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WO2018161135A1 - Colle de fibrine d'origine recombinante - Google Patents

Colle de fibrine d'origine recombinante Download PDF

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Publication number
WO2018161135A1
WO2018161135A1 PCT/BR2017/050242 BR2017050242W WO2018161135A1 WO 2018161135 A1 WO2018161135 A1 WO 2018161135A1 BR 2017050242 W BR2017050242 W BR 2017050242W WO 2018161135 A1 WO2018161135 A1 WO 2018161135A1
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Prior art keywords
fibrinogen
expression
thrombin
recombinant
cells
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Portuguese (pt)
Inventor
Marco Aurélio KRIEGER
Rafael Luis KESSLER
Henrique PRETI
Adriana LUDWIG
Luis Roberto Benghi SOARES
Leon SU
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Instituto De Biologia Molecular Do Parana - Ibmp
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Instituto De Biologia Molecular Do Parana - Ibmp
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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
    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue

Definitions

  • 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.
  • 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).
  • 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.
  • 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).
  • fibrin sealants 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).
  • 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.
  • fibrin sealants 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 Kansas, 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).
  • 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.
  • 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.
  • DHFR dihydrofolate reductase
  • 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).
  • the fibrin glue produced by the University of Kansas 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.
  • KAKETSUKEN 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 makes use of FACS (fluorescence activated cell sorting) for selection of highly stable positive cells.
  • the present invention (iii) utilizes the co-expression of chaperone ERp57 to achieve high levels of fibrinogen expression.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 consists of:
  • 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 ⁇ , ⁇ and ⁇ subunits (PLos ONE. 2013 Set; 8 (9): e74580).
  • 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.
  • Figure 6 shows a western blot against thrombin expressed and purified by the present invention.
  • 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;
  • 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.
  • 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).
  • UTR unfolded protein response
  • Atf6-Xbp1 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.
  • Fig. 1 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).
  • the cells were transformed with the vectors described in Fig. 1.
  • FACS selection Fig. 2
  • 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.
  • Figure 3 shows an example of selection of highly protein-secreting homogeneous populations of interest for Thrombin protein.
  • Fibrinogen is produced in the liver from the expression of three genes: FGA, FGB and FGG.
  • 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.
  • mRNA human liver messenger RNA
  • 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.
  • 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.
  • 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
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the ERp57 protein has been described as essential for assembling the hexameric protein fibrinogen from two trimers of the ⁇ , ⁇ and ⁇ 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).
  • the product has two distinct solutions in separate containers.
  • the first tube we have fibrinogen and Factor XIII as active substances.
  • the second container we have thrombin.
  • 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):
  • 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.

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Abstract

La présente invention concerne i) la construction de gènes synthétiques pour composants d'une colle de fibrine recombinante : fibrinogène A, fibrinogène B, fibrinogène G, facteur XIIIA pro-thrombine, écarine et ERp57 ; l'expression de ces gènes dans des cellules CHO génétiquement modifiées pour atteindre de hauts niveaux d'expression ; iii) l'augmentation de l'expression de fibrinogène par co-expression d'ERp57 ; iv) l'échelonnement de ce système d'expression dans un bioréacteur ; v) la purification des composants de la colle au moyen de procédés chromatographiques ; vi) la formulation des composants de la colle pour application comme complément chirurgical.
PCT/BR2017/050242 2017-03-09 2017-08-24 Colle de fibrine d'origine recombinante Ceased WO2018161135A1 (fr)

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BR102017004788-1A BR102017004788B1 (pt) 2017-03-09 2017-03-09 Método de produção de cola de fibrina de origem recombinante
BRBR1020170047881 2017-03-09

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020084528A1 (fr) * 2018-10-24 2020-04-30 Selexis Sa Systèmes d'expression, cellules recombinantes et leurs utilisations
WO2023222770A1 (fr) 2022-05-17 2023-11-23 Julius-Maximilians-Universitaet Wuerzburg Nouveaux variants de fibrinogène recombinant pour produits d'étanchéité à base de fibrine pour le soin des plaies chirurgicales

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