation at 37° C. At several points in time samples were taken and mixed immediately with SDS-PAGE sample buffer (NuPAGE LDS sample buffer, Invitrogen, cat #NP0007).
  • results, as shown in FIG. 4 indicate that the plasmin mediated digestion of ⁇ -ext rhFib is significantly slower than for plasma derived fibrinogen.
  • ⁇ -ext rhFib this takes more than 120 minutes and only the overnight incubation shows substantial amounts of fragment E generation, which are already present in the digest of the plasma derived fibrinogen after 30 minutes.

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