WO2018161135A1 - Fibrin glue of recombinant origin - Google Patents

Abstract

The present invention describes i) the construction of synthetic genes for components of a recombinant fibrin glue: fibrinogen A, fibrinogen B, fibrinogen G, factor XIIIA, prothrombin, ecarin and ERp57; ii) the expression of said genes in genetically modified CHO cells to achieve high levels of expression; iii) the increase in fibrinogen expression by the coexpression of ERp57; iv) the scaling of said expression system in a bioreactor; v) the purification of the glue components using chromatographic methods; vi) the formulation of the glue components for use as a surgical adjunct.

Description

 DESCRIPTIVE REPORT Recombinant fibrin glue

Application Field

The present invention relates to the field of biotechnology, more specifically in the field of mutation or genetic engineering, more specifically genetic manipulation of eukaryotic cells.

Background of the invention and prior art analysis

Blood coagulation is an essential pathway for hemostasis in humans. This pathway is formed by a complex enzymatic cascade that culminates in the formation of a fibrin network that, together with adhesion and platelet aggregation, form a clot aimed at stopping bleeding in the injured tissue (Rev. Bras. Hematol. Hemoter. 2010; 32 (5): 416-421).

[0003] Three proteins are critical in the final stages of the coagulation pathway (Blood. 2013 Mar 7; 121 (10): 1712-9): fibrinogen (factor I), prothrombin (factor II), and factor XIII. Pro-thrombin is a serine protease that, when activated by the prothrombinase complex (factor Xa + Va), becomes enzymatically active (thrombin) and proteolytically cleaves fibrinogen and factor XIII to form fibrin and factor Xllla molecules, respectively. Fibrin molecules spontaneously polymerize to form protofilaments which, after factor III-catalyzed crosslinking, form a stable three-dimensional network. Therefore, activation of thrombin is a key step in the formation of a clot.

Fibrin molecules are the basic units for filament formation that form a clot, and are therefore the preponderant protein in this structure. Fibrinogen is ranked as the third most abundant protein in human plasma, with a circulating concentration of 2-4g / L. [0005] Various techniques have been used to isolate and concentrate fibrinogen and thrombin from a human plasma pool, including precipitation (Blood separation and plasma fractionation, Wiley-Liss Pub, New York. Pp. 349-383), cryoprecipitation ( Methods Mol Biol, 201 1, 728, 259-265) and chromatographic purification (US patent 7,550,567. February 25, 2005; US patent 7,816,495. January 22, 2004).

Knowledge of the coagulation cascade and proteins critical for fibrin clot formation, together with methods for isolating these proteins from human plasma, has led to the development of blood products for the treatment of hemorrhage (Production of Plasma Proteins for Therapeutic). Use, John Wiley & Sons, 2013). Products known as fibrin sealants or fibrin glue are based on the local application of fibrinogen and thrombin molecules (may contain factor XIII) to form a biological sealant. This type of biological sealant, or fibrin glue, is widely used in surgery as an accessory agent for achieving hemostasis and also as surgical wound healing sealants (Drugs. 201 1 Oct 1; 71 (14): 1893-915). At the site of application, a fibrinogen-containing solution is mixed with another thrombin-containing solution that catalyzes the conversion of fibrinogen to fibrin monomers resulting in the formation of a tissue-adhering hydrogel, or fibrin clot.

Although commercial fibrin sealants have been available for over 30 years, the market continues to expand. Recent efforts have been made to develop fully recombinant human fibrin sealants in cell culture systems, with the great advantage of being free of any bloodborne pathogens. The proteins required for the preparation of a recombinant fibrin sealant (fibrinogen, thrombin and factor XIII) are extensively modified post-translationally (Blood. 2013 Mar 7; 121 (10): 1712-9). Therefore, their production is only viable in eukaryotic cells, discarding bacterial production systems.

Recombinant human thrombin produced in CHO cell is now available as an FDA licensed product (Recothrom, ZymoGenetics) and has recently also been expressed in CHO DHFR cells to achieve high levels of protein production (J Biosci Bioeng 2015 Oct; 120 (4): 432-7). Recombinant factor XIII, produced by fermentation in yeast, has recently been approved for the treatment of congenital factor IIll-A deficiency (Tretten, Novo Nordisk).

Fibrinogen, the most abundant protein in a clot and therefore critical in producing a recombinant fibrin glue, was primarily expressed in CHO cells (Blood, 1997, 89, 4407-4414). However, the production of high fibrinogen levels for commercialization of a biopharmaceutical has only recently been achieved. Dr. Willian H. Velabnder's research group from the University of Nebraska, USA, expressed fibrinogen in the milk of transgenic cows (Biomacromolecules. 2013 Jan 14; 14 (1): 169-78.) And recently published the first generation of a fully recombinant fibrin glue (J Surg Res. 2014 Mar; 187 (1): 334-42) containing fibrinogen obtained from cow's milk, factor XI MA produced in yeast and commercial recombinant thrombin (Recothrom, ZymoGenetics).

[0010] Another institute also leads the development of a fully recombinant fibrin glue: Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN of Japan. This group produces fibrinogen in CHO DG44 cells reaching more than 1 g / L of this molecule (J Biochem. 2016 Feb; 159 (2): 261-70). This was the first demonstration of recombinant fibrinogen production capable of meeting high industrial demand. However, the solution found by Hirashima and colleagues was the amplification of the copy number of fibrinogen genes through metatrexate amplification in dihydrofolate reductase (DHFR) deficient CHO cells. The same group recently also used the same expression method in deficient CHO DHFR cells to achieve high levels of thrombin protein production (J Biosci Bioeng. 2015 Oct; 120 (4): 432-7).

Although the gene amplification mechanism in DHFR-deficient cells has already been successfully used to produce several commercially important recombinant proteins (including erythropoietin, epogen, procrit, growth hormone, genentech and various monoclonal antibodies), the Metatrexate-mediated amplification has the problem of high genomic instability that may culminate with inhibition of gene expression of interest (Biotechnol Lett. 2013 Jul; 35 (7): 987-93; Biotechnol Bioeng. 201 Oct 1, 108 (10): 2434-46). In addition, after gene amplification with metatrexate, cultures should be continuously exposed to the drug to avoid gene silencing (Biotechnol Bioeng. 2009 Mar 1; 102 (4): 1 182-96), making production more expensive.

All the fibrin sealants described above, currently available as biopharmaceuticals and produced from human plasma, do not advance the present invention as a recombinant system for producing fibrin glue is used herein.

The fibrin glue produced by the University of Nebraska group in the USA (J Surg Res. 2014 Mar; 187 (1): 334-42) reveals an interesting method of expression and purification of fibrinogen from the milk of transgenic cows. . It is no use in the present invention to use expression systems other than those employed herein.

The Japanese Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN), together with the pharmaceutical company Teijin Pharma, produced fibrinogen and thrombin in CHO cells on a large scale for the first time by delivering the product on a bioabsorbable screen (KTF- 374). It is no use to analyze because (i) does not include factor XIIIA in its composition, (ii) by using a different recombinant expression system to achieve higher expression levels, including selection with antibiotics and metatrexate-mediated gene amplification, which implies genetic instability. The present invention, in turn, makes use of FACS (fluorescence activated cell sorting) for selection of highly stable positive cells. In addition, the present invention (iii) utilizes the co-expression of chaperone ERp57 to achieve high levels of fibrinogen expression.

Prior art problems

Several fibrin sealants are commercially available for the support of hemostasis in surgeries, all made from human plasma. Blood products have an intrinsic risk of contamination with viruses and prions. In addition, co-purification from the plasma of fibrinolytic factors that hinder the stabilization and functionality of fibrinogen molecules. In order to inhibit fibrinolysis, some manufacturers add plasminogen inhibitors to the formulation of blood-derived fibrin sealants. Tranexamic acid is used for this purpose, but because it is potentially neurotoxic, this type of sealant cannot be used in neurosurgery. Undesirable plasminogen can be removed by chromatographic techniques, without the use of plasminogen inhibitors, which makes the process more expensive. The central challenge in the production of fibrinogen from human plasma is to obtain a concentrated sterile solution after viral inactivation containing 50-120mg / ml fibrinogen that maintains its solubility and activity. This process is laborious and involves several steps of plasma manipulation.

Considering the above mentioned blood product problems, the main gap to be filled in the state of the art for producing fibrin-based sealants is the preparation of components fully produced in high efficiency recombinant mammalian cell systems. The main technological difficulties encountered in this field of recombinant protein production are directly associated with the low genetic stability of the cells used (Curr. Opin. Struct. Biol. 2015 Jun; 32: 81 -90), with negative implications on productivity.

Traditionally, recombinant protein expression is by cloning heterologous genes into plasmids containing genetic elements required for expression in host cells, such as compatible promoters, transcription termination signals, and elements selectable as antibiotic resistance genes. etc., which allow selection of cells containing the heterologous gene from other negative cells in the population.

However, it is necessary that the genetic set containing the heterologous gene and the selection gene be transmissible across cell generations and, for this purpose, the integration of such an assembly into the host cell genome is required. As genome integration is usually a rare and random event, clone generation is required, as distinct integrations have large scale variability in produce heterologous proteins because of the resistance of the genome to absorb new integrations, a fact often associated with viral infections or cancer. This resistance causes progressive inactivation of the heterologous genetic ensemble by DNA methylation processes, except that integrations are adjacent to essential genes. In practice, it is observed that there are no methods to predict the behavior of these cells during the processes of selection and elaboration of cell banks (Porter AJ, Racher AJ, Preziosi R, Dickson AJ. Biotechnology Progress 26 (5): 1455- 64 (2010)).

The only documented method for fibrinogen production (J Biochem. 2016 Feb; 159 (2): 261 -70) and thrombin (J Biosci Bioeng. 2015 Oct; 120 (4): 432-7) in CHO cells in Commercial scale uses metatrexate-mediated gene amplification in DHFR-deficient CHO cells. As previously stated, these strains are genetically unstable and do not ideally solve the problem of generating a stable cell population to maintain a master bank of producing cells.

Solution of the present invention

The present invention solves the above problems for producing a recombinant fibrin glue by producing the necessary proteins (Fibrinogen, Thrombin and Factor XIII) in a proprietary expression system for producing high stability recombinant cell lines, "Genetic platform for heterologous overexpression associated with the selection of highly protein producing cells" (patent application BR 10 2017 001876 8).

The genetic platform designed to achieve high degree of heterologous protein expression in eukaryotic cells, in short, consists of:

1 . Ensure high integration rate of transposase-mediated expression cassette into host cell chromosome. 2. Presence of genetic isolators flanking integration cassettes that ensure high transcription level regardless of chromosomal integration site.

3. Co-expression of a fluorescent reporter that allows real-time expression monitoring and selection of highly producing homogeneous clones or populations independent of antibiotic selection.

4. Development of proprietary cell lines by introducing optimized genes into the cell (Atf66-Xbp1 fusion), favoring the increase in the amount of viable protein while maintaining its biological integrity.

5. Possibility of doxycycline-regulated expression in a modified TetON system.

Such a genetic platform was used for recombinant expression in CHO cells of (i) fibrinogen, (ii) prothrombin, (iii) factor XIIIA, (iv) ecarina and (v) ERp57 proteins. Eccarin protein is a protease derived from the Echis carinatus snake venom that has the ability to activate prothrombin to thrombin through proteolytic cleavage, (Thromb Res. 1975 Jan; 6 (1): 57-63) eliminating the need of the prothrombinase complex (factor Xa + Va) for activated thrombin production. ERp57 protein is a chaperone that has homology to disulfide isomerases and is present in the endoplasmic reticulum. It has been described as essential for assembling the fibrinogen hexameric protein from two trimers of the α, β and γ subunits (PLos ONE. 2013 Set; 8 (9): e74580).

Coexpression of this protein with the α, β and γ subunits of fibrinogen in CHO cells leads to an increase in the concentration of fibrinogen secreted by these cells. The proteins expressed and secreted by CHO cells are then purified by chromatographic methods. The ecarina is added to the thrombin-containing supernatant for activation and is then completely removed from the mixture. Activated thrombin, fibrinogen and factor XIII produced by the above method are then used to formulate a recombinant fibrin glue. Description of the figures

Figure 1 shows the genetic constructs used for expression of each gene in CHO cells.

Figure 2 shows a schematic of the genetic platform used for transfection of genes of interest and selection of FACS positive populations. An example of the prothrombin gene is shown.

Figure 3 shows an example of selection of highly protein-secreting homogeneous populations of interest for thrombin protein.

Figure 4 shows Coomassie staining analysis of SDS-PAGE and western blot of total fibrinogen produced in CHO cells by the present invention. 1 = F-ABG, Post centricon, comassie staining; 2 = F-ABG, Post centricon, Western blot; 3 = F-ABG, Bioreactor Aliquot, IP: V5 Western Blot.

Figure 5 shows the 350nm turbimetry clotability tests. The polymerization and turbidity of recombinant fibrinogen is similar to that of standard human plasma-derived fibrinogen in the presence of activated human thrombin. 0.2 mg / ml FBG, 1 U / ml Thrombin (50 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM CaCl 2).

Figure 6 shows a western blot against thrombin expressed and purified by the present invention.

[0030] Figure 7 shows recombinant thrombin activity, activated by recombinant ecarina, by chromogenic assay. A1: Xa / Va factor activated commercial thrombin; B1, 2, 3: Ecarine (different purification fractions) + Commercial prothrombin; C1, 2, 3: Ecarine (different purification fractions) + Prothrombin; D1: Ecarin + Prothrombin E1: Ecarin + Commercial Prothrombin.

Figure 8 shows the binding of factor 11-catalyzed fibrin molecules thrombin analyzed by western blot against fibrinogen. Figure 9 shows the selection and expression of fibrinogen in CHO ERp57 - / + cells.

Detailed Methodology

The genetic platform for heterologous overexpression associated with the selection of highly protein producing cells (patent application BR 10 2017 001876 8) was employed for expression of the 4 proteins required for the production of recombinant fibrin glue. Fig. 1 shows the constructs used for the expression of each gene. Expression cassettes were inserted into the genome of proprietary cells produced by the group conditionally expressing the fusion of Atf6-Xbp1 genes under the control of tetracycline (or doxycycline). For further details of this system, see patent of the genetic platform (BR 10 2017 001876 8).

Atf6 and Xbp1 proteins are the main signal transducers in the unfolded protein response (UPR) stress pathway and, when activated, act on the cell nucleus by inducing the expression of a set of genes (chaperones, enzymes). involved in glycosylation, disulfide bridging and vesicle transport) capable of stabilizing a highly protein-secreting cellular phenotype (Celi. 2001 Dec 28; 107 (7): 881 - 91; Mol Immunol. 2004 Jul; 41 (9): 919-27; EP2316955A1). By fusing the active form of both proteins into a single Atf6-Xbp1 polypeptide, we built a simple and straightforward UPR pathway activation system that facilitates the induction of increased expression of recombinant proteins, as different proteins may have increased expression in the protein. presence of Atf6 or Xbp1. Basically, after cell culture reaches the stationary phase of growth (highest cell density point, -107 cells / ml) Atf6-Xbp1 expression is induced. Thus, the cells stop dividing and move to a cellular metabolism focused on protein synthesis and traffic.

For fibrinogen production, three recombinant gene platform plasmids, each containing a gene for a fibrinogen chain A, B, or G - and a different reporter gene (Fig. 1). sequentially integrated into the CHO-S host cell genome via transposase by the Sleeping Beauty transposase. The producer cells were then selected by cell sorting in flow cytometry by detecting the respective reporter gene product (Fig. 2). For the other proteins (prothrombin, factor XIII and ecarina), only one vector of the genetic platform was necessary since they are single chain proteins (Fig. 1).

Initially, the cells were transformed with the vectors described in Fig. 1. After FACS selection (Fig. 2) and significant cell culture growth (5-7 days post-transfection), the transformed culture underwent the first round of selection, where cells that were detectably fluorescent compared to non-transfected cells were collected. transfected. At this time, about 5% of the culture cells had this expression profile. However, after 14 days of transformation (7 days after the first round of cell sorting) more than 50% of cells have high levels of production of exogenous recombinant proteins, as measured by the reporter gene. Figure 3 shows an example of selection of highly protein-secreting homogeneous populations of interest for Thrombin protein.

After the second round of cell selection and even keeping these cells for more than 12 days growing (average life of this cell type in bioreactors), the results showed that over 98 percent of the cells still had high expression rate. , measured by the reporter system used (Fig. 3)

Cloning and expression of recombinant fibrin glue genes

Fibrinogen is produced in the liver from the expression of three genes: FGA, FGB and FGG. After processing in the endoplasmic reticulum and Golgi, the three proteins form a straight trimer in the A2B2G2 composition that is secreted into the bloodstream. Therefore, complementary DNA (cDNA) was prepared from human liver messenger RNA (mRNA) and the three genes were PCR amplified and sequenced. The complete sequences of FGA, FGB and FGG (without introns) were then synthetically prepared for use in the present invention: FGA (SEQ ID NO: 1), FGB (SEQ ID NO: 2) and FGG (SEQ ID NO: 3), present in the sequence listing. The proteins used to expression of the following: CD20 for FGA, CD25 for FGG and GFP for FGB (Fig. 1).

Amino acid sequences have been deduced from nucleotide sequences, and have 100% homology to the deduced sequence of NCBI templates (NM_021871 / NM_001 18471 / NM_021870.2)

Factor XIII is produced in the liver from the expression of two distinct genes, F13A1 and F13B. After processing, the protein is secreted into the circulation as a hetero tetramer composed of two A chains and two B chains. The F13A (2) homimer has latent catalytic activity and the F13B (2) homimer serves as a plasma carrier but is not necessary for the biological activity of Factor XIII. Once proteolytically processed by thrombin, F13A (2) and F13B (2) separate and F13A (2) exerts its biochemical activity as a transglutaminase producing a lateral covalent crosslink over fibrin polymers, imparting structural robustness to the fibrinocytic clot.

The F13A1 gene was amplified from human liver cDNA and sequenced, and the CDS region synthesized for cloning into the genetic platform. The synthetic sequence used (SEQ ID NO: 4) was cloned into the CD25 gene platform expression vector (Fig. 1). Amino acid sequences have been deduced from the nucleotide sequences shown below and are 100% homologous to the deduced sequence of NCBI templates NM_000129.3

Thrombin is produced in the liver from gene expression

F2 and is secreted into the bloodstream as a pro-enzyme with latent serine protease activity. Prothrombin is proteolytically processed by components of the blood coagulation cascade converting it into an active serine protease (thrombin) acting primarily on fibrinogen to produce fibrin polymers, Factor XIII (revealing its transglutaminase activity) and factor VIII, converting This in Villa factor, an essential amplifier in the coagulation cascade. Pre-thrombin (F2) was PCR amplified from human liver cDNA and sequenced. The complete CDS was synthesized (SEQ ID NO: 5) and cloned into the CD25 gene platform plasmid (Fig. 1) and also into the RFP plasmid. The amino acid sequences were deduced from the nucleotide sequences shown below and have 100% homology to the deduced sequence of NCBI templates NM_000506.3

The eccarin protein gene (SEQ ID NO: 6) and ERp57 (SEQ ID NO: 7) were similarly obtained synthetically and then subcloned into the gene platform vectors containing the GFP and RFP reporters, respectively. (Fig. 1).

Groups of cells whose protein expression remained constant relative to pre-freeze expression were maintained as a master bank. Cells obtained by this selective process were used for growth in a 1.7 liter bioreactor (Fig. 2) using the CHO Freestyle, 8 mM Glutamax, penicillin / streptomycin batch strategy. Cell growth and viability are monitored daily and culture is stopped at the first sign of loss of viability. The cells are then centrifuged and the fibrinogen-containing supernatant is concentrated by tangential filtration and diafiltered in an appropriate buffer to be used in the subsequent purification process. The same transfection, selection, expansion, storage, and production processes were performed for the other two components, Factor XIII and prothrombin, and ecarina used to activate prothrombin.

Component Purification

Following concentration of the fibrinogen-containing CHO cell supernatant and diafiltration in Tris buffer, the obtained solution is loaded onto a pre-equilibrated DEAE ion exchange column in the same buffer in an operation controlled by an FPLC system. Fibrinogen is eluted in a continuous gradient system of NaCI in Tris and collected automatically in a fraction collector. After analysis of western blot and ELISA fractions to determine the presence, integrity and concentration of fibrinogen, the selected fractions are mixed, concentrated and desalted to Citrate / NaCI buffer by selective filter centrifugation and subjected to gel filtration operated on the FPLC. The fibrinogen-containing fraction is collected and stored in a freezer at -20 ° C. A small sample of the fraction is retested for integrity and fibrinogen concentration. The supernatants of ecarina and prothrombin-containing CHO cells undergo the process of concentration by tangential filtration and are then mixed at a ratio of 1: 10 (ecarina: thrombin) and incubated at 37 ° C for 16 hours. The solution is then diafiltered to sodium citrate buffer and the sample is loaded onto a pre-equilibrated ion exchange column with the same buffer. Column-bound protein is washed with wash buffer and eluted with linear gradient of NaCl in Citrate.

For FXIII, concentration and diafiltration are performed in the same system in Tris buffer. The sample is then loaded onto an ion exchange column and equilibrated in the same buffer. Bound protein is washed with 5 volumes of wash buffer and eluted by continuous NaCl gradient in Tris. Samples collected after the ion exchange column (DEAE and QAE) are analyzed by western blot and ELISA for the presence, integrity and concentration with specific anti-thrombin and anti-FXIII antibodies. Fractions containing the desired protein are mixed and desalted into Citrate and NaCI (pro-Thrombin) or Tris and NaCI (FXIII) buffer by selective filter centrifugation and subjected to gel filtration operated on the FPLC. Fractions containing prothrombin or FXIII are collected and stored in a freezer.

Analytical Results

Coomassie staining analysis of SDS-PAGE and western blot of total fibrinogen (Fig. 4) demonstrated intact fibrinogen composed of individual A, B and G chains with expected molecular masses (Fig. 4). But the most important feature of any fibrinogen source is the measure of the clotability sensitive ratio. To measure clotability, fibrinogen is incubated with thrombin which proteolytically processes the terminal regions of fibrinogen A and B chains resulting in the polymerization of fibrin monomers into monomeric chains (fibrin) that are insoluble in the physiological environment. This insolubility causes the formation of an optically cloudy initial clot that absorbs light at a wavelength between 340 and 350 nm.

Therefore, this 350 nm turbimetry assay is used to measure the clotability of any fibrinogen sample using a standard sample with known clotability for comparison purposes. The The results from Fig. 5 show that the polymerization and turbidity of recombinant fibrinogen are similar to that of standard human plasma-derived fibrinogen in the presence of activated human thrombin.

Western blot analysis of the prothrombin produced in our system shows a predominant band with the expected molecular mass of 75 kDa (Fig. 6). Physiologically, pro-Thrombin is converted to active thrombin by the action of factor Xa protease in combination with cofactor Va (prothrombinase complex). Activation generates a covalently associated disulfide heterodimer composed of beta chain (37 kDa) and alpha chain (variable, 3-6 kDa). Thrombin activity can be assessed by absorbance assay using a chromogenic substrate (beta-Ala-Gly-Arg para-nitroanilide) which, when cleaved by thrombin releases the p-nitroanilide group which can be detected by absorbance at 405nm. Fig. 7 shows the results of recombinant thrombin activity analysis (measured by p-nitroanilide release) in the presence or absence of recombinant ecarina. The results show that recombinant eccarin is capable of activating recombinant prothrombin for thrombin, generating absorbance signals similar to factor Xa / Va activated human plasma thrombin. The results of Fig. 7 indicate that both proteins produced in the present invention, eccarin and prothrombin, are enzymatically active.

The initial clot formed by the insolubility of fibrin monomers has no rigid structure due to the absence of chemical bond between the monomers. Active thrombin acts on native Factor XIII by cleaving the N-terminal region from the Arg 37 residue, causing FX III transglutaminase mode activation. Active FXIII (FXIIIa) catalyzes a covalent reaction between Lysine and Glutamine residues in parallel fibrin molecules. This results in the structural and biochemical stability of the fibrin clot. This transglutaminase activity can be visualized on SDS-PAGE gels due to the appearance of SDS-resistant alpha chain (gamma-gamma) and gamma chain dimers and reducing agents (Fig. 8). The western blot results (using anti-fibrinogen antibody) of Fig. 8 show the formation of α-oligomers and γ-dimers only when in the presence of FXIII and activated thrombin, evidenced in denaturing gel. In non-denaturing gel (Fig. 8), it is possible to observe the formation of high-weight complexes. due to crosslinking between factor 11 catalysed fibrin molecules.

The ERp57 protein has been described as essential for assembling the hexameric protein fibrinogen from two trimers of the α, β and γ subunits (PLos ONE. 2013 Set; 8 (9): e74580). Comparison of fibrinogen expression levels in ERp57- vs ERp57 + cells showed that co-expression and ERp57 increases recombinant fibrinogen production by more than 5-fold (Fig. 9).

Formulation of fibrin glue components for single or joint applications

The product has two distinct solutions in separate containers. In the first tube we have fibrinogen and Factor XIII as active substances. In the second container we have thrombin. By mixing the contents of the two containers, we thus enable the activation of the components by obtaining a biological fibrin sealant for local application.

In addition, each protein may be prepared in individual tubes for independent delivery of each coagulation factor, which may be used for various purposes.

Tube A (Fibrinogen + Factor XIII):

- Fibrinogen: 50 - 120 mg / ml.

- Coagulation factor XIII: 10 - 90 U / ml (1 unit (U) is equivalent to factor XIII activity of 1 mL fresh citrated plasma (plasma pool) from healthy donors).

Excipients: arginine hydrochloride, sodium chloride (11 mg / ml), sodium citrate (4.8 - 9.7 mg / ml), glycine, calcium chloride (10-50 pmol / ml), and water for injection.

Tube B (Thrombin):

- Thrombin: 400 - 1200 IU / ml.

Excipients: calcium chloride (5 - 7 mg / ml), human albumin (10 - 20 mg), mannitol, sodium acetate, and water for injection.

Claims

1 . Recombinant fibrin glue, characterized in that it contains fibrinogen, thrombin and factor XIII. Recombinant fibrin glue according to claim 1, produced on a genetic platform for heterologous overexpression associated with the selection of highly protein producing cells. Reference cell banks according to claim 2, characterized in that they contain cells producing each of the proteins essential for recombinant fibrin glue. Fibrinogen production method according to claim 2, characterized by the co-expression of ERp57.