Novel Recombinant Fibrinogen Variant for Fibrin Sealants for Surgical Wound Care

Abstract

The present invention relates to a newly isolated recombinant fibrinogen, a eukaryotic cell producing said newly recombinant fibrinogen, and a fibrin sealant and a fibrin sealant kit comprising the newly isolated recombinant fibrinogen. Furthermore, the present invention relates to a new method for the production of recombinant fibrinogen using eukaryotic cells, and a new method for identifying a newly recombinant fibrinogen variant with reduced susceptibility to protein cleavage by plasmin without loss of their suitability as fibrinogen components in fibrin sealants.

Description

Background Art

[0001] An estimated 313 million surgeries are performed worldwide each year. 1 As the population increases to approximately 9.073 billion in the future, the number of required surgical procedures will also increase. 2 A major drawback of these medical interventions is the risk of massive bleeding, which can lead to an increase in morbidity and mortality. 3 This risk is particularly high in soft tissue surgeries, of which approximately 7% result in massive bleeding. 4、5 Although there have been significant developments in surgical techniques over the past few decades, uncontrollable bleeding remains a major problem, particularly in more extensive procedures such as liver resections. 6~10 For example, 2 - 6% of all patients who undergo heart surgery show bleeding during or after the operation, resulting in an increased mortality rate. However, bleeding during or after surgery is a burden not only for the patient but also for the entire healthcare system, as it is associated with significant costs due to the potential for a second surgery and an extended hospital stay. 11~13

[0002] A variety of different methods and applications are used to control bleeding and improve hemostasis during medical procedures. Standard methods, with an estimated annual sales of up to $15 billion in 2024, include the use of sutures, clips, and staples. 4 These methods have been established for a long time and show the desired effect in many cases, but there are still problems and drawbacks. In more difficult procedures, up to 30% of patients experience fluid leakage after surgery. 14 In addition, sutures used for surgical wound management are often affected by bacterial growth and can lead to microbial infections at the surgical site. 15、16

[0003] Over the past 20 years, various tissue sealants, hemostatic agents, and tissue adhesives have been developed as alternative technologies to address these challenges. To date, these approaches have proven to be a valuable addition to the current toolbox. The emergence of the importance of adhesives and sealants is reflected by a market size with a current value of $10.03 billion in 2020 and a predicted compound annual growth rate of 5.6%. 4 These hemostatic materials are mainly based on approaches that mimic and support the natural mechanisms of hemostasis in the body.

[0004] Over the years, various new technologies such as compression bandages, gels, and foams have been developed, especially for use in superficial wounds. 17~21 However, this trend has not currently emerged for soft tissue and internal wound procedures. In these applications, new materials must withstand advanced challenges such as a wet environment and dynamic forces. For these types of surgeries, blood transfusion remains the standard method. 22 However, the use of blood transfusion is associated with several problems, such as limited donor availability, potential immunogenicity, or the risk of infectious diseases. 23~25 Although more and more new approaches are under development, the transition from the laboratory to patient use is a major problem.

[0005] In 1998, the first such material approved by the US Food and Drug Administration (FDA) as a hemostatic agent was the so-called fibrin sealant. 26 This material functions by mimicking and supporting the natural mechanism of hemostasis. Fibrinogen and thrombin form the basic and main components. These two proteins play a central role in the final step of the blood coagulation cascade in the body, resulting in the formation of a fibrin clot and thus closing the wound.

[0006] Fibrinogen is a longitudinal hexameric protein whose shape closely resembles a dumbbell. The soluble protein is formed using three subunit α, β, and γ chains. In its native form, the protein forms a hexamer of two units of each of the described chains, having a total molecular weight of approximately 340 kDa (Aα chain (66.5 kDa), Bβ chain (52 kDa), and γ chain (46.5 kDa)). 30、36~37 Naturally, fibrinogen exists as a heterogeneous population of different forms. Both the alpha and gamma chains (α and γ chains) have different isoforms formed due to alternative splicing. The α chain includes two different forms, isoform E (αE), also known as isoform 1 (molecular weight: about 93 kDa) and referred to herein as "isoform alpha - E" or "isoform αE", and isoform 2, generally referred to as the α chain and referred to herein as "isoform alpha" or "isoform α". Fibrinogen containing isoform αE accounts for approximately 1 - 2% of the fibrinogen population in the human body. There are also indications that fibrinogen containing isoform αE may be less sensitive to proteolysis, making it an interesting candidate for increasing the half - life of fibrin clots against plasmin. 38 The term "Aα" or "A alpha" refers to the secreted form of the alpha chain isoform alpha, which lacks the N - terminal amino acids of the signal peptide present in the full - length isoform alpha but still contains "fibrinopeptide A" consisting of amino acids 20 - 35 of the full - length amino acid sequence of isoform alpha. Similarly, the term "AαE" or "A alpha - E" refers to the secreted form of the alpha chain isoform alpha - E, which lacks the N - terminal amino acids of the signal peptide present in the full - length amino acid sequence of isoform alpha - E but still contains "fibrinopeptide A" or (FpA) consisting of amino acids 20 - 35 of the full - length amino acid sequence of isoform alpha - E.

[0007] The γ chain also exists naturally in two different isoforms. The first isoform is called gamma or gamma A (γ or γA; also simply called the gamma chain, or gamma A chain). The second isoform is called gamma prime or gamma B (γ’ or γB; also simply called the gamma prime chain, or gamma B chain). Approximately 10 - 15% of the fibrinogen in the body consists of the heterodimer (γ / γ’), and only about 0.5% consists of the homodimer γ’ / γ’. However, the presence of the isoform γ’ can have an impact on the structure and function of fibrinogen and fibrin clots. 39

[0008] During hemostasis, the N - termini of both the α and β chains are processed by the enzyme thrombin, which results in the formation of the fibrin network. 40、41 Exposure of the protein to thrombin results in the cleavage of the so - called fibrinopeptide A (or FpA) sequence at the N - terminus of the α chain and the so - called fibrinopeptide B (FpB) at the N - terminus of the β chain, initiating the formation of the protein complex that leads to wound closure. 31 Due to its natural role and mechanism, fibrin has been comprehensively studied over the past few decades and has been used as a biomaterial for different pharmaceutical fields. 42、43 Use in fibrin sealants to facilitate wound closure in surgeries with acute bleeding is one of the most important applications of fibrinogen. 18、44~45

[0009] In the current generation of fibrin sealants (or fibrin glues), fibrinogen and thrombin are separately filled into a pre - filled dual - syringe system and applied directly to the bleeding site, where only the two components are directly mixed at the site of action. 27 Upon contact with the wound, fibrinogen and thrombin interact to form fibrin, which results in a hemostatic effect. 28The success of fibrin adhesion is based on many properties, such as biocompatibility, the natural occurrence of proteins in the body, resorption, and the fact that they do not cause inflammation or necrosis, and the possible addition of biologically active compounds (e.g., growth factors) by their native protein binding sites. 18、44~47

[0010] Nevertheless, these materials have some disadvantages. Fibrinogen is usually obtained from a large pool of human plasma. This creates the potential for the transmission of viral diseases. For example, there is research from Japan indicating that parvovirus B19 transmission can occur in up to 20% of treated patients. 29 However, one of the major problems with fibrin sealants, especially during substantial bleeding, is the rapid resorption of fibrin in vivo due to enzymatic degradation (i.e., fibrinolysis) by the serine protease plasmin. Plasmin is the most prominent protease during fibrinolysis. Fibrinolysis is an essential process in the body to prevent the formation of intravascular blood clots or the breakdown of blood clots during tissue regeneration, but the proteases transported by the blood result in a reduction in the stability of the wound sealant.

[0011] Therefore, the degradation of fibrin clots is mediated by plasmin, an endogenous serine protease. Fibrinogen has plasmin cleavage sites in all three chains for this purpose. 32、33 Active plasmin is a serine protease derived from its zymogen, plasminogen. 48 After activation, plasmin rapidly degrades the fibrin clot. The fibrinogen used in fibrin sealants often originates from pooled human plasma and thus already contains small amounts of plasminogen. Upon contact of the sealant with the injury site, this plasminogen can be activated by proteases released from cells and combined with endogenous plasmin, potentially amplifying the resorption of the sealant. 49

[0012] Over the years, different approaches have been developed to address this problem. Currently, the most successful approach is the addition of protease inhibitors to the sealant, which locally restricts plasmin activity. The most common plasmin inhibitor used for these applications is aprotinin (bovine pancreatic trypsin inhibitor). Aprotinin is a peptide serine protease inhibitor with a molecular weight of 6.5 kDa, which has been shown to successfully inhibit different proteases, including plasmin. It is also the only inhibitor currently used in fibrin sealant formulations (e.g., TISSEEL®, Baxter) that have already been approved by the FDA.

[0013] However, the use of aprotinin also has major drawbacks, such as the risk of allergy and anaphylactic shock. 27 In addition, aprotinin rapidly diffuses from the site of injury. 49 Therefore, plasmin inhibition has a relatively short time frame.

[0014] Considering the above drawbacks of available fibrin sealants, an object of the present invention described below is to provide a fibrin sealant having an extended half-life in the presence of the protease plasmin without the need for the inclusion of a plasmin inhibitor. In particular, A) the physiological advantages of fibrin sealants in hemostasis; B) prevention of potential viral transmission by product application; and C) extension of the fibrin half-life without potential allergic reactions due to the addition of aprotinin, a plasmin inhibitor There is a need for a fibrin sealant that combines these.

[0015] In addition, production steps and costs must be minimized by reducing the components of this system.

[0016] Wypasek et al., Thrombosis Research, Vol. 182, Oct 2019, p. 133-140 discloses a screening study for mutants in Polish patients with hemorrhagic diseases, and describes the genetic and clinical characterization of congenital fibrinogen mutations in individual strands in patients using concentration determination, polymerase chain reaction (PCR), and Sanger sequencing. Wypasek et al. analyzed patients with hemorrhagic diseases and their association with different mutations, thereby disclosing which point mutations in patients with hemorrhagic diseases correlate with disease activity. Wypasek et al. are not related to recombinant fibrinogen. The fibrinogen from the plasma preparation disclosed by Wypasek et al. is not equivalent to recombinant fibrinogen because the former may contain protein impurities (plasminogen) and / or viral contaminants. Furthermore, fibrinogen naturally exists as a heterogeneous population of distinct isoforms, resulting in altered properties. Wypasek et al. are not related to identifying sites that extend the stability of fibrin clot / glue, and do not provide information on how the wild type (without mutation) should be changed to obtain a longer-lasting fibrin glue. Furthermore, there is no indication in Wypasek et al. on how fibrinogen can be recombinantly engineered to exhibit sustained stability compared to the wild type. Instead, Wypasek et al. are concerned with hemorrhagic diseases that result in altered amounts and / or qualities of circulating fibrinogen, but fibrinolysis of fibrinogen variants has not been examined. Summary of the Invention

[0017] The inventors have considered a method for generating a modified wound adhesive having improved properties, including an increase in the half-life in the presence of plasmin, a protease, thereby ensuring less resorption of the adhesive and, as a result, a more persistent hemostatic effect. In doing so, the inventors have found a novel recombinant fibrinogen variant using a novel approach based on molecular manipulation that limits plasmin-induced cleavage of fibrinogen, which is less sensitive to plasmin-dependent degradation and is therefore more stable or stable for a longer period in the presence of plasmin. Therefore, the novel recombinant fibrinogen variant can be advantageously used in fibrin sealants without the need to add a plasmin protease inhibitor such as aprotinin. Therefore, the novel recombinant fibrinogen variant overcomes the limitations of the prior art fibrin sealants described above.

[0018] Furthermore, the inventors have found a new method for the production of isolated recombinant fibrinogen variants using eukaryotic cells, preferably mammalian cell culture cells. This method advantageously prevents the degradation of rFbg during cell culture-based expression, thereby increasing the yield of recombinant fibrinogen variants that can be produced and isolated using a cell culture-based production system, ensuring the recombinant production of fully intact fibrinogen, especially the fibrinogen alpha chain.

[0019] Finally, the inventors have developed a novel time-resolved screening method that enables the elucidation of plasmin cleavage sites for possible amino acid exchanges in order to find new molecularly engineered fibrinogen variants. Therefore, this method is advantageously used to create a new generation of fibrin sealants with improved stability against plasmin degradation. By changing both the number of mutations at the plasmin cleavage site and their location in the quaternary structure of the protein, fibrin gel resorption can be fine-tuned for specific applications.

[0020] The present invention thus relates to the following embodiments. [1] An isolated recombinant fibrinogen comprising two fibrinogen alpha chains, two fibrinogen beta chains and two fibrinogen gamma chains, and having an amino acid sequence derived from human fibrinogen, wherein; (a) In the fibrinogen alpha chain, with respect to each position in SEQ ID NO: 2, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R38, R42, K97, K100, R114, R123, R129, R135, K210, R216, R218, K225, K227, K238, K243, K249, R258, R271, R287, R308, R353, R367, R425, R426, K432, K437, K440, R443, K446, K448, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, K581, R591, K599, K602, K620, R621, K625, R687 and R847 are substituted or deleted, preferably substituted; and / or (b) In the fibrinogen beta chain, with respect to each position in SEQ ID NO: 3, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R44, K51, K52, R53, R60, R72, K77, K83, R87, K152, R158, K160, K163, K178, R285, K328, R334, K348, K353, K367, K374, K458 and K471 are substituted or deleted, preferably substituted; and / or (c) In the fibrinogen gamma chain, with respect to each position in SEQ ID NO: 4, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R40, K79, K84, K88, K111, K114, K185, K199, K238, K292, R301, K382 and K399 are substituted or deleted, preferably substituted An isolated recombinant fibrinogen, characterized in that... [2]One or more substituted or deleted K and R residues in the fibrinogen alpha chain comprise or consist of one or more of R135, K210, K243, R287, R308, R425, R426, K432, K437, K440, K446, K448, R458, R459, K463, K467, K480, R512, R547, K558, R573, K575, R591, K599, K620, R621, K625 and combinations thereof; and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain comprise or consist of one or more of R44, K51, R53, K77, R158, K160, K178, R334, K348, K353, K367, K374, K458, K471, and combinations thereof; and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain comprise or consist of K292, R301, or both, the isolated recombinant fibrinogen according to item 1. [3]One or more substituted or deleted K and R residues in the fibrinogen alpha chain comprise or consist of one or more of R38, R42, K97, K100, R114, R123, R129, R135, R216, K225, K238, K249, R258, R271, R443, R510, K527 and K602; and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain comprise or consist of one or more of K52, R72, K83, K152 and K163; and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain comprise or consist of K79, K84, K88, K111, K114, K185, K382 and K399, preferably one or more of K88, K111, K382 and K399, the isolated recombinant fibrinogen according to item 1 or 2. [4] One or more substituted or deleted K and R residues in the fibrinogen alpha chain are K97, K100, R114, R123, R129, R135, K210, R216, K225, K238, K243, K249, R258, R271, R287, R308, R425, R426, K432, K437, K440, R443, K446, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, R591, K599, K602, K620, K621, K625 and combinations thereof, preferably K97, K100, R123, K225, K238, K243, R510, R512, K527, K599, K602, K620, R621, K625 and combinations thereof, more preferably K100, R123, K225, K238, R510, K527, K602, K620 and combinations thereof, or consists of; and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain are R44, K51, K52, R53, R72, K77, K83, K152, R158, K160, K163, K178, R334, K348, K353, K367, K374, K458, K471 and combinations thereof, preferably K51, K52, R53, R72, K77, K152, R158, K160, K163, and combinations thereof, more preferably K52, R72, K152, K160 and combinations thereof, or consists of; and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain are K79, K84, K88, K111, K114, K382 and K399, and combinations thereof, preferably K79, K84, K88, K111, K382 and K399, and combinations, more preferably K79, K88, K111, K382, K399 and combinations thereof, or one or more of K88, K111, K382, K399 and combinations thereof, or consists of, an isolated recombinant fibrinogen according to any of the preceding items. [5] The isolated recombinant fibrinogen according to any of the preceding items, wherein one or more substituted or deleted K and R residues in the fibrinogen alpha chain comprise or consist of K100, R114, R123, R129, R135, K210, R216, K225, K238, K243, K249, R258, R271, R287, R308, R425, R426, K432, K437, K440, R443, K446, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, R591, K599, K602, K620, K621 and combinations thereof. [6] The isolated recombinant fibrinogen according to any of the preceding items, wherein one or more substituted or deleted K and R residues in the fibrinogen alpha chain comprise or consist of K100, R114, R123, K225, K238, K249, R258, R271, R287, R308, R425, R426, K432, R443, R458, K467, R510, K527, R547, K558, K575, K599, K602, K621 and combinations thereof; and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain comprise or consist of R44, K51, K52, R72, K83, K152, R158, K160, K163 and combinations thereof; and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain comprise or consist of one or more of K79, K88, K111, K382, K399 and combinations thereof. [7] In one, or preferably both, of the fibrinogen alpha chain and / or fibrinogen beta chain and / or fibrinogen gamma chain, at least two, preferably at least three, more preferably at least four, even more preferably at least five, or at least six, or at least seven, or at least eight, or at least nine, etc., or all of said K and R residues are deleted or substituted, preferably substituted, the isolated recombinant fibrinogen according to any of the preceding items. [8] All K and R residues shown in item 2, and / or item 3, and / or item 4, and / or item 5, and / or item 6 are deleted or substituted, preferably substituted, the isolated recombinant fibrinogen according to item 1. [9] The fibrinogen alpha, beta, and gamma chains other than the K and R residues defined in any of items 1 to 8 do not contain further mutations at the K and R residues, the isolated recombinant fibrinogen according to any of the preceding items.

[10] One or more K or R residues are alanine (A), histidine (H), N6-[(2-azidoethoxy)carbonyl]-L-lysine, propargyl-L-lysine, azidohomoalanine, trans-cycloocta-2-ene-L-lysine, exo BCN-L-lysine, trans-cycloocta-4-ene-L-lysine, pyrrolysine and pyrrolysine analogs (e.g., N ε -cyclopentyloxycarbonyl-L-lysine), lysine-nitrobenzyl-oxycarbonyl-N εAn isolated recombinant fibrinogen as described in any of the preceding items, substituted with an amino acid or an isotope of these amino acids, selected from the group consisting of -L-lysine, O-methyl-L-tyrosine and analogs, methionine (M), isoleucine (I), leucine (L), phenylalanine (F), tryptophan (W); preferably selected from alanine (A), histidine (H), N6-[(2-azidoethoxy)carbonyl]-L-lysine, propargyl-L-lysine, azidohomoalanine, methionine (M), isoleucine (I), leucine (L); more preferably selected from alanine (A) or histidine (H).

[11] An isolated recombinant fibrinogen as described in any of the preceding items, wherein one or more of these K or R residues located in the α-helix region are substituted with an amino acid or an isotope of these amino acids selected from the group consisting of alanine (A), histidine (H), N6-[(2-azidoethoxy)carbonyl]-L-lysine, propargyl-L-lysine, azidohomoalanine, methionine (M), isoleucine (I), leucine (L); preferably alanine (A) or histidine (H).

[12] An isolated recombinant fibrinogen as described in any of the preceding items, wherein the fibrinogen alpha, beta, and gamma chains contain only substitutions of K or R residues and no deletions.

[13] In addition to one or more substituted or deleted K and R residues in the fibrinogen alpha chain, and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain, and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain, all K residues within 2, preferably 5, more preferably 10, or 15, or 20 amino acids upstream and / or downstream of (some or all of) said one or more substituted or deleted K and R residues, and optionally all R residues as well, are substituted or deleted, preferably substituted, an isolated recombinant fibrinogen as described in any of the preceding items.

[14] In addition to one or more substituted or deleted K and R residues in the fibrinogen alpha chain, and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain, and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain, one or more, for example, 1, 2, 3 or 4 amino acids within 3, preferably 2 amino acids upstream and / or downstream of (some or all of) said one or more substituted or deleted K and R residues are also substituted or deleted, preferably substituted, the isolated recombinant fibrinogen according to any of the preceding items.

[15] In addition to one or more substituted or deleted K and R residues in the fibrinogen alpha chain, and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain, and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain, one or both of the amino acids immediately adjacent to (some or all of) said one or more substituted or deleted K and R residues are also substituted or deleted, preferably substituted, the isolated recombinant fibrinogen according to any of the preceding items.

[16] One or both of the amino acids immediately adjacent directly to (some or all of) said one or more substituted or deleted K and R residues, which are amino acids other than one or more substituted or deleted K and R residues in the fibrinogen alpha chain, and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain, and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain, are not substituted or deleted, the isolated recombinant fibrinogen according to any of the preceding items.

[17] i. At least one, preferably each, of the fibrinogen alpha chains, preferably excluding K and R residues that are preferably substituted or deleted, has an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7% identical to amino acids 20 - 629 of SEQ ID NO: 1 and comprises or consists of; and / or ii. At least one, preferably each, of the fibrinogen beta chains, preferably excluding K and R residues that are preferably substituted or deleted, has an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7% identical to amino acids 31 - 491 of SEQ ID NO: 3 and comprises or consists of; and / or iii. At least one, preferably each, of the fibrinogen gamma chains, preferably excluding K and R residues that are preferably substituted or deleted, has an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7% identical to amino acids 27 - 433 of SEQ ID NO: 4 and comprises or consists of, The isolated recombinant fibrinogen according to any one of the preceding items.

[18] (i) Each fibrinogen alpha chain preferably excludes one or more substituted or deleted K and R residues and comprises, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7% identity to any of SEQ ID NOs: 6, 7, 8, 41, 42, 43, 45, 46, 47, 48, 49, and 50, preferably to SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 41, or SEQ ID NO: 42, or SEQ ID NO: 43, more preferably to SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; and / or (ii) Each fibrinogen beta chain preferably excludes one or more substituted or deleted K and R residues and comprises, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7% identity to SEQ ID NO: 9 or SEQ ID NO: 44, preferably to SEQ ID NO: 9; and / or (iii) Each fibrinogen gamma chain preferably excludes one or more substituted or deleted K and R residues and comprises, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7% identity to SEQ ID NO: 10 or SEQ ID NO: 11. An isolated recombinant fibrinogen as described in any of the preceding items.

[19] i. Each fibrinogen alpha chain preferably comprises, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% identity with SEQ ID NO: 6 or SEQ ID NO: 7, preferably SEQ ID NO: 7, excluding K and R residues that are preferably one or more substituted or deleted; and ii. Each fibrinogen beta chain preferably comprises, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% identity with SEQ ID NO: 9, excluding K and R residues that are preferably one or more substituted or deleted; and iii. Each fibrinogen gamma chain preferably comprises, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% identity with SEQ ID NO: 10, excluding K and R residues that are preferably one or more substituted or deleted. An isolated recombinant fibrinogen as described in any of the preceding items.

[20] Each fibrinogen alpha chain contains, or consists of, an amino acid sequence having the sequence of SEQ ID NO: 6, 7, 8, 41, 42, 43, 45, 46, 47, 48, 49, and 50, preferably the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 41, or SEQ ID NO: 42, or SEQ ID NO: 43, more preferably any of the sequences of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, in addition to one or more substituted or deleted K and R residues; and / or each fibrinogen beta chain contains, or consists of, an amino acid sequence having the sequence of SEQ ID NO: 9, or SEQ ID NO: 44, preferably the sequence of SEQ ID NO: 9, in addition to one or more substituted or deleted K and R residues; and / or each fibrinogen gamma chain contains, or consists of, an amino acid sequence having the sequence of SEQ ID NO: 10, or SEQ ID NO: 11, in addition to one or more substituted or deleted K and R residues, the isolated recombinant fibrinogen according to any of the preceding items.

[21] Each fibrinogen alpha chain further contains one or more (e.g., 1, 2, 3, or 4) substitutions or deletions within 4, or 3, or 2 amino acids immediately upstream and / or downstream of at least one (e.g., 1, or 2, or 3, etc.) metalloproteinase (MMP) cleavage site (e.g., at least one MMP cleavage site is K432 with respect to each position in SEQ ID NO: 2), the isolated recombinant fibrinogen according to any of the preceding items.

[22] One or more substitutions or deletions within 4, or 3, or 2 amino acids immediately upstream and / or downstream of at least one metalloproteinase (MMP) cleavage site in the fibrinogen alpha chain include, or consist of, the substitutions of L433 and / or T435, preferably L433H and / or T435R, with respect to each position in SEQ ID NO: 2, the isolated recombinant fibrinogen according to item 21.

[23] Preferably, the isolated recombinant fibrinogen according to any of the preceding items, which is a soluble hexamer containing two fibrinogen alpha chains, two beta chains, and two gamma chains.

[24] An isolated recombinant fibrinogen as described in any of the preceding items, which is functional.

[25] An isolated recombinant fibrinogen as described in any of the preceding items, wherein "functional" means that the recombinant fibrinogen is biologically active.

[26] An isolated recombinant fibrinogen as described in any of the preceding items, wherein the recombinant fibrinogen is intact.

[27] An isolated recombinant fibrinogen as described in any of items 1 to 26, and optionally, a fibrin sealant comprising thrombin.

[28] A fibrin sealant comprising a functional recombinant fibrinogen, wherein each recombinant fibrinogen preferably comprises two fibrinogen alpha chains, two fibrinogen beta chains, and two fibrinogen gamma chains with an amino acid sequence derived from human fibrinogen; the recombinant fibrinogen is i. Preferably, except for one or more substituted or deleted K and R residues, any of SEQ ID NOs: 6, 7, 8, 41, 42, 43, 45, 46, 47, 48, 49, and 50, preferably SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 41, or SEQ ID NO: 42, or SEQ ID NO: 43, more preferably SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, and has an amino acid sequence with at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% identity, preferably each comprising or consisting of, a fibrinogen alpha chain; and / or ii. Preferably, except for one or more substituted or deleted K and R residues, preferably, the fibrinogen beta chain comprises, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% identity to SEQ ID NO: 9, or SEQ ID NO: 44, SEQ ID NO: 9; and / or iii. Preferably, except for one or more substituted or deleted K and R residues, the fibrinogen gamma chain comprises, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% identity to SEQ ID NO: 10, or SEQ ID NO: 11; comprising (a) In one or preferably both of the fibrinogen alpha chains, with respect to each position in SEQ ID NO: 2, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R38, R42, K97, K100, R114, R123, R129, R135, K210, R216, R218, K225, K227, K238, K243, K249, R258, R271, R287, R308, R353, R367, R425, R426, K432, K437, K440, R443, K446, K448, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, K581, R591, K599, K602, K620, R621, K625, R687 and R847 are substituted or deleted; and / or (b) In one or, preferably, both of the fibrinogen beta chains, with respect to each position in SEQ ID NO: 3, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R44, K51, K52, R53, R60, R72, K77, K83, R87, K152, R158, K160, K163, K178, R285, K328, R334, K348, K353, K367, K374, K458 and K471 are substituted or deleted; and / or (c) In one or, preferably, both of the fibrinogen gamma chains, with respect to each position in SEQ ID NO: 4, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R40, K79, K84, K88, K111, K114, K185, K199, K238, K292, R301, K382 and K399 are substituted or deleted A fibrin sealant, characterized in that.

[29] Preferably, thrombin from human or bovine, more preferably human; and optionally, one or more additives selected from the group consisting of prototransglutaminase, preferably factor XIII (FXIII), calcium chloride, polyphosphate (PolyP), Zn2+, fibronectin, and hydroxyapatite are further included in the fibrin sealant according to item 27 or 28.

[30] Each recombinant fibrinogen protein is preferably a soluble hexamer containing two fibrinogen alpha chains, two beta chains and two gamma chains, in the fibrin sealant according to any one of items 27 to 29.

[31] The isolated recombinant fibrinogen is functional, in the fibrin sealant according to any one of items 27 to 30.

[32] Functional means that the recombinant fibrinogen is biologically active, in the fibrin sealant according to any one of items 27 to 31.

[33] The recombinant fibrinogen is intact, in the fibrin sealant according to any one of items 27 to 32.

[34] A fibrin sealant according to any one of items 27 to 33, which does not contain aprotinin.

[35] A fibrin sealant according to any one of items 27 to 34, which does not contain a plasmin inhibitor.

[36] A fibrin sealant according to any one of items 27 to 35, which does not contain a protease inhibitor.

[37] A fibrin sealant according to any one of items 27 to 36, for use in sealing a defective site or incision surface of an organ and tissue, or for a sealing joint between incised tissues or between an incised tissue and a prosthesis material.

[38] i) A container containing the isolated recombinant fibrinogen according to any one of items 1 to 26; and ii) A container containing thrombin, preferably derived from human or bovine, more preferably human A fibrin sealant kit comprising.

[39] i) A container containing a functional recombinant fibrinogen, wherein each recombinant fibrinogen protein contains two fibrinogen alpha chains, two fibrinogen beta chains and two fibrinogen gamma chains; and ii) A container containing thrombin, preferably derived from human or bovine, more preferably human comprising; (a) In one, preferably both, of the fibrinogen alpha chains, with respect to each position in SEQ ID NO: 2, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R38, R42, K97, K100, R114, R123, R129, R135, K210, R216, R218, K225, K227, K238, K243, K249, R258, R271, R287, R308, R353, R367, R425, R426, K432, K437, K440, R443, K446, K448, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, K581, R591, K599, K602, K620, R621, K625, R687 and R847 are substituted or deleted; and / or (b) In one, preferably both, of the fibrinogen beta chains, with respect to each position in SEQ ID NO: 3, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R44, K51, K52, R53, R60, R72, K77, K83, R87, K152, R158, K160, K163, K178, R285, K328, R334, K348, K353, K367, K374, K458 and K471 are substituted or deleted; and / or (c) In one, preferably both, of the fibrinogen gamma chains, with respect to each position in SEQ ID NO: 4, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R40, K79, K84, K88, K111, K114, K185, K199, K238, K292, R301, K382 and K399 are substituted or deleted A fibrin sealant kit, characterized by the above.

[40] The fibrin sealant kit according to item 38 or 39, further comprising at least one additive selected from the group consisting of prototransglutaminase, preferably factor XIII (FXIII), one or more calcium salts, preferably calcium chloride, polyphosphate (PolyP), Zn2+, fibronectin, hydroxyapatite, growth factor, preferably VEGF, one or more monosaccharides and / or polysaccharides, and cells, preferably stem cells.

[41] The fibrin sealant kit according to item 40, wherein the factor XIII is contained in a container containing fibrinogen.

[42] The fibrin sealant kit according to any one of items 38 to 41, which does not contain aprotinin.

[43] The fibrin sealant kit according to any one of items 38 to 42, which does not contain a plasmin inhibitor.

[44] The fibrin sealant kit according to any one of items 38 to 43, which does not contain a protease inhibitor.

[45] The fibrin sealant according to any one of items 27 to 37 or the fibrin sealant kit according to any one of items 38 to 44 for use in soft tissue procedures and / or internal wound procedures.

[46] The fibrin sealant according to any one of items 27 to 37 or the fibrin sealant kit according to any one of items 38 to 44 for use as a hemostatic agent, tissue sealant, or wound adhesive.

[45] Use of the fibrin sealant according to any one of items 27 to 37 or the fibrin sealant kit according to any one of items 38 to 44 in soft tissue procedures and / or internal wound procedures.

[48] In the fibrin sealant, or the fibrin sealant kit i. Preferably, except for one or more substituted or deleted K and R residues, preferably any of SEQ ID NO: 6, 7, 8, 41, 42, 43, 45, 46, 47, 48, 49, and 50, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 41, or SEQ ID NO: 42, or SEQ ID NO: 43, preferably having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% identity with SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, an amino acid sequence, preferably each containing or consisting of, a fibrinogen alpha chain; and / or ii. Preferably, except for one or more substituted or deleted K and R residues, preferably SEQ ID NO: 9, or SEQ ID NO: 44, having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% identity with SEQ ID NO: 9, an amino acid sequence, preferably each containing or consisting of, a fibrinogen beta chain; and / or iii. Preferably, except for one or more substituted or deleted K and R residues, having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% identity with SEQ ID NO: 10 or SEQ ID NO: 11, an amino acid sequence, preferably each containing or consisting of, a fibrinogen gamma chain; comprising; (a) In one or, preferably, both of the fibrinogen alpha chains, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R38, R42, K97, K100, R114, R123, R129, R135, K210, R216, R218, K225, K227, K238, K243, K249, R258, R271, R287, R308, R353, R367, R425, R426, K432, K437, K440, R443, K446, K448, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, K581, R591, K599, K602, K620, R621, K625, R687 and R847 are substituted or deleted; and / or (b) In one or, preferably, both of the fibrinogen beta chains, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R44, K51, K52, R53, R60, R72, K77, K83, R87, K152, R158, K160, K163, K178, R285, K328, R334, K348, K353, K367, K374, K458 and K471 are substituted or deleted; and / or (c) In one or, preferably, both of the fibrinogen gamma chains, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R40, K79, K84, K88, K111, K114, K185, K199, K238, K292, R301, K382 and K399 are substituted or deleted; Use of an isolated recombinant fibrinogen, characterized in that.

[49] A eukaryotic cell comprising an exogenous nucleotide sequence encoding one or more recombinant fibrinogen alpha chains, one or more recombinant fibrinogen beta chains, and one or more recombinant fibrinogen gamma chains; i. One or more fibrinogen alpha chains, preferably each of which, preferably excluding K and R residues that are preferably one or more substituted or deleted, is any of SEQ ID NOs: 6, 7, 8, 41, 42, 43, 45, 46, 47, 48, 49, and 50, preferably SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 41, or SEQ ID NO: 42, or SEQ ID NO: 43, more preferably SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, and has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.7% identity with an amino acid sequence, or consists of such; and / or ii. One or more fibrinogen beta chains, preferably each of which, preferably excluding K and R residues that are preferably one or more substituted or deleted, is SEQ ID NO: 9, or SEQ ID NO: 44, preferably SEQ ID NO: 9, and has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.7% identity with an amino acid sequence, or consists of such; and / or iii. One or more fibrinogen gamma chains, preferably each of which, preferably excluding K and R residues that are preferably one or more substituted or deleted, is SEQ ID NO: 10, or SEQ ID NO: 11, and has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.7% identity with an amino acid sequence, or consists of such; (a) In one or, preferably, both of the fibrinogen alpha chains, with respect to each position in SEQ ID NO: 2, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R38, R42, K97, K100, R114, R123, R129, R135, K210, R216, R218, K225, K227, K238, K243, K249, R258, R271, R287, R308, R353, R367, R425, R426, K432, K437, K440, R443, K446, K448, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, K581, R591, K599, K602, K620, R621, K625, R687 and R847 are substituted or deleted; and / or (b) In one or, preferably, both of the fibrinogen beta chains, with respect to each position in SEQ ID NO: 3, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R44, K51, K52, R53, R60, R72, K77, K83, R87, K152, R158, K160, K163, K178, R285, K328, R334, K348, K353, K367, K374, K458 and K471 are substituted or deleted; and / or (c) In one or, preferably, both of the fibrinogen gamma chains, with respect to each position in SEQ ID NO: 4, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R40, K79, K84, K88, K111, K114, K185, K199, K238, K292, R301, K382 and K399 are substituted or deleted A eukaryotic cell, characterized by the above.

[50] The eukaryotic cell according to item 49, which produces a recombinant fibrinogen, preferably functional, when cultured under conditions where fibrinogen is produced.

[51] A eukaryotic cell according to item 49 or 50, which is a mammalian cell culture cell, preferably a Chinese hamster ovary (CHO) cell selected from CHO-K1, CHO-DG44, CHO-Pro minus and CHO-S cells, PER.C6 cells, or a human embryonic kidney (HEK) cell selected from HEK 293, HEK 293T, HEK 293S and HEK 293 EBNA cells, or a mouse myeloma cell, preferably a mouse myeloma cell selected from NS0 cells, NS-1 cells and Sp2 / 0 cells, or a baby hamster kidney (BHK) cell, preferably BHK-21 cell, or a rat myeloma cell, preferably YB2 / 0 cell or YB2 / 3HL cell.

[52] Each fibrinogen alpha chain further comprises one or more (e.g., 1, 2, 3 or 4) substitutions or deletions within 4, or 3, or 2 amino acids immediately upstream and / or downstream of at least one (e.g., 1, or 2, or 3, etc.) metalloproteinase (MMP) cleavage site (e.g., at least one MMP cleavage site is K432 with respect to each position in SEQ ID NO: 2); preferably, one or more substitutions or deletions within 4, or 3, or 2 amino acids immediately upstream and / or downstream of at least one metalloproteinase (MMP) cleavage site in the fibrinogen alpha chain comprises or consists of substitutions of L433 and / or T435, preferably L433H and / or T435R, with respect to each position in SEQ ID NO: 2; a fibrin sealant according to any one of items 27-37, or a fibrin sealant kit according to any one of items 38-44, or a fibrin sealant or fibrin sealant kit for use according to item 45 or 46, or the use according to item 47 or 48, or the eukaryotic cell according to any one of items 49-51.

[53] A method for the production of an isolated recombinant fibrinogen having one or more site-specific mutations, preferably compared to human wild-type fibrinogen, comprising: A) Preferably, a eukaryotic cell comprising an exogenous nucleotide sequence encoding at least one fibrinogen alpha chain, at least one fibrinogen beta chain, and at least one fibrinogen gamma chain, derived preferably from a mammal, more preferably from human fibrinogen, is cultured in a culture medium under conditions under which fibrinogen is produced; and B) recovering the produced recombinant fibrinogen comprising, wherein step A) is preferably carried out in the presence of at least one matrix metalloproteinase (MMP) inhibitor selected from the group consisting of 1,10-phenanthroline, UK 370106, GM6001, and combinations thereof, preferably UK 370106, Method.

[54] A method for the production of an isolated recombinant fibrinogen having one or more site-specific mutations, preferably compared to human wild-type fibrinogen, A) Preferably, a eukaryotic cell comprising an exogenous nucleotide sequence encoding at least one fibrinogen alpha chain, at least one fibrinogen beta chain, and at least one fibrinogen gamma chain, derived preferably from a mammal, more preferably from human fibrinogen, is cultured in a culture medium under conditions under which fibrinogen is produced; and B) recovering the produced recombinant fibrinogen comprising; each fibrinogen alpha chain comprises one or more (e.g., 1, 2, 3, or 4) substitutions or deletions within 4, or 3, or 2 amino acids immediately upstream and / or downstream of at least one (e.g., 1, or 2, or 3, etc.) matrix metalloproteinase (MMP) cleavage site (e.g., at least one MMP cleavage site is K432 with respect to each position in SEQ ID NO: 2); Preferably, one or more substitutions or deletions within 4, or 3, or 2 amino acids immediately upstream and / or downstream of at least one metalloproteinase (MMP) cleavage site in the fibrinogen alpha chain, for each position in SEQ ID NO:2, comprise or consist of the substitutions L433 and / or T435, preferably L433H and / or T435R, Method.

[55] Step B) preferably comprises collecting at least a portion of the culture medium containing recombinant fibrinogen that is greater than 0.5 μg / ml, more preferably greater than 1 μg / ml, even more preferably greater than 2 μg / ml, even more preferably greater than 5 μg / ml, even more preferably greater than 10 μg / ml, even more preferably greater than 20 μg / ml, for example greater than 50 μg / ml, or for example greater than 100 μg / ml, for the production of the isolated recombinant fibrinogen according to item 53 or 54.

[56] Step B) optionally comprises concentrating fibrinogen from the culture medium to form a concentrated medium and preferably purifying the recombinant fibrinogen from the culture medium, for the production of the isolated recombinant fibrinogen according to any one of items 53 to 55.

[57] The purification is performed by a chromatography method, preferably affinity chromatography, for the production of the isolated recombinant fibrinogen according to item 56.

[58] A method for producing the isolated recombinant fibrinogen according to any one of items 53 to 57, wherein the eukaryotic cell is selected from mammalian cell culture cells, preferably Chinese hamster ovary (CHO) cells, preferably CHO-K1, CHO-DG44, CHO-Pro minus and CHO-S cells, Chinese hamster ovary (CHO) cells selected from PER.C6 cells, or human embryonic kidney (HEK) cells, preferably human embryonic kidney (HEK) cells selected from HEK 293, HEK 293T, HEK 293S or HEK 293 EBNA cells, or mouse myeloma cells, preferably mouse myeloma cells selected from NS0 cells, NS-1 cells and Sp2 / 0 cells, or baby hamster kidney (BHK) cells, preferably BHK-21 cells, or rat myeloma cells, preferably YB2 / 0 cells or YB2 / 3HL cells.

[59] A method for producing the isolated recombinant fibrinogen according to any one of items 53 to 58, wherein at least one of the exogenous nucleotide sequences, preferably each, is optimized for expression in mammalian cell culture cells under conditions where fibrinogen is produced, or the eukaryotic cell according to any one of items 47 to 49.

[60] A method for producing the isolated recombinant fibrinogen according to any one of items 53 to 59, wherein the cell comprises a single nucleotide construct comprising nucleotide sequences encoding the fibrinogen alpha, beta and gamma chains, or the eukaryotic cell according to any one of items 47 to 49.

[61] A method for producing the isolated recombinant fibrinogen according to any one of items 53 to 60, wherein the isolated recombinant fibrinogen is the isolated recombinant fibrinogen according to any one of items 1 to 26.

[62] i. Preferably, each of one or more fibrinogen alpha chains, preferably excluding one or more substituted or deleted K and R residues, is any of SEQ ID NO: 6, 7, 8, 41, 42, 43, 45, 46, 47, 48, 49, and 50, preferably SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 41, or SEQ ID NO: 42, or SEQ ID NO: 43, more preferably SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, and has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% amino acid sequence identity; and / or ii. Preferably, each of one or more fibrinogen beta chains, preferably excluding one or more substituted or deleted K and R residues, is SEQ ID NO: 9, or SEQ ID NO: 44, preferably SEQ ID NO: 9, and has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% amino acid sequence identity; and / or iii. Preferably, each of one or more fibrinogen gamma chains, preferably excluding one or more substituted or deleted K and R residues, is SEQ ID NO: 10, or SEQ ID NO: 11, and has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% amino acid sequence identity; (a) In one, preferably both, of the fibrinogen alpha chains, with respect to each position in SEQ ID NO: 2, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R38, R42, K97, K100, R114, R123, R129, R135, K210, R216, R218, K225, K227, K238, K243, K249, R258, R271, R287, R308, R353, R367, R425, R426, K432, K437, K440, R443, K446, K448, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, K581, R591, K599, K602, K620, R621, K625, R687 and R847 are substituted or deleted; and / or (b) In one, preferably both, of the fibrinogen beta chains, with respect to each position in SEQ ID NO: 3, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R44, K51, K52, R53, R60, R72, K77, K83, R87, K152, R158, K160, K163, K178, R285, K328, R334, K348, K353, K367, K374, K458 and K471 are substituted or deleted; and / or (c) In one, preferably both, of the fibrinogen gamma chains, with respect to each position in SEQ ID NO: 4, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R40, K79, K84, K88, K111, K114, K185, K199, K238, K292, R301, K382 and K399 are substituted or deleted A method for the production of an isolated recombinant fibrinogen according to any one of items 53 to 60, characterized in that.

[63] A method for identifying a plasmin-resistant recombinant fibrinogen variant, comprising A) i) Preferably, incubate by covering a fibrin gel with a density of 10-30 mg / ml, more preferably 20 mg / ml, preferably with a plasmin solution at 0.001-0.01 mU / ml; ii) Collect the supernatant at different time points after the addition of the plasmin solution, and optionally, preferably by the addition of PSMF and / or heat, inactivate the plasmin; iii) Determine the cleavage sites in fibrinogen to obtain a time-resolved cleavage pattern of fibrinogen, preferably using liquid chromatography mass spectrometry or solid phase extraction (SPE) mass spectrometry, more preferably using high performance liquid chromatography / electrospray ionization mass spectrometry (HPLC / ESI-MS); and iv) Identify the lysine (K) and / or arginine (R) residues immediately and optionally immediately following the cleavage sites in the amino acid sequence of fibrinogen Steps for identifying plasmin cleavage sites in fibrinogen; and B) Optionally, select one or more of the identified lysine (K) and / or arginine (R) residues in the amino acid sequence of fibrinogen for substitution or deletion, preferably based on the time-resolved cleavage pattern, to obtain a new recombinant fibrinogen variant; and optionally, incubate the new recombinant fibrinogen variant with plasmin and test for sensitivity to protein cleavage by plasmin A method comprising.

[64] The method according to item 63, wherein in step A) iii), an internal standard, preferably angiotensin II, is used to analyze the cleavage pattern.

[65] The method according to item 63 or 64, wherein in step iii), the collected supernatant is purified using liquid chromatography, preferably HPLC, or solid phase extraction (SPE).

[66] The method according to any one of items 63-65, wherein the fibrinogen is wild-type fibrinogen.

[67] A method according to any one of items 63 to 65, wherein fibrinogen is a mutant fibrinogen, preferably selected from the isolated recombinant fibrinogens according to any one of items 1 to 26.

Brief Description of Drawings

[0021]

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Mode for Carrying Out the Invention

[0022] The present invention relates to a new isolated recombinant fibrinogen variant having improved stability in the presence of plasmin, a fibrin sealant and a fibrin sealant kit comprising said new isolated recombinant fibrinogen variant, and a eukaryotic cell expressing said new recombinant fibrinogen variant. The present invention further relates to a method for the production of an isolated recombinant fibrinogen variant using cell culture cells, and a method for identifying a plasmin-resistant recombinant fibrinogen variant.

[0023] In a first aspect, the present invention is directed to an isolated recombinant fibrinogen comprising two fibrinogen alpha chains, two fibrinogen beta chains and two fibrinogen gamma chains and having an amino acid sequence derived from human fibrinogen.

[0024] The term "fibrinogen" is known to those skilled in the art and, as used herein, refers to a hexameric protein comprising two fibrinogen alpha chains, two fibrinogen beta chains and two fibrinogen gamma chains. The terms "recombinant fibrinogen" and "isolated recombinant fibrinogen" are also known to those skilled in the art and refer to fibrinogen produced by genetic manipulation of cells, preferably using an expression construct encoding a fibrinogen chain and / or a fibrinogen chain precursor. Methods for the production of recombinant fibrinogen are described, for example, in the following patents and patent applications: WO9607728A1, WO2018161135A1, US6037457A, US6083902A, US2010151522A1, US2010159512A1, US2017037108A1 and US10208101B2.

[0025] The term "fibrinogen alpha chain", which is known to those skilled in the art, refers to one of the three subunits of the hexameric fibrinogen protein. As described above, it is generally referred to as the alpha chain and is referred to herein as "isoform alpha" or "isoform α", isoform 2; and isoform E (αE), also known as isoform 1, which is referred to herein as "isoform alpha-E" or "isoform αE", there are two human wild-type isoforms of the fibrinogen alpha chain. The full-length human wild-type fibrinogen alpha chain isoform alpha before post-translational modification (P(trademark)) has, for example, the amino acid sequence shown in SEQ ID NO: 1 (see also Table 1). The full-length human wild-type fibrinogen alpha chain isoform alpha-E before post-translational modification has, for example, the amino acid sequence shown in SEQ ID NO: 2 (see also Table 1).

[0026] The term "Aα" or "A alpha" (having, for example, the wild-type sequence shown in SEQ ID NO: 6) refers to the secreted form of the alpha chain isoform alpha, which lacks the 19-amino acid-long "signal peptide" or "signal sequence" present at the N-terminus, for example, in the full-length alpha chain before post-translational modification. The Aα form still contains "fibrinopeptide A", which consists of amino acids 20-35, for example, in the full-length amino acid sequence of isoform alpha before post-translational modification. Similarly, the term "AαE" or "A alpha-E" (having, for example, the wild-type sequence shown in SEQ ID NO: 8) refers to the secreted form of the alpha chain isoform alpha-E, which lacks the 19-amino acid-long "signal peptide" or "signal sequence" present at the N-terminus, for example, in the full-length amino acid sequence of isoform alpha-E before post-translational modification, but still contains "fibrinopeptide A" or (FpA), which consists of amino acids 20-35, for example, in the full-length amino acid sequence of isoform alpha-E before post-translational modification.

[0027] For example, for the 625 amino acids of the wild-type peptide, although synthesized as a precursor, the alpha chain isoform alpha (i.e., A alpha) exists in plasma as a polypeptide approximately 610 amino acids in length (e.g., having the wild-type sequence shown in SEQ ID NO: 7), which has lost some amino acid residues at the C-terminus compared to the full-length isoform alpha and Aα immediately after secretion (see also Table 1).

[0028] The term "fibrinogen beta chain", known to those skilled in the art, refers to the second of the three subunits of the hexameric fibrinogen protein. The fibrinogen beta chain is generally referred to herein as the "beta chain" or "β chain". In its full-length human wild-type fibrinogen beta chain before post-translational modification, for example, it has the amino acid sequence shown in SEQ ID NO: 3 (see also Table 1). The term "Bβ" or "B beta" (e.g., having the wild-type sequence shown in SEQ ID NO: 9) refers to the secreted form of the beta chain after post-translational modification, which lacks the "signal peptide" or "signal sequence" at the N-terminus that is, for example, 30 amino acids in length and present in the full-length beta chain before post-translational modification. The Bβ form still contains "fibrinopeptide B", which consists of amino acids 31-44 of the full-length amino acid sequence of the wild-type Bβ, for example, the fibrinogen beta chain before post-translational modification.

[0029] The term "fibrinogen gamma chain", which is known to those skilled in the art, refers to the third of the three subunits of the hexameric fibrinogen protein. As described above, there are two human wild-type isoforms of the fibrinogen gamma chain. The first isoform is known as gamma or gamma A (γ or γA; also simply referred to as the gamma chain, or the gamma A chain), and in this specification, it is referred to as the gamma chain "isoform gamma" or "isoform γ". The second isoform is known as gamma' or gamma B (γ' or γB; also simply referred to as the gamma' chain, or the gamma B chain), and in this specification, it is referred to as the gamma chain "isoform gamma'" or "isoform γ'". The full-length human wild-type fibrinogen gamma chain isoform gamma before post-translational modification has, for example, the amino acid sequence shown in SEQ ID NO: 4 (see also Table 1). The full-length human wild-type fibrinogen gamma chain isoform gamma' before post-translational modification has, for example, the amino acid sequence shown in SEQ ID NO: 5 (see also Table 1). The gamma chain isoform gamma (for example, having the wild-type sequence shown in SEQ ID NO: 10) and the gamma chain isoform gamma' (for example, having the wild-type sequence shown in SEQ ID NO: 11) after post-translational modification of their secreted forms lack the N-terminal, for example, the 26-amino acid-long "signal peptide" or "signal sequence" present in the full-length gamma chain before post-translational modification.

[0030] The fibrinogen alpha, beta, and gamma chains of the isolated recombinant fibrinogen of the present invention have amino acid sequences derived from human fibrinogen. This means that the fibrinogen amino acid sequences are of human origin, preferably the amino acid sequences of wild-type human fibrinogen (see also Table 1 below), as shown, for example, in SEQ ID NOs: 1-11 (prior to any molecular manipulations). One or several of the amino acid sequences of the individual peptides of the hexameric isolated recombinant fibrinogen may be as shown in SEQ ID NOs: 1-11, provided that at least one of the peptides of the isolated recombinant fibrinogen contains one or more modifications, as detailed below. Also, these sequences are of human origin, but they may be modified, for example, by substitution, deletion, insertion, or post-translational modification (beyond any modifications at the lysine (K) and / or arginine (R) residues described below).

[0031] In addition, it should be understood that the wild-type sequences of the fibrinogen alpha, beta, and gamma chains are also subject to sequence variations, resulting in differences in amino acid sequences. Thus, these fibrinogen alpha, beta, and / or gamma chains of the isolated recombinant fibrinogen of the present invention that do not contain one or more substitutions or deletions at one or more lysine (K) and / or arginine (R) residues, as shown below according to the present invention, may contain sequence variations that include one or several amino acids that result in differences to the amino acid sequences of SEQ ID NOs: 1-11.

Table 1A

Table 1B

Table 1C

Table 1D

Table 1E

[0032] The isolated recombinant fibrinogen of the present invention is (a) In the fibrinogen alpha chain, for each position in SEQ ID NO: 2 (or SEQ ID NO: 1 for all residues except R687 and R847 which are not found in SEQ ID NO: 1), one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R38, R42, K97, K100, R114, R123, R129, R135, K210, R216, R218, K225, K227, K238, K243, K249, R258, R271, R287, R308, R353, R367, R425, R426, K432, K437, K440, R443, K446, K448, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, K581, R591, K599, K602, K620, R621, K625, R687 and R847 are substituted or deleted; and / or (b) In the fibrinogen beta chain, for each position in SEQ ID NO: 3, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R44, K51, K52, R53, R60, R72, K77, K83, R87, K152, R158, K160, K163, K178, R285, K328, R334, K348, K353, K367, K374, K458 and K471 are substituted or deleted; and / or (c) In the fibrinogen gamma chain, for each position in SEQ ID NO: 4, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R40, K79, K84, K88, K111, K114, K185, K199, K238, K292, R301, K382 and K399 are substituted or deleted characterized by.

[0033] In the context of the present invention, the term "with respect to each position in SEQ ID NO..." should be understood to mean only that the so-called amino acid sequence functions as a reference sequence for each indicated K or R residue in each fibrinogen chain. In other words, SEQ ID NO:2 (or equivalently, SEQ ID NO:1 for all residues of the alpha chain except these residues not found in SEQ ID NO:1, i.e., R687 and R847), 3, and 4 in the context of this term simply act as a reference system for clearly identifying K and R residues without limiting the sequences of the alpha, beta, and gamma chains of the isolated recombinant fibrinogen of the present invention in any way beyond this specific identification of the positions of the K and R residues.

[0034] For example, it does not mean that each alpha, beta, and / or gamma chain of the isolated recombinant fibrinogen of the present invention defined in the same manner contains all of such reference sequences, i.e., all of SEQ ID NO:1, 3, and 4. On the contrary, in many preferred embodiments of the present invention, the alpha, beta, and / or gamma chains of the isolated recombinant fibrinogen of the present invention contain only fragments of the amino acid sequences shown in SEQ ID NO:1, 3, and 4, or variants of such fragments. Such fragments of the full-length wild-type sequence are shown, for example, in SEQ ID NOs:6 - 11. Furthermore, the amino acid sequences of the alpha, beta, and / or gamma chains of the isolated recombinant fibrinogen of the present invention may contain one or more sequence variations (other than these K and R residues listed above), even when compared to one of these alpha, beta, and / or gamma chain fragments of SEQ ID NOs:6 - 11.

[0035] The isolated recombinant fibrinogen of the present invention may contain one or more substitutions or deletions at one or more of the K and R residues, as shown above, or as shown in any of the more specific embodiments below, in the fibrinogen alpha chain, and / or fibrinogen beta chain, and / or fibrinogen gamma chain. This means that the isolated recombinant fibrinogen contains one or more substitutions or deletions of the K and R residues shown above, such as one, or preferably two, gamma chains, and / or one, or preferably two, beta chains, and / or one, or preferably two, alpha chains of the recombinant fibrinogen, in one, or two, or three, or four, or five, or six, or seven, or eight, or nine, etc.

[0036] For example, in one embodiment of the present invention, the isolated recombinant fibrinogen comprises, in the gamma chain of the isolated recombinant fibrinogen, as shown above, in the gamma chain sequence, for example, 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 substitutions or deletions at K and / or R residues. In another embodiment, the isolated recombinant fibrinogen comprises, in the beta chain of the isolated recombinant fibrinogen, as shown above, in the beta chain sequence, for example, 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, ···, or 18, or 19 substitutions or deletions at K and / or R residues. In yet another embodiment, the isolated recombinant fibrinogen comprises, in the alpha chain of the isolated recombinant fibrinogen, as shown above, in the alpha chain sequence, for example, 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, ···, or 46, or 47 substitutions or deletions at K and / or R residues. In yet another embodiment, the isolated recombinant fibrinogen of the present invention comprises a combination of the above-described substitutions or deletions in the alpha, beta, and / or gamma chains. In a preferred embodiment, two alpha chains and / or two beta chains and / or two gamma chains of the isolated recombinant fibrinogen have the same substitution or deletion, preferably a substitution, at one or more of the K and R residues.

[0037] In a preferred embodiment, the isolated recombinant fibrinogen has, in one or, preferably, both gamma chains of the isolated recombinant fibrinogen, as shown above, in the gamma chain sequence, at least two, preferably at least three, more preferably at least four, for example five, or six substitutions or deletions at K and / or R residues, preferably including substitutions, and optionally no substitutions at the K and / or R residues identified above in the alpha and beta chains. Equivalent embodiments using the alpha chain, and equivalent embodiments using the beta chain, are alternatively preferred embodiments of the invention.

[0038] According to a specific embodiment of the invention, one or more substitutions or deletions in the alpha chain and / or beta chain and / or gamma chain are present in both alpha chains and / or both beta chains and / or both gamma chains of the isolated recombinant fibrinogen, such that, with respect to one or more substitutions or deletions, there is no difference between the two alpha chains and / or two beta chains and / or two gamma chains.

[0039] According to one embodiment, the isolated recombinant fibrinogen has one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R38, R42, K97, K100, R114, R123, R129, R135, K210, R216, K225, K238, K243, K249, R258, R271, R287, R308, R425, R426, K432, K437, K440, R443, K446, K448, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, R591, K599, K602, K620, R621 and K625 substituted or deleted, preferably substituted; and / or in one or both fibrinogen beta chains, for each position in SEQ ID NO: 3, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R44, K51, K52, R53, R72, K77, K83, K152, R158, K160, K163, K178, R334, K348, K353, K367, K374, K458 and K471 are substituted or deleted, preferably substituted; and / or in one or both fibrinogen gamma chains, for each position in SEQ ID NO: 4, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of K79, K84, K88, K111, K114, K185, K292, R301, K382 and K399 are substituted or deleted, preferably substituted.

[0040] According to a preferred embodiment of the present invention, one or more substituted or deleted K and R residues in the fibrinogen alpha chain, with respect to each position in SEQ ID NO: 1, include or consist of one or more of R135, K210, K243, R287, R308, R425, R426, K432, K437, K440, K446, K448, R458, R459, K463, K467, K480, R512, R547, K558, R573, K575, R591, K599, K620, R621, K625 and combinations thereof. Additionally, according to a preferred embodiment of the present invention, one or more substituted or deleted K and R residues in the fibrinogen beta chain, with respect to each position in SEQ ID NO: 3, include or consist of one or more of R44, K51, R53, K77, R158, K160, K178, R334, K348, K353, K367, K374, K458, K471, and combinations thereof. Additionally, according to a preferred embodiment of the present invention, one or more substituted or deleted K and R residues in the fibrinogen gamma chain, with respect to each position in SEQ ID NO: 4, include or consist of K292, R301, or both. In another preferred embodiment of the present invention, in addition to the one or more substituted or deleted K and R residues shown in this paragraph, the isolated recombinant fibrinogen of the present invention includes one or more K and R residues shown in the following paragraph.

[0041] According to another preferred embodiment of the present invention, one or more substituted or deleted K and R residues in the fibrinogen alpha chain, with respect to each position in SEQ ID NO: 1 (or SEQ ID NO: 2), include or consist of one or more of R38, R42, K97, K100, R114, R123, R129, R135, R216, K225, K238, K249, R258, R271, R443, R510, K527, and K602; and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain, with respect to each position in SEQ ID NO: 3, include or consist of one or more of K52, R72, K83, K152, and K163; and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain, with respect to each position in SEQ ID NO: 4, include or consist of one or more of K79, K84, K88, K111, K114, K185, K382, and K399, preferably one or more of K88, K111, K382, and K399. According to yet another preferred embodiment, one or more substituted or deleted K and R residues in the fibrinogen alpha chain, with respect to each position in SEQ ID NO: 1 (or SEQ ID NO: 2), include or consist of K100, R114, R123, R129, R135, K210, R216, K225, K238, K243, K249, R258, R271, R287, R308, R425, R426, K432, K437, K440, R443, K446, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, R591, K599, K602, K620, K621, and combinations thereof.

[0042] According to yet another preferred embodiment of the present invention, one or more substituted or deleted K and R residues in the fibrinogen alpha chain, with respect to each position in SEQ ID NO: 1 (or SEQ ID NO: 2), are K97, K100, R114, R123, R129, R135, K210, R216, K225, K238, K243, K249, R258, R271, R287, R308, R425, R426, K432, K437, K440, R443, K446, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, R591, K599, K602, K620, K621, K625 and combinations thereof, preferably K97, K100, R123, K225, K238, K243, R510, R512, K527, K599, K602, K620, R621, K625 and combinations thereof, more preferably K100, R123, K225, K238, R510, K527, K602, K620 and combinations thereof, or consist of.

[0043] According to yet another preferred embodiment of the present invention, one or more substituted or deleted K and R residues in the fibrinogen beta chain, with respect to each position in SEQ ID NO: 3, are R44, K51, K52, R53, R72, K77, K83, K152, R158, K160, K163, K178, R334, K348, K353, K367, K374, K458, K471 and combinations thereof, preferably K51, K52, R53, R72, K77, K152, R158, K160, K163, and combinations thereof, more preferably K52, R72, K152, K160 and combinations thereof, or consist of.

[0044] According to yet another preferred embodiment of the present invention, one or more substituted or deleted K and R residues in the fibrinogen gamma chain, with respect to each position in SEQ ID NO: 4, are K79, K84, K88, K111, K114, K382, and K399, and combinations thereof, preferably K79, K84, K88, K111, K382, and K399, and combinations, more preferably K79, K88, K111, K382, K399, and combinations thereof, or one or more of K88, K111, K382, K399, and combinations thereof, or consist of the foregoing.

[0045] According to a more preferred embodiment of the present invention, one or more substituted or deleted K and R residues in the fibrinogen alpha chain, with respect to each position in SEQ ID NO: 1 (or SEQ ID NO: 2), include or consist of K100, R114, R123, K225, K238, K249, R258, R271, R287, R308, R425, R426, K432, R443, R458, K467, R510, K527, R547, K558, K575, K599, K602, K621, and combinations thereof; and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain, with respect to each position in SEQ ID NO: 3, include or consist of R44, K51, K52, R72, K83, K152, R158, K160, K163, and combinations thereof; and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain, with respect to each position in SEQ ID NO: 4, include or consist of one or more of K79, K88, K111, K382, K399, and combinations thereof.

[0046] In a specific embodiment, the isolated recombinant fibrinogen of the present invention, with respect to each position in SEQ ID NO: 4, contains two, or three, or four, or all of the K and R residues K79, K88, K111, K382, and K399 in one or both fibrinogen gamma chains. According to another specific embodiment, all of the K and R residues shown in any of the above embodiments are deleted or substituted, preferably substituted. In some embodiments, the fibrinogen alpha, beta, and gamma chains do not contain additional mutations in the K and R residues other than the K and R residues defined in any of the above embodiments.

[0047] One or more of the K and R residues defined above can be substituted or deleted. Methods for making such amino acid substitutions and deletions are known in the art. According to a preferred embodiment, one or more of the K and R residues defined above are substituted. For example, the fibrinogen alpha, beta, and gamma chains of the isolated recombinant fibrinogen of the present invention can contain only substitutions of K or R residues and may not contain deletions.

[0048] The one or more amino acids used to substitute the K and R residues shown above are not particularly limited. According to a preferred embodiment, one or more of the K or R residues are alanine (A), histidine (H), N6-[(2-azidoethoxy)carbonyl]-L-lysine, propargyl-L-lysine, azidohomoalanine, trans-cyclooct-2-ene-L-lysine, exo BCN-L-lysine, trans-cyclooct-4-ene-L-lysine, pyrrolysine and pyrrolysine analogs (e.g., N ε -cyclopentyloxycarbonyl-L-lysine), lysine-nitrobenzyl-oxycarbonyl-N ε-L-lysine, O-methyl-L-tyrosine and analogs, methionine (M), isoleucine (I), leucine (L), phenylalanine (F), tryptophan (W), preferably selected from the group consisting of alanine (A), histidine (H), N6-[(2-azidoethoxy)carbonyl]-L-lysine, propargyl-L-lysine, azidohomoalanine, methionine (M), isoleucine (I), leucine (L); more preferably selected from alanine (A) or histidine (H), substituted with an amino acid or an isotope of these amino acids. A particularly preferred amino acid is histidine (H). According to a specific embodiment, one or more of these K or R residues located in the α-helix region of the alpha, beta, and / or gamma chains are alanine (A), histidine (H), N6-[(2-azidoethoxy)carbonyl]-L-lysine, propargyl-L-lysine, azidohomoalanine, trans-cycloocta-2-ene-L-lysine, exo BCN-L-lysine, trans-cycloocta-4-ene-L-lysine, pyrrolysine and pyrrolysine analogs (e.g., N ε -cyclopentyloxycarbonyl-L-lysine), lysine-nitrobenzyl-oxycarbonyl-N ε -L-lysine, O-methyl-L-tyrosine and analogs, methionine (M), isoleucine (I), leucine (L); preferably alanine (A), histidine (H), N6-[(2-azidoethoxy)carbonyl]-L-lysine, propargyl-L-lysine, or azidohomoalanine; more preferably selected from the group consisting of alanine (A) or histidine (H), with an amino acid or an isotope of these amino acids; most preferably substituted with histidine (H).

[0049] According to another preferred embodiment of the present invention, one or more substituted or deleted K and R residues in the fibrinogen gamma chain include, or consist of, K88, K111, K382, and K399 with respect to each position in SEQ ID NO: 4, and these residues are substituted with histidine (H). According to yet another preferred embodiment of the present invention, one or more substituted or deleted K and R residues in the fibrinogen gamma chain include, or consist of, K88, K111, K382, and K399 with respect to each position in SEQ ID NO: 4, and these residues are substituted with alanine (A).

[0050] In addition to the one or more K and R residues, other amino acids in the immediate vicinity of the one or more K and R residues may also be mutated to further enhance the effect on plasmin digestion. According to one embodiment, in addition to the one or more K and R residues that are substituted or deleted in the fibrinogen alpha chain, and / or the one or more K and R residues that are substituted or deleted in the fibrinogen beta chain, and / or the one or more K and R residues that are substituted or deleted in the fibrinogen gamma chain, one or more, for example, 1, 2, 3 or 4 amino acids within 3, preferably 2 amino acids upstream and / or downstream in the reference sequence of the one or more substituted or deleted K and R residues (part or all thereof) (i.e., in SEQ ID NO: 1, 3, and / or 4) are also substituted or deleted, preferably substituted. According to another embodiment, in addition to the one or more K and R residues that are substituted or deleted in the fibrinogen alpha chain, and / or the one or more K and R residues that are substituted or deleted in the fibrinogen beta chain, and / or the one or more K and R residues that are substituted or deleted in the fibrinogen gamma chain, all K residues, or all R residues, or all K and R residues within 2, preferably 3, more preferably 4, even more preferably 5, even more preferably 7, or 10, or 12, or 15, or 20 amino acids upstream and / or downstream in the reference sequence of the one or more substituted or deleted K and R residues (part or all thereof) (i.e., in SEQ ID NO: 1, 3, and / or 4) are also substituted or deleted, preferably substituted.

[0051] According to yet another embodiment of the present invention, in addition to one or more substituted or deleted K and R residues in the fibrinogen alpha chain, and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain, and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain, one or both of the amino acids immediately adjacent to the reference sequence of said one or more substituted or deleted K and R residues (part or all thereof) (i.e., in SEQ ID NO: 1, 3, and / or 4) are also substituted or deleted, preferably substituted.

[0052] According to yet another embodiment, in the reference sequence (i.e., in SEQ ID NO: 1, 3, and / or 4), one or more substituted or deleted K and R residues in the fibrinogen alpha chain, and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain, and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain, which are immediately adjacent to or within two amino acids of said one or more substituted or deleted K and R residues (part or all thereof), are not substituted or deleted.

[0053] The amino acid sequences of the alpha, beta, and gamma chains of the isolated recombinant fibrinogen of the present invention, other than one or more substitutions or deletions of the K and R residues shown above, which are derived from human fibrinogen, are not particularly limited. The isolated recombinant fibrinogen of the present invention may include, for example, one isoform or two isoforms of the alpha chain and / or gamma chain, i.e., for example, homodimers of the alpha chain and gamma chain such as Aα / Aα - Bβ / Bβ - γ / γ or AαE / AαE - Bβ / Bβ - γ’ / γ’ or Aα / Aα - Bβ / Bβ - γ / γ, etc., or heterodimers of the alpha chain and / or gamma chain such as Aα / AαE - Bβ / Bβ - γ / γ or Aα / AαE - Bβ / Bβ - γ’ / γ or AαE / AαE - Bβ / Bβ - γ’ / γ, etc.

[0054] In addition, each of the three subunits of the isolated recombinant fibrinogen may contain one wild-type form or two wild-type forms, provided that at least one of the indicated lysine (K) and / or arginine (R) residues in at least one of the fibrinogen alpha chains, or at least one of the fibrinogen beta chains, or at least one of the fibrinogen gamma chains is substituted or deleted. Additionally, the isolated recombinant fibrinogen of the present invention may contain two different variants of the fibrinogen alpha chain, and / or the fibrinogen beta chain, and / or the fibrinogen gamma chain, for example, two different variants of the same fibrinogen alpha chain isoform, and / or the same fibrinogen gamma chain isoform.

[0055] The alpha, beta and / or gamma chain variants may be of the length of the sequence corresponding to each wild-type chain sequence in the secreted form, or not, or may be a shortened version of such a chain, and may further include sequence variations in the form of substitutions, deletions, and insertions compared to the wild sequence, beyond those at the K and R residues of the present invention. In the case of any such shortened and / or otherwise mutated variant of a fibrinogen chain, preferably such a variant still forms intact fibrinogen in the sense that the fibrinogen is correctly folded and not enzymatically cleaved other than any intended post-translational modifications, and is preferably functional, which preferably means that the isolated recombinant fibrinogen formed from such a variant is biologically active and can polymerize to form a stable fibrin polymer (fibrin clot) upon contact with a photoinitiator. Thus, as used herein, "functional fibrinogen" preferably means a fibrinogen having an activity qualitatively the same as the physiological activity of wild-type fibrinogen, although quantitative factors such as the level of activity, molecular weight, etc. may vary.

[0056] In the isolated recombinant fibrinogen of the present invention, according to a preferred embodiment, i. at least one, or preferably each fibrinogen alpha chain, preferably excluding one or more substituted or deleted K and R residues, is any of SEQ ID NOs: 6, 7, 8, 41, 42, 43, 45, 46, 47, 48, 49, and 50, preferably SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 41, or SEQ ID NO: 42, or SEQ ID NO: 43, more preferably SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, and has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% amino acid sequence identity; and / or ii. at least one, or preferably each fibrinogen beta chain, preferably excluding one or more substituted or deleted K and R residues, is SEQ ID NO: 9, or SEQ ID NO: 44, preferably SEQ ID NO: 9, and has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% amino acid sequence identity; and / or iii. At least one, or preferably each fibrinogen gamma chain, preferably comprises or consists of an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% identity with SEQ ID NO: 10 or SEQ ID NO: 11, excluding one or more substituted or deleted K and R residues.

[0057] The term "excluding one or more substituted or deleted K and R residues" in the context above means that sequence identity is calculated after exclusion of any one or more K and R residues that have been substituted or deleted in accordance with the teachings of the invention (i.e., excluding these one or more K and R residues that have been substituted or deleted to achieve the effect of increasing plasmin resistance as taught herein).

[0058] According to another preferred embodiment, in the isolated recombinant fibrinogen, i. each fibrinogen alpha chain preferably contains, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% identity with SEQ ID NO: 6 or SEQ ID NO: 7, preferably SEQ ID NO: 7, excluding one or more substituted or deleted K and R residues; and ii. each fibrinogen beta chain preferably contains, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% identity with SEQ ID NO: 9, excluding one or more substituted or deleted K and R residues; and iii. each fibrinogen gamma chain preferably contains, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% identity with SEQ ID NO: 10, excluding one or more substituted or deleted K and R residues.

[0059] According to yet another preferred embodiment, in an isolated recombinant fibrinogen, i. each fibrinogen alpha chain preferably contains, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% identity with SEQ ID NO: 8, except for one or more substituted or deleted K and R residues (i.e., these K and R residues substituted or deleted to achieve the effect of increasing plasmin resistance as taught herein); and ii. each fibrinogen beta chain preferably contains, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% identity with SEQ ID NO: 9, or SEQ ID NO: 44, preferably SEQ ID NO: 9, except for one or more substituted or deleted K and R residues; and iii. each fibrinogen gamma chain preferably contains, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%, or 99.6%, or 99.7%, or 100% identity with SEQ ID NO: 11, except for one or more substituted or deleted K and R residues.

[0060] According to another preferred embodiment, in the isolated recombinant fibrinogen of the present invention, one, or preferably each fibrinogen alpha chain, in addition to one or more substituted or deleted K and R residues (i.e., these K and R residues substituted or deleted to achieve the effect of increasing plasmin resistance as taught herein), comprises or consists of an amino acid sequence having the sequence of any of SEQ ID NOs: 6, 7, 8, 41, 42, 43, 45, 46, 47, 48, 49, and 50, preferably the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 41, or SEQ ID NO: 42, or SEQ ID NO: 43, more preferably the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; and / or one, or preferably each fibrinogen beta chain, in addition to one or more substituted or deleted K and R residues, comprises or consists of an amino acid sequence having the sequence of SEQ ID NO: 9, or SEQ ID NO: 44, preferably the sequence of SEQ ID NO: 9; and / or one, or preferably each fibrinogen gamma chain, in addition to one or more substituted or deleted K and R residues, comprises or consists of an amino acid sequence having the sequence of SEQ ID NO: 10, or SEQ ID NO: 11.

[0061] Furthermore, the isolated recombinant fibrinogen may contain post-translational modifications. Examples of such post-translational modifications are phosphorylation, glycosylation, hydroxylation, pyrrolidone carboxylic acid formation, lipidation, methylation, acetylation, oxidation or sulfation.

[0062] Preferably, the isolated recombinant fibrinogen of the present invention is soluble. Also, the isolated recombinant fibrinogen is preferably functional, where functional means, for example, that the recombinant fibrinogen is biologically active and that the polymerization behavior of the recombinant fibrinogen is not qualitatively different (although it may be different), and at least is not substantially altered, compared to human wild-type fibrinogen. Thus, as used herein, "functional fibrinogen" can preferably refer to fibrinogen having substantially the same physiological activity as wild-type fibrinogen, although quantitative factors such as the level of activity, molecular weight, etc. may be different.

[0063] In a second aspect, the present invention is directed to an isolated recombinant fibrinogen of the first aspect of the present invention detailed in any of the embodiments described above, and optionally, a novel fibrin sealant comprising thrombin. The terms "fibrin sealant" or "fibrin glue" are known in the art and preferably refer to a biological adhesive comprising fibrinogen or a fibrinogen derivative, the effect of which is to limit the final stage of coagulation to form a fibrin polymer. This makes the fibrin sealant useful in surgery, for example, to control bleeding or to adhere two tissues to each other. Fibrin sealants, as well as methods and processes for preparing such fibrin sealants, are known in the art.

[0064] A very common type of fibrin sealant uses fibrinogen and contains two components. One component contains concentrated human fibrinogen, (e.g., bovine) aprotinin, and factor XIII (see, e.g., US8821861B2, US2007 / 0231372A1, and US2014 / 0205636A1). The second component contains (e.g., bovine or human) thrombin and calcium chloride. Application of this type of sealant can be performed, for example, using a double-barrel syringe, which allows simultaneous delivery of both components to the desired site of fibrin clot formation. Mixing of the two components at the target site generates a fibrin clot via a series of reactions.

[0065] A second type of fibrin sealant uses a composition consisting mainly of fibrin I and / or fibrin II monomers (see, e.g., US6083902A and EP0592242A1). In these types of sealants, fibrin I monomers and / or fibrin II monomers and / or desBB fibrin monomers are prepared from fibrinogen using a suitable proteolytic enzyme such as thrombin prior to sealant application. The fibrin monomers are maintained in a soluble form using a suitable buffer. Fibrin I monomers, fibrin II monomers, or desBB fibrin monomers in such solutions can be converted to fibrin polymers by mixing the solution with a second solution under conditions that allow spontaneous polymerization of the fibrin monomers to form fibrin clots.

[0066] Preferably, the fibrin sealant further comprises thrombin, preferably human or bovine thrombin, more preferably human thrombin, as a second component for initiating fibrin polymerization when the recombinant fibrinogen contacts thrombin. Methods and processes for preparing thrombin are known in the art. As an alternative to thrombin, the fibrin sealant may comprise one or more other components that initiate and drive fibrin polymerization when it contacts fibrinogen.

[0067] The fibrin sealant may further comprise additional components such as, for example, prototransglutaminase, calcium chloride, polyphosphate (PolyP), Zn 2+ , fibronectin, and / or hydroxyapatite. Preferably, the prototransglutaminase is factor XIII (FXIII). Thus, according to a preferred embodiment, the fibrin sealant of the present invention preferably comprises human or bovine, more preferably human thrombin; and optionally, prototransglutaminase, preferably factor XIII (FXIII), one or more calcium salts (preferably calcium chloride), polyphosphate (PolyP), Zn 2+ , fibronectin, hydroxyapatite, growth factors (e.g., VEGF), one or more monosaccharides and / or polysaccharides, and sugars and polyhydric alcohols (preferably selected from mannitol, sorbitol, trehalose), and one or more additives selected from the group consisting of cells (genetically modified or not; e.g., genetically modified or non-genetically modified stem cells). According to a specific embodiment, the fibrin sealant comprises genetically modified (GMO) cells, preferably genetically modified stem cells. In yet another embodiment, the fibrin sealant further comprises one or more active ingredients (e.g., antibiotics, cytokines, antibodies or growth factors).

[0068] The new fibrin sealant of the present invention containing the isolated recombinant fibrinogen of the present invention has the advantage that the addition of an inhibitor of plasmin protease, particularly aprotinin, is no longer necessary to ensure the persistent stability of the fibrin clot once formed. Thus, in a preferred embodiment, the fibrin sealant of the present invention does not contain aprotinin. According to another preferred embodiment, the fibrin sealant of the present invention does not contain any plasmin inhibitor. According to yet another embodiment, the fibrin sealant of the present invention does not contain any protease inhibitor.

[0069] In a third aspect, the present invention relates to i) a container containing the isolated recombinant fibrinogen of the first aspect of the present invention detailed in any of the embodiments described above; and ii) a container containing thrombin, preferably of human or bovine origin, more preferably human and relates to a fibrin sealant kit comprising the same.

[0070] The fibrin sealant kit may contain additional components such as, for example, prototransglutaminase, calcium chloride, polyphosphate (PolyP), Zn 2+ , fibronectin, and hydroxyapatite. Preferably, the prototransglutaminase is factor XIII (FXIII). Therefore, according to a preferred embodiment, the fibrin sealant kit of the present invention comprises prototransglutaminase, preferably factor XIII (FXIII), one or more calcium salts (preferably calcium chloride), polyphosphate (PolyP), Zn 2+, selected from the group consisting of fibronectin, hydroxyapatite, growth factors (e.g., VEGF), one or more monosaccharides, disaccharides, polysaccharides, and sugars and polyhydric alcohols (preferably selected from mannitol, sorbitol, trehalose), and cells (recombinant or not; e.g., recombinant or non-recombinant stem cells). According to a specific embodiment, the fibrin sealant kit contains recombinant (GMO) cells, preferably recombinant stem cells. In yet another embodiment, the fibrin sealant kit further contains one or more active ingredients, preferably selected from the group consisting of active pharmaceutical ingredients (e.g., antibiotics, cytokines, antibodies or growth factors).

[0071] According to a preferred embodiment, factor XIII is contained in the container of the fibrin sealant kit containing recombinant fibrinogen. According to another preferred embodiment, the container of the fibrin sealant kit containing thrombin further contains calcium chloride.

[0072] The new fibrin sealant kit of the present invention containing the isolated recombinant fibrinogen of the present invention has the advantage that an inhibitor of plasmin protease, particularly aprotinin, is no longer necessary to ensure the persistent stability of the fibrin clot once formed. Thus, in a preferred embodiment, the fibrin sealant kit of the present invention does not contain aprotinin. According to another preferred embodiment, the fibrin sealant kit of the present invention does not contain any plasmin inhibitor. According to yet another embodiment, the fibrin sealant kit of the present invention does not contain any protease inhibitor.

[0073] The fibrin sealant and fibrin sealant kit of the present invention can be used, for example, as a hemostatic agent, a tissue sealant, or a wound adhesive. Thus, according to one embodiment of the present invention, the fibrin sealant, or the fibrin sealant kit, is for use as a hemostatic agent, a tissue sealant, or a wound adhesive. Furthermore, the fibrin sealant and the fibrin sealant kit are useful in soft tissue procedures and internal wound procedures. Thus, according to another embodiment, the fibrin sealant, or the fibrin sealant kit of the present invention, is for use in soft tissue procedures and / or internal wound procedures.

[0074] The fibrin sealant and fibrin sealant kit of the present invention can be used, for example, for sealing defective sites or incised surfaces of organs and tissues, or for sealing joints between incised tissues or between incised tissues and prosthesis materials. Therefore, according to a specific embodiment, the fibrin sealant of the present invention, and the fibrin sealant kit of the present invention, are for use in sealing defective sites or incised surfaces of organs and tissues, or for sealing joints between incised tissues or between incised tissues and prosthesis materials.

[0075] According to a fourth aspect, the present invention provides a eukaryotic cell comprising an exogenous nucleotide sequence encoding one or more recombinant fibrinogen alpha chains, one or more recombinant fibrinogen beta chains, and one or more recombinant fibrinogen gamma chains, (a) In the fibrinogen alpha chain, with respect to each position in SEQ ID NO: 2, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R38, R42, K97, K100, R114, R123, R129, R135, K210, R216, K225, K238, K243, K249, R258, R271, R287, R308, R425, R426, K432, K437, K440, R443, K446, K448, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, R591, K599, K602, K620, R621, K625, R687 and R847 are substituted or deleted; and / or (b) In the fibrinogen beta chain, with respect to each position in SEQ ID NO: 3, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R44, K51, K52, R53, R60, R72, K77, K83, R87, K152, R158, K160, K163, K178, R285, K328, R334, K348, K353, K367, K374, K458 and K471 are substituted or deleted; and / or (c) In the fibrinogen gamma chain, with respect to each position in SEQ ID NO: 4, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R40, K79, K84, K88, K111, K114, K185, K199, K238, K292, R301, K382 and K399 are substituted or deleted Characterized in that it is directed to eukaryotic cells.

[0076] The term "exogenous" in this context means that at least a portion of one or more nucleotide sequences encoding one or more recombinant fibrinogen alpha chains, one or more recombinant fibrinogen beta chains, recombinant fibrinogen gamma chains, and / or one or more promoter sequences driving their expression has been introduced by means of molecular manipulation rather than being part of the original eukaryotic cell. According to a preferred embodiment, the eukaryotic cell comprises an exogenous nucleotide sequence encoding two or fewer recombinant fibrinogen alpha chains, recombinant fibrinogen beta chains, and recombinant fibrinogen gamma chains. According to another preferred embodiment, the eukaryotic cell comprises an exogenous nucleotide sequence encoding exactly one recombinant fibrinogen alpha chain, and / or exactly one recombinant fibrinogen beta chain, and / or exactly one recombinant fibrinogen gamma chain.

[0077] With respect to one or more substituted or deleted lysine (K) and / or arginine (R) residues in the fibrinogen alpha, beta, and / or gamma chains, embodiments relating to the isolated recombinant fibrinogen of the first aspect of the invention described above are applied with modifications where necessary. Similarly, the sequences of one or more fibrinogen alpha chains, one or more recombinant fibrinogen beta chains, and one or more recombinant fibrinogen gamma chains of the isolated recombinant fibrinogen are as defined in embodiments relating to the isolated recombinant fibrinogen of the first aspect of the invention described above.

[0078] Eukaryotic cells are not particularly limited as long as genetic manipulation can be performed to yield eukaryotic cells containing an exogenous nucleotide sequence encoding one or more recombinant fibrinogen alpha chains, one or more recombinant fibrinogen beta chains, and / or one or more recombinant fibrinogen gamma chains according to the present invention. According to a preferred embodiment, the eukaryotic cells are mammalian cell culture cells. Methods for preparing eukaryotic cells, particularly mammalian cell culture cells, and recombinant proteins, and specifically such cells that express recombinant fibrinogen, are known in the art. See, for example, U.S. Patents US10,208,101 and US6,037,457, and U.S. Patent Applications US2010 / 0151522A1, US2010 / 0159512A1, and US2017 / 0037108A1.

[0079] The eukaryotic cells of the present invention preferably produce recombinant fibrinogen when cultured under conditions where fibrinogen is produced. The fibrinogen thus produced is preferably intact, containing correctly folded polypeptides and not digested, and is preferably functional, such that it can be used in a fibrin sealant.

[0080] According to a preferred embodiment, the eukaryotic cell is a mammalian cell culture cell, preferably a Chinese hamster ovary (CHO) cell, or a human embryonic kidney (HEK) cell, more preferably a Chinese hamster ovary (CHO) cell. According to a preferred embodiment, the eukaryotic cell is a human embryonic kidney (HEK) cell selected from HEK 293, HEK 293T, HEK 293S, and HEK 293 EBNA cells. According to another preferred embodiment, the eukaryotic cell is a mouse myeloma cell, preferably a mouse myeloma cell selected from NS0 cells, NS-1 cells, and Sp2 / 0 cells. According to yet another preferred embodiment, the eukaryotic cell is a baby hamster kidney (BHK) cell, preferably a BHK-21 cell. According to yet another preferred embodiment, the eukaryotic cell is a rat myeloma cell, preferably a YB2 / 0 cell or a YB2 / 3HL cell. According to a particularly preferred embodiment, the eukaryotic cell is a CHO cell selected from CHO-K1, CHO-DG44, CHO-Pro minus, and CHO-S cells, and PER.C6 cells, more preferably a CHO-S cell. A commercially available example of a suitable CHO-S cell is the ExpiCHO-S cell line manufactured by Thermo Fisher.

[0081] Exogenous nucleotide sequences encoding alpha, beta, and gamma chain variants of fibrinogen can be introduced into eukaryotic cells, for example, by using an expression vector. Expression vectors using eukaryotic cells, preferably mammalian cell culture cells, as hosts are not particularly limited, and expression vectors known in the art, such as plasmid vectors, viral vectors, etc., can be appropriately selected. The promoter contained in such a fibrinogen expression vector is not particularly limited as long as it functions efficiently in the eukaryotic host cell used and ultimately results in fibrinogen, preferably functional fibrinogen. Examples include the SV40 promoter, cytomegalovirus (CMV) promoter, RSV promoter, beta-actin promoter, etc. It is also possible to combine the promoter with a suitable enhancer. Such fibrinogen expression vectors may optionally contain one or more selectable marker genes, which are known in the art and are not particularly limited. Other construction elements (such as terminators, etc.) optionally contained in the fibrinogen expression vector are not particularly limited.

[0082] Other methods are envisioned that use known methods of molecular manipulation to introduce an exogenous nucleotide sequence encoding an alpha, beta, and gamma chain variant of fibrinogen into a eukaryotic host cell, or to introduce an exogenous nucleotide sequence to modify the alpha, beta, and gamma chain sequences already present in a eukaryotic host cell (and, for example, to cause the expression of human-derived fibrinogen prior to the introduction of the exogenous nucleotide sequence). According to another preferred embodiment, at least one, and preferably each, of the exogenous nucleotide sequences is optimized for expression in mammalian cell culture cells under conditions where fibrinogen is produced.

[0083] In one embodiment, one fibrinogen expression vector is used to introduce an exogenous nucleotide sequence, such that, for example, a single expression vector contains all of the genes encoding the alpha, beta, and gamma chains of fibrinogen. In another embodiment, the fibrinogen expression vector is composed of an expression vector having two of the genes encoding the alpha, beta, and gamma chains of fibrinogen (such as the alpha and gamma chains, the beta and gamma chains, etc.), and an expression vector having the remaining one. In yet another embodiment, the fibrinogen expression vector is composed of three expression vectors each containing the gene encoding the alpha, beta, and gamma chains of fibrinogen. When fibrinogen is expressed using two or more expression vectors, each expression vector may be introduced into eukaryotic cells simultaneously, or may be introduced separately at different times, for example, by using different selection markers, where the order of introduction is not particularly limited. In a preferred embodiment, the fibrinogen expression vector is a single expression vector containing all of the genes encoding the alpha, beta, and gamma chains of fibrinogen, which, for example, results in expression at a composition ratio of 1:1:1, preferably at a composition ratio of 1:1:1 in eukaryotic cells.

[0084] A preferred example of a single expression vector containing all of the genes of the alpha, beta, and gamma chains of fibrinogen is one having three expression cassettes in which the respective genes encoding the alpha, beta, and gamma chains of fibrinogen are under the control of different promoters. The promoters regulating the expression of each gene may be the same or different, and the same promoter (such as the CMV promoter) is preferably used. See, for example, FIGS. 2 and 9 and Table 2.

[0085] Alternatively, two or more of the genes encoding the alpha, beta, and gamma chains of fibrinogen may be under the control of a single promoter. In this case, sequences that enable polycistronic expression (e.g., IRES sequences, 2A sequences derived from foot-and-mouth disease virus, etc.) can be inserted between the respective genes under the control of a single promoter.

[0086] According to a fifth aspect, the present invention is a method for producing an isolated recombinant fibrinogen having one or more site-specific mutations, preferably compared to human wild-type fibrinogen, A) culturing in a culture medium, under conditions in which fibrinogen is produced, a eukaryotic cell comprising an exogenous nucleotide sequence encoding at least one fibrinogen alpha chain, at least one fibrinogen beta chain, and at least one fibrinogen gamma chain, preferably derived from mammalian, more preferably human, fibrinogen; and B) recovering the produced recombinant fibrinogen comprising, wherein step A) is preferably performed in the presence of at least one matrix metalloproteinase (MMP) inhibitor selected from the group consisting of 1,10-phenanthroline, UK 370106, GM6001, and combinations thereof, preferably UK 370106. The method is targeted.

[0087] It should be understood that for the MMP inhibitor to be suitable for the method of this fifth aspect, it must be compatible with the use of eukaryotic cells during the culturing step A) (e.g., not cause excessive cytotoxicity). Preferably, the MMP inhibitor used in this method is selected from the group consisting of 1,10-phenanthroline, UK 370106, GM6001, and combinations thereof. A particularly preferred MMP inhibitor is UK 370106.

[0088] UK 370106, which has (βR)-β-[[[(1S)-1-[[[(1S)-2-methoxy-1-phenylethyl]amino]carbonyl]-2,2-dimethylpropyl]amino]carbonyl]-2-methyl-[1,1'-biphenyl]-4-hexanoic acid, is a highly selective MMP-3 and MMP-12 inhibitor. GM6001, also known as ilomastat or N-[(2R)-2-(hydroxyamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide, is a broad-spectrum matrix metalloprotease (MMP) inhibitor.

[0089] In step A), the MMP inhibitor can be added directly to the culture medium, and in step B), the MMP inhibitor is already present in the solution, and the culture medium from step A) containing the recombinant fibrinogen produced in step A) can be recovered. Step B) may be carried out in the presence of EDTA.

[0090] The methods of using 1,10-phenanthroline, UK 370106, and GM6001 and particularly preferred concentrations are known in the art. For UK 370106, preferred concentrations are, for example, at least 0.3 μM and up to 150 μM, preferably at least 0.5 μM and up to 100 μM, more preferably at least 1 μM and up to 80 μM, even more preferably at least 5 μM and up to 60 μM, even more preferably at least 10 μM and up to 50 μM, even more preferably at least 20 μM and up to 40 μM, for example, about 25 μM, or about 30 μM, or about 35 μM, preferably about 25 μM. For 1,10-phenanthroline, preferred concentrations are, for example, at least 0.1 mM and up to 10 mM, preferably at least 0.2 mM and up to 5 mM, more preferably at least 0.5 mM and up to 2 mM, for example, about 0.6, or 0.8 or 1, or 1.2, or 1.4 mM, preferably about 1 mM. For GM6001, preferred concentrations are, for example, at least 250 nM and up to 250 μM, preferably at least 2.5 μM and up to 100 μM, more preferably at least 5 μM and up to 50 μM, even more preferably 10 μM - 40 μM, or 10 μM - 25 μM, or 10 μM - 25 μM, for example, about 10 or 15, or 20, or 25, or 30 μM, preferably about 25 μM.

[0091] According to a preferred embodiment, the method of the present invention in step B) preferably comprises collecting at least a portion of the culture medium containing more than 0.5 μg / ml, more preferably more than 1 μg / ml, even more preferably more than 2 μg / ml, even more preferably more than 5 μg / ml, even more preferably more than 10 μg / ml, even more preferably more than 20 μg / ml, even more preferably more than 50 μg / ml, even more preferably more than 100 μg / ml of recombinant fibrinogen.

[0092] According to another preferred embodiment, the method of the present invention, in step B), optionally, concentrates fibrinogen from the culture medium to form a concentrated medium, and preferably purifies recombinant fibrinogen from the culture medium. According to a specific preferred embodiment, the purification is performed by a chromatography method, preferably affinity chromatography.

[0093] The eukaryotic cells used in the method of the present invention are not particularly limited as long as they enable the production of recombinant fibrinogen. According to a preferred embodiment, the eukaryotic cells are mammalian cell culture cells, preferably Chinese hamster ovary (CHO) cells, preferably CHO-K1, CHO-DG44, CHO-Pro minus and CHO-S cells, Chinese hamster ovary (CHO) cells selected from PER.C6 cells, or human embryonic kidney (HEK) cells, preferably human embryonic kidney (HEK) cells selected from HEK 293, HEK 293T, HEK 293S or HEK 293 EBNA cells, or mouse myeloma cells, preferably mouse myeloma cells selected from NS0 cells, NS-1 cells and Sp2 / 0 cells, or baby hamster kidney (BHK) cells, preferably BHK-21 cells, or rat myeloma cells, preferably YB2 / 0 cells or YB2 / 3HL cells. Preferably, the eukaryotic cells are CHO cells such as CHO-S cells. A commercially available example of suitable CHO-S cells is the ExpiCHO-S cell line manufactured by Thermo Fisher.

[0094] According to another preferred embodiment, at least one, and preferably all, of the exogenous nucleotide sequences are optimized for expression in mammalian cell culture cells under conditions where fibrinogen is produced. Regarding the eukaryotic cells and their preparation, the embodiments described under the fourth aspect of the present invention are applied with modifications where necessary.

[0095] The method of this fifth aspect is preferably directed to the production of an isolated recombinant fibrinogen having one or more site-specific mutations compared to the sequence of human wild-type fibrinogen. Thus, the isolated recombinant fibrinogen produced is, according to a preferred embodiment, a mutant isolated recombinant fibrinogen that is different from the amino acid sequence of human wild-type fibrinogen or a fragment thereof (e.g., as defined in Table 1) in one or more amino acids, preferably by substitution or deletion, preferably by substitution. According to a specific embodiment of the method for the production of an isolated recombinant fibrinogen, the isolated recombinant fibrinogen is an isolated recombinant fibrinogen according to any of the embodiments described above in the context of the first aspect of the invention, and / or the eukaryotic cell is a cell according to any of the embodiments described above in the context of the fourth aspect of the invention.

[0096] According to a sixth aspect, the invention is a method for identifying a plasmin-resistant recombinant fibrinogen variant, comprising A) i) incubating a fibrin gel, preferably at a density of 10 - 30 mg / ml, more preferably 20 mg / ml, with a plasmin solution having an activity of preferably 0.001 - 0.01 mU / ml; ii) collecting the supernatant at different times after addition of the plasmin solution and optionally inactivating the plasmin, preferably by addition of PSMF and / or heat; iii) determining the cleavage sites in the fibrinogen to obtain a time-resolved cleavage pattern of the fibrinogen, preferably using liquid chromatography mass spectrometry or solid phase extraction (SPE), more preferably high performance liquid chromatography / electrospray ionization mass spectrometry (HPLC / ESI-MS); and iv) identifying lysine (K) and / or arginine (R) residues immediately before and optionally immediately after the cleavage sites in the amino acid sequence of the fibrinogen Steps for identifying the plasmin cleavage sites in fibrinogen according to; and B) Optionally, for substitutions or deletions, preferably based on a time-resolved cleavage pattern, select one or more of the identified lysine (K) and / or arginine (R) residues in the amino acid sequence of fibrinogen to obtain a new recombinant fibrinogen variant; and optionally, incubate the new recombinant fibrinogen variant with plasmin and test for sensitivity to protein cleavage by plasmin A method comprising the above is the subject of this invention.

[0097] In step A)i), for example, a fibrin gel at a density of 10-30 mg / ml, preferably about 20 mg / ml, is incubated with a plasmin solution, preferably by covering the fibrin gel with the plasmin solution. The plasmin solution preferably has an activity of 0.0005-0.5 mU / ml, more preferably 0.0005-0.1 mU / ml, even more preferably 0.001-0.01 mU / ml; more preferably an activity of 0.002-0.02 mU / ml, for example, containing plasmin equal to about 0.5 mU / ml. The plasmin is preferably human plasmin. The plasmin solution may contain one or more other enzymes, for example, one or more additional proteases.

[0098] Methods for preparing fibrin gels are known in the art. Fibrin gels can be prepared by dialyzing fibrinogen against a suitable buffer (10 mM Tris / HCl, 150 mM NaCl, pH 7.5) at room temperature (RT). Gelation of fibrinogen at a concentration in the range of 0.5-106 mg / ml can be initiated by the addition of thrombin (1 U / ml), preferably also adding factor XIIIa (50 mU / ml) and CaCl2 (10 mM). The fibrinogen solution can be incubated at RT for at least 2 hours.

[0099] In step A) ii), the supernatant containing the plasmin solution and digested fibrin is collected at different time points after the addition of the plasmin solution. For example, the first time point for collection can be t = 0, for example, immediately before or after the addition of the plasmin solution (in this case, where the plasmin solution is used), and can be used as a reference sample. Further time points for collection may be at equal intervals, for example, every 30 minutes, or every 20 minutes, or every 15 minutes, or every 10 minutes, etc. After collection, the plasmin is preferably inactivated by the addition of a plasmin inhibitor such as phenylmethylsulfonyl fluoride (PMSF) and / or by using heat, preferably immediately after sample collection. Heat inactivation can be performed by incubation of the collected sample at 95 °C for, for example, 10 minutes. PSMF can be used, for example, at 0.5 - 2 mM, or about 1 mM. Preferably, the plasmin is inactivated using both PSMF and heat.

[0100] In step A) iii), the cleavage sites in fibrinogen are determined in order to obtain the time-resolved cleavage pattern of fibrinogen. The method for doing so is not particularly limited. Preferably, liquid chromatography mass spectrometry or solid phase extraction (SPE) mass spectrometry is used. In a specific preferred embodiment, high performance liquid chromatography / electrospray ionization mass spectrometry (HPLC / ESI-MS) is used to determine the cleavage sites. Determination of the cleavage sites may also involve quantification of plasmin-dependent cleavage of fibrinogen. For this quantification, according to a specific embodiment, an internal standard, for example, angiotensin II, is used in A) iii) to analyze the cleavage pattern.

[0101] In step A) iv), the lysine (K) and / or arginine (R) residues immediately before and after the cleavage sites in the amino acid sequence of fibrinogen are identified, for example, using bioinformatics tools known in the art.

[0102] Finally, in optional step B), one or more of the identified lysine (K) and / or arginine (R) residues in the amino acid sequence of fibrinogen are selected. The selection can be based, for example, on a time-resolved cleavage pattern. One or more of the identified lysine (K) and arginine (R) are substituted or deleted, preferably substituted, to obtain a new recombinant fibrinogen variant; and optionally, the new recombinant fibrinogen variant (e.g., in solution and / or as part of a fibrin gel) is incubated with plasmin to test its sensitivity to protein cleavage by plasmin.

[0103] According to one embodiment, the fibrin in the fibrin gel is preferably exclusively based on wild-type fibrinogen. According to another embodiment, the fibrin in the fibrin gel is preferably exclusively selected from one of the isolated recombinant fibrinogens according to any of the embodiments of the first aspect of the invention as shown above, based on mutant fibrinogen.

Examples

[0104] Example 1 Materials and Methods Material Restriction enzymes were purchased from New England Biolabs (NEB, Frankfurt a.M., Germany) and used according to the manufacturer's instructions. For PCR, Phusion™ High-Fidelity DNA Polymerase from ThermoFisher Scientific (Waltham, USA) was also used according to the manufacturer's instructions. Custom oligonucleotides were purchased from SigmaAldrich / Merck (Darmstadt, Germany), desalted and lyophilized upon arrival, and dissolved in H20 at 100 μM. The ExpiCHO™ expression system containing Gibco ExpiCHO-S cells, Gibco ExpiCHO™ expression medium, Gibco ExpiFectamine™ CHO reagent, ExpiCHO™ feed, ExpiFectamine™ enhancer, and Gibco OptiPRO™ serum-free complex medium were purchased from ThermoFisher Scientific (Waltham, USA). Spectra™ Prestained Multicolor High Range protein ladder (40 - 300 kDa), Spectra™ Prestained Multicolor Broad Range protein ladder (40 - 300 kDa) and PageRuler™ (10 - 170 kDa), Coomassie Brilliant Blue G250, SuperSignal™ West Pico PLUS chemiluminescent substrate, Bradford protein assay kit, mammalian cell lysis buffer, 1-Step™ UltraTMB ELISA substrate solution were from Thermo Fischer Scientific (Dreieich, Germany). HiTrap™ Capto MMC, Superdex™ 200 Hiload 16 / 600 and Superdex™ 75 10 / 300 GL were from Cytiva (Buckinghamshire, GB).

[0105] Method Site-directed mutagenesis The γ-chain vector DNA was amplified using PCR with primers designed to insert mutations at different positions in the sequence (FGG-Lys88-S-FW: 5’-GCAGCTGATC AGCGCCATCC AGCTGACCTA CAACCCCGAC GAGG-3’; FGG-Lys88-S-RV: 5’-GGTCAGCTGG ATGGCGCTGA TCAGCTGCTT CACTTCGCTG GTC-3’; FGG-Lys111-S-FW: 5’-CCGCCACCCT GAGCTCCCGG AAGATGCTGG AAGAGATC-3’; FGG-Lys111-S-RV: 5’-CGGGAGCTCA GGGTGGCGGC GTCGATCATG TTG-3’; FGG-Lys356-S-FW: 5’-CTACAGCAGC GCCAGCACCC CCAACGGCTA CGACAAC-3’; FGG-Lys356-S-RV: 5’- GTGCTGGCGC TGCTGTAGGT GCCGCCCTGG TAGTACACG-3’; FGG-Lys373-S-FW: 5’-CACCTGGAGC ACCCGGTGGT ACAGCATGAA GAAAACCAC-3’; FGG-Lys373-S-RV: 5’-CCGGGTGCTC CAGGTGGCCC AGATGATGCC GTTGT-3’). The amplified cDNA was transformed into E. coli dH5α, and the mutations of the target residues were verified by Sanger sequencing.

Table 2

Table 3

Table 4

[0106] Gibson Assembly For the insertion of different mutations into the γ-chain, a portion of the DNA sequence of the γ-chain was purchased from ThermoFisher Scientific (Waltham, USA). To perform Gibson assembly, primer pairs for both vectors containing the γ-chain and the gene to be synthesized were designed (FGG insert Fw: 5’-gagaacaaga ccagcgaagt gaa-3’; FGG insert Rv: 5’-cttcatggtg gttttcttca tgc-3’: FGG vector Fw: 5’-gtacagcatg aagaaaacca cca-3’; FGG vector Rv: 5’-cttcacttcg ctggtcttgt tct-3’). The amplified cDNA was purified using a PCR purification kit (E.Z.N.A® kit, Omega Bio-tek, Inc., Norcross, USA), inserted into the vector pcDNA3, and transformed into E. coli dH5α. The success of the cloning was verified by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany).

[0107] Construction of the expression plasmid All cloning steps were performed by SLIC using PCR-amplified inserts, and the vectors were opened with restriction enzymes. First, the open reading frame (ORF) for the fibrinogen (Fbg) α-chain was cloned into the XbaI-open pcDNA™4 / TO / myc-His A vector either without a stop codon incorporating the vector-encoded myc-His tag or with a stop codon for the untagged version. The ORFs for the fibrinogen β and γ chains were cloned into the EcoRI-open pcDNA™3 vector. In a second step, the FGB- and FGG-ORFs were PCR-amplified together with the surrounding CMV promoter and bGH poly(A) signal (using oligonucleotides #7 and #8, or #9 and #10, respectively) and inserted into the pcDNA4 / TO / FGA construct in a single SLIC reaction and opened by restriction with the SapI enzyme (cut in the vector backbone between the ColE1 origin of replication and the bleomycin resistance gene). Clones were screened by a combination of colony PCR and analytical restriction digestion and verified by sequencing of all three ORFs.

[0108] Cell culture ExpiCHO-S™ cells were cultured according to the manufacturer's protocol. Briefly, the cells were maintained without the use of antibiotics in 125 mL flasks in ExpiCHO™ expression medium at 37 °C, 8% CO2, and 130 rpm. For general maintenance, the cells were passaged every 3 - 4 days when they reached a density of approximately 6 × 106 cells / mL.

[0109] Production of recombinant fibrinogen Fibrinogen variants were expressed according to the user manual (Thermo Scientific, Waltham, USA). Briefly, ExpiCHO-S™ cells (Thermo Scientific, Waltham, USA) were cultured to a final density of 10×106 cells / mL. Plasmids encoding different fibrinogen variants (0.8 μg / culture mL) and ExpiFectamine™ CHO (Thermo Scientific, Waltham, USA) were diluted in 5 mL of cold OptiPro™ medium (Thermo Scientific, Waltham, USA) and incubated for 5 minutes at RT. The ExpiFectamine™ CHO / DNA complex was transferred to a shaker flask and incubated at 37 °C and 8% CO2 and 130 rpm. After 24 hours of incubation, ExpiFectamine™ CHO enhancer and ExpiFectamine™ CHO feed (Thermo Scientific, Waltham, USA) were added to the culture flask. The culture flask was transferred to an incubator at 32 °C and 8% CO2 while shaking at 130 rpm. This step was repeated 4 days after incubation. After another incubation step of up to 7 days at 32 °C and 8% CO2 and 130 rpm, the cells were harvested and centrifuged at 3000 rpm for 15 minutes.

[0110] For the production of recombinant fibrinogen (rFbg) with the addition of UK 370106, the inhibitor was added to the medium to a final concentration of 25 μM. Daily, the cells were centrifuged at 1100 rpm for 4 minutes. The supernatant was removed, EDTA was added to a final concentration of 2 mM, and this was stored at -80 °C until further use. Purification was performed as previously described by the addition of 2 mM EDTA to all buffers used during the process.

[0111] Purification of fibrinogen Recombinant fibrinogen WT and mutants were purified from the supernatant of ExpiCHO-S cells. After filtration of the supernatant using a precision filtration membrane (Filtropur S 0.2 μM, polyethersulfone membrane, Sarstedt, Nuembrecht, Germany), the solution containing the recombinant protein was purified by ion exchange chromatography using an FPLC system with a Capto MMC column (GE Healthcare Akta Explorer, Life sciences, Freiburg, Germany) equilibrated in 20 mM phosphate buffer (pH 7) containing 150 mM NaCl. The protein was eluted from the column using a linear gradient with 200 mM arginine buffer (pH 8.5) and 500 mM NaCl. After buffer exchange to 10 mM arginine buffer (pH 8.5) containing 150 mM NaCl, recombinant fibrinogen was purified in a second step by size exclusion chromatography (Hiload 16 / 600 Superdex 200 PG, GE Healthcare, Life sciences, Freiburg, Germany) using 10 mM arginine buffer (pH 8.5) containing 150 mM NaCl. Protein concentration was determined using the BCA or Bradford assay according to the manufacturer's manual. The recombinant protein was stored at -80 °C.

[0112] SDS-Page The purified proteins and conjugates were analyzed using Tris-glycine SDS-PAGE with or without the addition of β-mercaptoethanol as previously described. Gels were stained with Coomassie Brilliant Blue G250 and recorded using a FluorChem FC2 imaging system (Protein Simple, Santa Clara, USA).

[0113] Fibrin gel formation Lyophilized fibrinogen (SigmaAldrich) was dissolved in ddH2O and dialyzed against 10 mM Tris / HCl, 150 mM NaCl (pH 7.5) overnight at RT. Gelation of fibrinogen (20 mg / mL) was initiated by the addition of thrombin (Merck) (1 U / mL), factor XIIIa (fibrogamin®, CSL Behring) (50 mU / mL) and CaCl2 (10 mM). 100 μL of the fibrinogen solution was transferred to a 96-well plate and incubated at RT for 2 hours. Thereafter, the gel was stored at 4 °C for further experiments.

[0114] Plasmin digestion To analyze plasmin digestion of the mutated restriction sites, 100 μL of human plasmin (Merck) (0.5 U / mL) was added to 100 μg of Fbg and rFbg (Ala and His), and incubated at 37 °C and 130 rpm for 24 hours to ensure complete cleavage. Thereafter, the samples were stored at -80 °C. For the determination of the time-dependent cleavage sites of plasmin and the analysis of quantification, fibrin gels (20 mg / mL) were incubated with different plasmin concentrations ranging from 0.005 mU / mL to 0.5 U / mL. Samples were taken at different time points from 1 minute to 24 hours. PMSF (1 mM) was added to the samples and heated at 95 °C for 10 minutes to ensure the inactivation of plasmin. The samples were stored at -80 °C.

[0115] High-resolution mass spectrometry of digestion in gel Samples were diluted with Nupage Sample buffer™. In the next step, dithiothreitol (DTT) was added to the samples to a final concentration of 50 mM for the reduction of disulfide bonds. After protein denaturation at 70 °C for 10 minutes, iodoacetamide (IAA) was added to a final concentration of 120 mM and the samples were incubated in the dark for 20 minutes. SDS-PAGE was performed (see above), the bands of interest were cut from the gel and stored at -80 °C until further use.

[0116] For in-gel digestion, the excised gel slices were desalted with 30% acetonitrile, shrunk with 100% acetonitrile, and dried in a vacuum concentrator.

[0117] Trypsin digestion was carried out overnight at 37 °C in 0.05 M NH4HCO3 (pH 8) using 0.1 μg of protease per slice. Peptides were extracted from the gel slices using 5% formic acid.

[0118] ProAlanase digestion was performed by incubating the gel slices with 0.1% formic acid containing 0.2 μg of ProAlanase for 1.5 hours. Peptides were extracted as described above.

[0119] Mass spectrometry analysis Sample preparation and in-solution digestion: Proteins were digested either with plasmin alone (see above) or with plasmin and ProAlanase (ProAla) (Promega) with slight differences in handling. For all digestions, all samples (WT / HIS / ALA) were brought to a concentration of 0.2 μg / μL in 50 mM tris buffer. For "plasmin only", the samples were purified immediately (see below). For ProAla (Promega), 50 μL of 8 M guanidinium hydrochloride was added to 10 μg (50 μL) of the protein stock solution and briefly heated to ensure denaturation. To enable specific ProAla digestion, the solution was acidified using 900 μL of 32 mM HCl. After checking that the pH was in the range of 1 - 1.5, ProAla was added (1:50 ProAla:protein; 0.25 μg) and proteolysis was continued for 2.5 hours. Then, the pH was set slightly basic by the addition of 0.5 - 1 mL of 2 M Tris buffer. In the next step, the samples were purified immediately.

[0120] Before purification, the samples were reduced and alkylated as follows. Tris(2-carboxyethyl)phosphine (TCEP) was added to a final concentration of 20 mM. The sample was heated to 60 °C for 5 minutes, and IAA was added to a final concentration of 40 mM during cooling. The sample was cooled completely in the dark for 20 minutes. Immediately afterwards, the sample was loaded onto a 1 mL Strata X 33 syringe column (10 mg bed; Phenomenex, Torrance, CA). For all steps, only gravity flow was used. The column was activated with 3 mL of 100% ACN and then washed with 3 mL of 100% deionized water. Then the sample was added. Subsequently, the column was washed with 5 mL of 0.4% formic acid in ddH2O. Elution was performed using 0.5 mL of 0.4% formic acid in 80% ACN. The eluted sample was frozen in liquid nitrogen and lyophilized. For analysis, the sample was resuspended in 50 μL of 0.2% formic acid, 2% acetonitrile and used immediately for LC MS / MS analysis.

[0121] Screening of inhibitors for Fbg expression ExpiCHO-S cells were cultured to a density of 6×106 cells / mL. Then the cells were centrifuged at 1000 rpm for 4 minutes at RT and the supernatant was collected. Commercially available fibrinogen (SigmaAldrich) was mixed with the supernatant to a final concentration of 3.3 mg / mL and a volume of 200 μl. The mixture was incubated at 37 °C and 750 rpm, and samples were taken at 0, 24 and 48 hours. For screening, different inhibitors were added to the mixture (final concentrations: PMSF: 1 mM; aprotinin: 50 μg / mL; GM6001: 25 μM; EDTA: 4 mM; 1,10-phenanthroline: 1 mM; UK 370106: 100 μM). As a control, fibrinogen was incubated under the same conditions in 10 mM Tris / HCl, 150 mM NaCl (pH 7.5). For visualization of fibrinogen degradation, 20 μL of the mixture was analyzed using SDS-PAGE.

[0122] LC-MS / Data analysis NanoLC-MS / MS analysis was performed on an LTQ-Orbitrap Velos Pro (Thermo Fisher Scientific, Darmstadt, Germany) connected to an EASY-nLC 1000 (Thermo Fisher Scientific, Darmstadt, Germany) equipped with a PicoView Ion Source (New Objective, Littleton, USA). Peptides were loaded onto a pre-column (trap column, 2 cm × 150 μM ID) packed with 3 μm C18 ReproSil and then eluted onto a self-packed capillary column (30 cm × 150 μm ID) with ReproSil-Pur 120 C18-AQ, 1.9 μm and separated with a linear gradient from 3 - 30% acetonitrile and 0.1% formic acid for 30, 60, 120 minutes, and at a flow rate of 500 nL / min.

[0123] The gradients were used as follows: a 30-minute gradient was used for samples digested in gel, a 60-minute gradient was used for the determination and quantification of time-resolved plasmin cleavage sites, and a 120-minute gradient was used for all other in-solution digestions.

[0124] MS scans were acquired on the Orbitrap analyzer at a resolution of 30,000 at m / z 400, and MS / MS scans were acquired on the Orbitrap analyzer at a resolution of 75,000 at m / z 400 using HCD fragmentation with 30% normalized collision energy. The TOP5 data-dependent MS / MS method was used, and dynamic exclusion was applied with a repeat count of 1 and an execution duration of 7 seconds to exclude precursors with slightly changed masses. The minimum signal threshold for precursor selection was set to 50,000. Predictive automatic gain control (AGC) was used with AGC targets of 1×106 for MS scans and 5×104 for MS / MS scans. The lock mass option was applied for internal calibration in all runs using background ions from protonated decamethylcyclopentasiloxane (m / z 371.10124).

[0125] Data analysis Data analysis was performed using PMI-Byos (Protein Metrics inc., Cupertino United States). The data was searched against a database (uniport reference proteome) containing all human proteins for quality control (data not shown), and once the target protein was identified, the searches detailed below were performed against a custom database containing either the WT sequences of the α, β, and γ chains of fibrinogen, or in the case of the analysis of the γ chain, only the γ sequence. The isoform sequence (P02671-2|FIBA_HUMAN isoform 2) from uniport was used to determine the success of expression after inhibitor addition. Samples were first searched using PMI-Preview (Protein Metrics inc., Cupertino United States), and the suggested modifications were included in the main search. Decoys were added in all cases. The results were filtered, and only peptide matches with a scoring >100 were considered.

[0126] The settings for each individual search were verified and were as follows:

[0127] Determination of cleavage efficiency in mutants All data files were searched in three batches (separated by the protease used for digestion) using the settings described below.

[0128] Digestion with plasmin only (γ): 15 ppm precursor tolerance, 20 ppm fragment mass tolerance, C-terminal digestion of K (non-specific), modifications are concurrent with the recommendations of PMI-Byos for the sequence variant analysis workflow (two rare and common modifications are allowed).

[0129] Digestion with plasmin and ProAlanase (γ): 15 ppm precursor tolerance, 20 ppm fragment mass tolerance, digestion of the C-termini of A and P (non-specific), modifications are concurrent with PMI-Byos recommendations for the sequence variant analysis workflow (two rare and common modifications are allowed).

[0130] In addition, an “experimental quality control” assessment was performed. Both sets of digests were searched against a database containing all three fibrinogen chains. The settings were as follows: 5 ppm precursor and 20 ppm fragment tolerance, digestion of the C-terminus of APKR, semi-specifically set with up to 5 missed cleavages (data not shown). Modifications were dramatically reduced compared to the gamma chain search. Data were filtered as described above.

[0131] In-gel digestion of the α-chain Trypsin digestion: A custom database of all three chains, their mutants and other proteins was used. Settings: 5 ppm precursor and 20 ppm fragment tolerance, digestion of the C-terminus of R / K, fully specific with up to 2 missed cleavages allowed, 1 common and 1 rare modification allowed.

[0132] ProAlanase digestion: A custom database of all three chains, their mutants and other proteins was used. Settings: 5 ppm precursor and 20 ppm fragment tolerance, digestion of the C-terminus of P / A, non-specific with up to 2 missed cleavages allowed, 1 common and 1 rare modification allowed.

[0133] α-chain expression by in-gel digestion of protease inhibitors The database contained the alpha chain and isoform 2 of the other two chains. Settings: 5 ppm precursor and 20 ppm fragment tolerance, digestion of the C-terminus of R / K, fully specific with up to 3 missed cleavages allowed, 3 common and 2 rare modifications allowed.

[0134] Determination and quantification of time-resolved cleavage sites This search was performed using MaxQuant (V1.6.17.0) with a database containing all three Fbg chains, and in addition to plasminogen, prothrombin, factor FXIIIa, and angiotensin peptides, common contaminants were added. Data files for six consecutive time points were searched in batch. Using instrument-specific standard settings, up to seven missed cleavages were allowed to account for differences between plasmid and trypsin, but specific trypsin digestion was performed, and up to five modifications were allowed for the characteristics of the match between runs. The allowed modifications were oxidation (M), acetyl (N-terminus), deamidation (NQ), phospho (STY), and fixed carbamidomethyl (C). Bioinformatics analysis was performed using Perseus (V4.1.3). The results were filtered as follows: removal of contaminants, at least one intensity > 0. Then, all intensities were normalized against the angiotensin intensity of each data file. Since the angiotensin intensity was too low to confirm, the last two time points were excluded. Then, information for each of the Fbg chains was extracted. For each chain separately and for each time point, the intensities of all peptides were summed with respect to either their disclosure or end position. The sum of the intensities for all peptides starting or ending at a particular cleavage site enabled us to generate time points that depend not on a single peptide but on a particular cleavage site.

[0135] Results Design of recombinant fibrinogen for improved stability against plasmin Fibrinolysis has been studied for a long time, and multiple cleavage sites of plasmin have been described in all chains (α, β, and γ chains). Furthermore, the plasmin cleavage sites are distributed and located in different parts throughout the quaternary structure of fibrinogen. To examine the effects of mutations (AA exchanges) on proteolysis and the effects of their locations on the complex structure, plasmin cleavage sites in different parts of the quaternary structure of fibrinogen were selected. The mutation sites are located in the α-helix domain and the random coil domain (known as D domain 44; Figure 1A). This was selected to insert mutations only in the gamma chain relative to the distribution of mutations throughout all chains. The goal was to facilitate the improved degradation stability of the complex by keeping one composite chain intact while minimizing changes. The gamma chain was selected because it has the smallest number of cleavage sites compared to the alpha and beta chains and facilitates cross-linking by factor XIIIa. Four different cleavage sites in either the α-helix or the D domain also enabled us to study their effects on the structure of fibrinogen. The mutation sites are QLI(K)AIQ; ATL(K)SRM (both in the alpha helix domain); TYS(K)AST; ATW(K)TRW (both in the D domain) (Figure 1A, B, Figures 7 - 8). The ideal substrate for plasmin contains either an arginine (Arg, R) or a lysine (Lys, K) residue at the P1 position. To test the effect of AA exchanges of these sequences on plasmin cleavage, four different sequences were synthesized by changing K to the polar AA serine (Ser, S) as a proof of principle by using SPPS. After purification, the peptides were incubated with plasmin and the cleavage efficiency was analyzed using HPLC. Peptide sequences containing S instead of K showed a significant reduction in cleavage efficiency compared to the original sequences. After this proof of principle, the aforementioned mutations were introduced into the gamma chain by using site-directed mutagenesis. Furthermore, two different expression plasmids were constructed to test the effects of different fibrinogen forms.One contains Aα, Bβ and γ chains (recombinant fibrinogen, rFbg), and the other contains αE, Bβ and γ' chains (recombinant fibrinogen isoform s, rFbgi).

[0136] Production of recombinant fibrinogen Production of recombinant fibrinogen was carried out using eukaryotic protein expression in CHO cells. Construction of the expression plasmid pcDNA4-TO-FBG was carried out using ligation-independent cloning (SLIC; Figure 9). The plasmid encodes all three chains of the α, β and γ chains. All chains were set in series and are under the control of the human cytomegalovirus (CMV) promoter, a promoter frequently used for eukaryotic protein expression due to high levels of production. A bovine growth hormone (bGH) polyadenylation signal was added at the end of each gene to ensure transcription termination (Figure 2). Expression plasmids were constructed for both the wild type (WT) and variants with all four mutations.

[0137] CHO cells were transfected with the expression plasmid and incubated for up to 12 days. Subsequently, the cell suspension was centrifuged and the supernatant was collected. Purification of the recombinant protein was performed by anion exchange chromatography (AiEx) using multimodal functionality on a Capto MMC column for high volume throughput and enhanced binding capacity. After expression of rFbgi, non-reducing SDS-PAGE analysis showed bands above 300 kDa for WT that could be attributed to the native hexameric form of fibrinogen. However, high molecular weight bands were visualized for the expression of Ser-fibrinogen, and the introduction of S instead of K at the four mutation sites resulted in either degradation or shortening of the γ-chain and thus showed prevention of fibrinogen production and assembly (Figures 10A and B). S has been described in the literature to act as a helix-breaking substance in the secondary protein structure in rare cases. Since two of the four mutations are located in the center of the α-helix of fibrinogen, these mutations result in helix disruption and thus interfere with quaternary structure formation.

[0138] To mimic the physicochemical properties of target AA, histidine (His, H) and alanine (Ala, A) were selected as substitutions for K. Ala is known to be abundant in the α-helix and is considered the most stabilizing AA for the helix. His is also known to stabilize the α-helix due to hydrogen bonding with the CO group in the helix backbone. Furthermore, histidine mimics the positive charge of the native lysine residue and thus may stabilize (or at least not interfere with) the structure of fibrinogen. After insertion of the new mutations into the plasmid using Gibson assembly and SLIC, new mutants of both rFbg and rFbgi were produced in CHO cells and showed high purity after size-exclusion chromatography (SEC, Figures 3A, B). Non-reducing SDS-PAGE analysis showed bands for all four high-molecular-weight variants (His-rFbgi, Ala-rFbgi, rFbg, His-rFbg, Ala-rFbg) above 300 kDa (Figures 3B, 10D, E). This suggests that the insertion of these mutations impaired protein expression and that fibrinogen hexamers were constructed.

[0139] Structural Analysis of Recombinant Fibrinogen To further verify the structure of the recombinant fibrinogen mutants, reduced SDS-PAGE was performed. Both the β and γ chains were visualized as gels with molecular weights of approximately 52 kDa and 46.5 kDa, respectively, and in their native form exceeding 300 kDa (Figure 3B, Figures 10C - E). Furthermore, the presence and sequence of the different chains were confirmed by high-resolution ESI-MS (Figures 11 - 16). This indicates that the introduction of different mutations does not impair gene expression, that the γ chain can be produced, and that a general approach can be used in the future. However, the bands of the α chain (α chain: 66.5 kDa; αE: 93 kDa) were not visible in SDS-PAGE under reducing conditions. Instead, another band appeared at approximately 44 kDa for both rFbg and rFbgi, indicating shortening of the α chain during the production or purification process (Figure 3B, Figures 10C - E). Furthermore, the band of native fibrinogen was slightly lower than the control, also indicating shortening of the protein (Figure 3B).

[0140] The fibrinogen α chain, particularly the C-terminus, is generally more sensitive to proteolysis. CHO cells, which are known to secrete proteases during cell culture, can cause proteolysis of the alpha chain. However, all CHO cell lines have their own unique protease expression patterns, which makes it difficult to identify the specific protease responsible. For optimization of the approach, since this form of fibrinogen is generally used as part of the fibrin glue, this was focused on rFbg (Aα, Bβ, and γ chains) from this point of view.

[0141] To verify that the new band of approximately 44 kDa is a truncated form of the α-chain and to find the exact site of truncation, in-gel digestion for characterization of the fragment by mass spectrometry was performed. High-resolution ESI-MS analysis showed high alignment with both the native α-chain and truncation of the chain at K432, as confirmed by both trypsin digestion and ProAlanase digestion (data not shown) (Figure 4A, B, C, Figure 17). This result also matches the molecular weight of the band and the deletion of the AA sequence. Degradation of the α-chain during expression is often described in the literature as a result of protease cleavage in the supernatant. It has been previously described that the addition of aprotinin, a serine protease inhibitor, during CHO cell expression inhibited the proteolysis of the fibrinogen α-chain. 51 To limit this degradation, the same approach as previously described was applied, and K432 was exchanged for H in the α-gene cassette to inhibit protein cleavage at the mutation site (rFbg432). In addition, to prevent the emergence of new cleavage sites due to evolutionary pressure, an expression plasmid was constructed in which all Ks (K432, K437, K440, K446, K448: rFbg5xKtoH) within 20 AAs upstream / downstream of the cleavage site K432 were exchanged for H. However, SDS-PAGE analysis after CHO cell protein expression of both His-rFbg432 and His-rFbg5xKtoH revealed that the α-chain was still degraded during the process (Figure 18A). Another enzyme reported to degrade the α-chain is matrix metalloproteinase (MMP). Analysis of the effect of different MMPs on fibrinogen showed a degradation pattern similar to that of both rFbg and rFbgi, as well as potential cleavage sites at K432 (e.g., MMP2, 3, 12, 14).

[0142] Therefore, different serine proteases (suggested in the literature) and MMP inhibitors were screened for their effects on α-chain degradation to find suitable compounds for the inhibition of α-chain degradation. Commercially available human Fbg was incubated with the supernatant obtained from the CHO cells used for expression, and the protein was incubated at 37 °C for several days. Analysis using SDS-PAGE showed the same degradation pattern for Fbg as seen during expression. The truncated α-chain was also visualized after the addition of the serine protease inhibitor aprotinin, which is an explanation of why the insertion mutation at 432 had no effect on degradation (Figures 18B, C). However, intact α-chains were detected even several days after the addition of either EDTA, 1,10-phenanthroline or the MMP inhibitor UK 370106 (Figures 4D, 18B, C). This indicates that the degradation of rFbg during CHO cell expression may be the result of overexpression of MMPs in the supernatant and subsequent cleavage of the α-chain.

[0143] Therefore, His-rFbg432 was expressed by the addition of 25 μM of UK 370106. Reducing SDS-PAGE analysis showed three bands of approximately 68 kDa, 52 kDa and 46.5 kDa, similar to those of commercially available Fbg, indicating that the addition of UK 370106 resulted in the production of fully intact α-chains (Figure 4E). The slight difference in the molecular weight of the α-chain compared to the control may be due to potential differences in the complex glycosylation pattern of rFbg. The sequence of the α-chain was verified using high-resolution ESI-MS (Figure 19).

[0144] Analysis of plasmin digestion of the γ-chain of recombinant fibrinogen mutants The α-chain plays an important part in the fibrin gelation process. Due to the shortened α-chain, gel formation by rFbg was impossible as the production of the intact complex (compared to the above) still continued. Therefore, plasmin digestion in solution was performed with both WT as well as Ala- and His-rFbg mutants to examine whether the inserted mutations restricted the proteolytic efficiency of plasmin at each cleavage site. Plasmin was added to the fibrinogen solution (1 mg / mL) to a final concentration of 0.5 U / mL and the mixture was incubated for 24 h to ensure complete cleavage of fibrinogen. Four different cleavage sites K88, K111, K382, K399 in WT could be detected using high-resolution ESI-MS. However, for all four mutations, no cleavage was detected in either of the two mutants, indicating that the AA exchange from K to A or H restricted the proteolytic efficiency of plasmin in Fbg (Figs. 5, 11 - 12, 20). At some positions, both cleaved and uncleaved peptides were detected; however, the cleavage did not contain the mutation. This could be due to residual WT-Fbg peptides resulting from residual WT-Fbg either on the column (carry-over) or at any point in the post-treatment. The inventors consider that this cannot be an artefact of expression or cell line as CHO cells do not produce Fbg natively and the expression plasmid only contains the mutant form of the γ-chain.

[0145] Time-dependent analysis of plasmin digestion of fibrinogen After demonstrating that the exchange of AA from K to either H or A impairs plasmin-mediated degradation of the γ-chain, we analyzed which cleavage sites cut intact fibrin gels during the early stages of fibrinolysis. These sites could be potential targets for future site-directed mutagenesis in order to directly stabilize and limit fibrinogenolysis at the onset. Thus, fibrin gels (Sigma Aldrich, 20 mg / mL) were molded, plasmin solution (0.005 mU / ml) was added on top of the gels, and incubated for up to 2 hours. At different time points, supernatants were collected and plasmin was inactivated by the addition of PSMF and heating. The solutions were then purified using a C18 gravity column (Strata X, Phenomenex, Torrance, CA), and 100 fmol of ProteoMass™ angiotensin II MALDI-MS standard (Sigma-Aldrich, Steinheim, Germany) was added to each sample as an internal standard to quantify cleavage at the plasmin cleavage sites. The criminal changes in plasmin cleavage were analyzed using high-resolution ESI-MS. For the analysis, the specificity of plasmin described in the literature (cleaving the C-terminus of R / K, similar to trypsin) was used. However, an untargeted search revealed several non-tryptic cleavage sites. Therefore, plasmin appears to cleave both the C- and N-termini of R / K. In addition, some repetitive interference was observed, indicating AAs other than R / K at the P1 and P1’ positions.

[0146] The analysis shows that cleavage of the fibrinogen at an earlier stage is most predominant within the α-chain, which is consistent with reports from the literature. The data support the report that the cleavage is the most predominant cleavage at positions 602 (K|MADE) and 620 (K|RGHA) and starts in the αC region of fibrinogen. Next, cleavage sites further down in the αC region are cleaved, resulting in complete removal of this part of fibrinogen (e.g., positions 527 (K|TFPG), 510 (R|HRHP), 225 (K|MKPV), and 238 (K|SQLQ)). At the same time, cleavage starts in the α-helix region of the α-chain (e.g., positions 100 (K|DSHS) and 123 (R|DNTY)) (Figure 6A, D). However, the inventors were able to detect a number of different cleavage sites by this approach that were not previously reported in the literature (Figures 7 - 8). Furthermore, this approach enables much more sensitive readout and analysis compared to methods such as HPLC and SDS-PAGE. According to the literature, cleavage of the β-chain starts at the N-terminus, which is emphasized by positions 52 (K|REEA) and 72 (R|ARPA) (Figure 6B, E). Subsequently, the α-helix part between fragments D and E is cleaved by plasmin (e.g., 152 (K|DLWQ) and 160 (K|QVKD)) (Figure 6B, E).

[0147] Interestingly, the only additional cleavage site was found in this time-resolved assay for the γ-chain (Figure 6C, F), supporting the initial hypothesis regarding the use of the γ-chain for mutagenesis insertion. With only five cleavage sites detected during the early stages of plasminolysis, fewer mutations are required to potentially protect the full-length chains in the fibrin complex.

[0148] Conclusion The rapid resorption of fibrin sealants due to plasmin cleavage remains a major problem in the control of major bleeding. However, only a few approaches have been investigated over the past few decades. Here, we report a novel approach using molecular manipulations that limit plasmin-induced cleavage of fibrinogen. The plasmin cleavage sites in both the helical and random coil domains of fibrinogen were exchanged using site-directed mutagenesis. The γ-chain was successfully produced in CHO cells, indicating that the insertion of mutations in different structures of the protein did not affect its folding or expression. Mass spectrometry analysis showed that plasmin cleavage at these sites was restricted after AA, demonstrating that AA exchange at the plasmin restriction site limits enzymatic cleavage. In addition, a process for the production of rFbg in ExpiCHO-S™ cells was developed by screening different MMP inhibitors during expression and their subsequent use. Furthermore, a time-resolved screening protocol for the elucidation of plasmin cleavage sites for potential AA exchanges for the further generation of fibrinogen variants was developed. Using the approach for the production of molecularly engineered fibrinogen described herein, a new generation of fibrin sealants with improved stability against plasminolysis could be created. By changing both the number of mutations at the plasmin cleavage site and its location in the quaternary structure of the protein, fibrin gel resorption could potentially be fine-tuned for specific applications.

[0149] Example 2 Materials and Methods Turbidity Assay: The turbidity experiment was carried out at 37 °C in a Tecan Infinite M Plex plate reader. A 96-well plate with a final volume of 100 μl per well was used and the experiment was performed in triplicate. Thrombin-catalyzed fibrin polymerization of fibrinogen (0.1 mg / mL) was initiated by the addition of thrombin (0.05 U / mL), factor XIIIa (Fibrogammin®, CSL Behring; 50 mU / ml) and CaCl2 (10 mM). The change in turbidity at 350 nm was followed over time (0 - 3600 seconds).

[0150] Cross-linking of fibrin: To examine the cross-linking of fibrin, fibrinogen (0.1 mg / mL) was incubated at 37 °C with FXIIIa (0.5 U / mL) and human thrombin (final concentration 0.05 U / mL) in Tris buffer and CaCl2 (10 mM). The reaction was stopped at various times by the addition of an equal volume of sodium dodecyl sulfate (SDS) sample buffer with 2-mercaptoethanol and incubation at 90 °C (5 minutes). Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue.

[0151] Results Design and production of recombinant fibrinogen for improved stability during mammalian cell-based protein expression The degradation of fibrinogen during mammalian protein expression has long been recognized and several cleavage sites for MMPs have been described in all chains. 50,51 Specifically, the α-chain is affected by degradation and is thought to impair the functionality of the resulting fibrinogen. 52 Based on MS analysis of the degraded α-chain (see above), mutations were incorporated into the target sites for MMPs. The aim was to achieve improved proteolytic stability of the complex during cell-based protein expression by maintaining the α-chain intact while keeping the modifications to a minimum. The mutation sites were the sequence 429 HTEK(H)V(R)S436 Among them, they are spatially close (Figure 21). To test the effects of different forms of fibrinogen, expression plasmids were generated. The plasmids consist of Aα, Bβ- and γ chains with mutations only in the Aα and γ chains (His-rFbgH433R435).

[0152] After the insertion of new mutations into the plasmids by Gibson assembly and SLIC, the new mutants were produced in CHO cells and showed high purity after size exclusion chromatography. Non-reducing SDS-PAGE analysis revealed high molecular weight bands above 300 kDa (Figure 22). This indicates that the insertion of these mutations did not affect protein expression and that the fibrinogen hexamers were accurately assembled. Furthermore, reducing SDS-PAGE and in-gel digestion (Figures 22 - 25) detected the presence of all three chains. However, the degradation products of the α chain were undetectable in this variant. Therefore, it was confirmed that the mutations in the α chain enhanced the stability during recombinant expression in CHO cells.

[0153] Time-dependent analysis of fibrin polymerization After demonstrating that the exchange of AA substitutions impairs the proteolytic effect of the α chain during expression, the ability of the mutants to form fibrin fibers was examined. As shown in Figure 26, thrombin-induced fibrin polymerization of His-rFbgH433R435 was slightly reduced with a similar time shift and equivalent absorption compared to the control (pFbg). To further evaluate the functionality of the fibrinogen variant, FXIIIa-catalyzed cross-linking of the γ chain was performed (Figure 27). Here, the band of the cross-linked γ-γ dimer of rFbgH433R435 was visible after 5 minutes, indicating rapid lateral ligation. The intensity increased with longer incubation, but the intensities of the bands of the γ and Aα chains decreased after each incubation period. However, the experiment demonstrated that the mutant formed a functional fibrin gel and was thus potentially suitable as a wound adhesive.

[0154] References 1. Meara, J. G.; Leather, A. J. M.; Hagander, L.; Alkire, B. C.; Alonso, N.; Ameh, E. A.; Bickler, S. W.; Conteh, L.; Dare, A. J.; Davies, J.; Merisier, E. D.; El-Halabi, S.; Farmer, P. E.; Gawande, A.; Gillies, R.; Greenberg, S. L. M.; Grimes, C. E.; Gruen, R. L.; Ismail, E. A.; Kamara, T. B.; Lavy, C.; Lundeg, G.; Mkandawire, N. C.; Raykar, N. P.; Riesel, J. N.; Rodas, E.; Rose, J.; Roy, N.; Shrime, M. G.; Sullivan, R.; Verguet, S.; Watters, D.; Weiser, T. G.; Wilson, I. H.; Yamey, G.; Yip, W., Global Surgery 2030: evidence and solutions for achieving health, welfare, and economic development. The Lancet 2015, 386 (9993), 569-624. 2. Vollset, S. E.; Goren, E.; Yuan, C. W.; Cao, J.; Smith, A. E.; Hsiao, T.; Bisignano, C.; Azhar, G. S.; Castro, E.; Chalek, J.; Dolgert, A. J.; Frank, T.; Fukutaki, K.; Hay, S. I.; Lozano, R.; Mokdad, A. H.; Nandakumar, V.; Pierce, M.; Pletcher, M.; Robalik, T.; Steuben, K. M.; Wunrow, H. Y.; Zlavog, B. S.; Murray, C. J. L., Fertility, mortality, migration, and population scenarios for 195 countries and territories from 2017 to 2100: a forecasting analysis for the Global Burden of Disease Study. Lancet 2020, 396 (10258), 1285-1306. 3. Ramirez Manuel G, C. G. F., Ramirez Manuel A, The Economic Burden of Bleeds and Transfusions in Selected Surgeries: A Retrospective Multi Center Analysis from the Us Perspective. Am J Biomed Sci & Res. 2019, 2(5). 4. Reportlinker. Global Medical Adhesives and Sealants Industry. https: / / www.reportlinker.com / p05478477 / Global-Medical-Adhesives-and-Sealants- Industry.html?utm_source=GNW (accessed Nov 8, 2021). 6. Berrevoet, F.; de Hemptinne, B., Use of Topical Hemostatic Agents during Liver Resection. Digestive Surgery 2007, 24 (4), 288-293. 7. Aldrighetti, L.; Pulitano, C.; Arru, M.; Catena, M.; Finazzi, R.; Ferla, G., "Technological" approach versus clamp crushing technique for hepatic parenchymal transection: a comparative study. J Gastrointest Surg 2006, 10 (7), 974-9. 8. Martinez-Mier, G.; Esquivel-Torres, S.; Alvarado-Arenas, R. A.; Ortiz-Bayliss, A. B.; Lajud-Barquin, F. A.; Zilli-Hernandez, S., Liver resection morbidity, mortality, and risk factors at the departments of hepatobiliary surgery in Veracruz, Mexico. Rev Gastroenterol Mex 2016, 81 (4), 195-201. 9. Girardis M., B. S., Marietta M., Severe bleeding in critical care. Gullo A. (eds) Anaesthesia, Pain, Intensive Care and Emergency A.P.I.C.E.. Springer, Milano 2006. 10. Doci, R.; Gennari, L.; Bignami, P.; Montalto, F.; Morabito, A.; Bozzetti, F.; Bonalumi, M. G., Morbidity and mortality after hepatic resection of metastases from colorectal cancer. Br J Surg 1995, 82 (3), 377-81. 11. Christensen, M. C.; Krapf, S.; Kempel, A.; von Heymann, C., Costs of excessive postoperative hemorrhage in cardiac surgery. J Thorac Cardiovasc Surg 2009, 138 (3), 687-93. 12. Colson, P. H.; Gaudard, P.; Fellahi, J. L.; Bertet, H.; Faucanie, M.; Amour, J.; Blanloeil, Y.; Lanquetot, H.; Ouattara, A.; Picot, M. C., Active Bleeding after Cardiac Surgery: A Prospective Observational Multicenter Study. PLoS One 2016, 11 (9), e0162396. 13. Biancari, F.; Brascia, D.; Onorati, F.; Reichart, D.; Perrotti, A.; Ruggieri, V. G.; Santarpino, G.; Maselli, D.; Mariscalco, G.; Gherli, R.; Rubino, A. S.; De Feo, M.; Gatti, G.; Santini, F.; Dalen, M.; Saccocci, M.; Kinnunen, E. M.; Airaksinen, J. K.; D'Errigo, P.; Rosato, S.; Nicolini, F., Prediction of severe bleeding after coronary surgery: the WILL-BLEED Risk Score. Thromb Haemost 2017, 117 (3), 445-456. 14. Kim, Y. J.; Shin, S. K.; Lee, H. J.; Chung, H. S.; Lee, Y. C.; Park, J. C.; Hyung, W. J.; Noh, S. H.; Kim, C. B.; Lee, S. K., Endoscopic management of anastomotic leakage after gastrectomy for gastric cancer: how efficacious is it? Scandinavian Journal of Gastroenterology 2013, 48 (1), 111-118. 15. Matz, D.; Teuteberg, S.; Wiencierz, A.; Soysal, S. D.; Heizmann, O., Do antibacterial skin sutures reduce surgical site infections after elective open abdominal surgery? - Study protocol of a prospective, randomized controlled single center trial. Trials 2019, 20 (1), 390. 16. Edmiston, C. E.; Krepel, C. J.; Marks, R. M.; Rossi, P. J.; Sanger, J.; Goldblatt, M.; Graham, M. B.; Rothenburger, S.; Collier, J.; Seabrook, G. R., Microbiology of Explanted Suture Segments from Infected and Noninfected Surgical Patients. Journal of Clinical Microbiology 2013, 51 (2), 417. 17. Golling, M.; Schaudt, A.; Mehrabi, A.; Mood, Z. A.; Bechstein, W. O., Clinical application of soft polyglycolic acid felt for hemostasis and repair of a lacerated liver: report of two cases. Surg Today 2008, 38 (2), 188-92. 18. Spotnitz, W. D., Fibrin Sealant: The Only Approved Hemostat, Sealant, and Adhesive a Laboratory and Clinical Perspective. ISRN Surgery 2014, 2014, 203943. 19. FDA US Food and Drug Administration. Premarket approval (PMA) for Ethicon OMNEX surgical sealant; 2010. 20. FDA US Food and Drug Administration. Premarket approval (PMA) for DuraSeal dural sealant system; 2005. 21. Grau, A. E.; Duran, J. A., Treatment of a large corneal perforation with a multilayer of amniotic membrane and TachoSil. Cornea 2012, 31 (1), 98-100. 22. Caspers, M.; Maegele, M.; Frohlich, M., Current strategies for hemostatic control in acute trauma hemorrhage and trauma-induced coagulopathy. Expert Rev Hematol 2018, 11 (12), 987-995. 23. Hartmann, J.; Klein, H. G., Supply and demand for plasma-derived medicinal products - A critical reassessment amid the COVID-19 pandemic. Transfusion 2020, 60 (11), 2748-2752. 24. Baxter Package Insert. Tachosil; 2010. 25. Johnson, E. J. a. Package Insert. Evarrest; 2012. 26. FDA, Initial U.S. Approval. Tisseel by Baxter Healthcare Corporation. 1998. 27. Corporation, B. H. Baxter Advanced Surgery - Tisseel. 28. Palta, S.; Saroa, R.; Palta, A., Overview of the coagulation system. Indian J Anaesth 2014, 58 (5), 515-523. 29. Kawamura, M.; Sawafuji, M.; Watanabe, M.; Horinouchi, H.; Kobayashi, K., Frequency of transmission of human parvovirus B19 infection by fibrin sealant used during thoracic surgery. Ann Thorac Surg 2002, 73 (4), 1098-100. 30. Mosesson, M. W., Fibrinogen and fibrin structure and functions. J Thromb Haemost 2005, 3 (8), 1894-904. 31. Kattula, S.; Byrnes, J. R.; Wolberg, A. S., Fibrinogen and Fibrin in Hemostasis and Thrombosis. Arterioscler Thromb Vasc Biol 2017, 37 (3), e13-e21. 32. Yuan, H.; Vance, K. M.; Junge, C. E.; Geballe, M. T.; Snyder, J. P.; Hepler, J. R.; Yepes, M.; Low, C.-M.; Traynelis, S. F., The serine protease plasmin cleaves the amino-terminal domain of the NR2A subunit to relieve zinc inhibition of the N-methyl-D-aspartate receptors. J Biol Chem 2009, 284 (19), 12862-12873. 33. Bini, A.; Wu, D.; Schnuer, J.; Kudryk, B. J., Characterization of stromelysin 1 (MMP-3), matrilysin (MMP-7), and membrane type 1 matrix metalloproteinase (MT1-MMP) derived fibrin(ogen) fragments D-dimer and D-like monomer: NH2-terminal sequences of late-stage digest fragments. Biochemistry 1999, 38 (42), 13928-36. 34. Henschen, A.; McDonagh, J., Chapter 7 Fibrinogen, fibrin and factor XIII. In New Comprehensive Biochemistry, Neuberger, A.; van Deenen, L. L. M., Eds. Elsevier: 1986; Vol. 13, pp 171-241. 35. Hudson, N. E., Biophysical Mechanisms Mediating Fibrin Fiber Lysis. Biomed Res Int 2017, 2017, 2748340. 36. Kollman, J. M.; Pandi, L.; Sawaya, M. R.; Riley, M.; Doolittle, R. F., Crystal Structure of Human Fibrinogen. Biochemistry 2009, 48 (18), 3877-3886. 37. Roberts, I. V.; Bukhary, D.; Valdivieso, C. Y. L.; Tirelli, N., Fibrin Matrices as (Injectable) Biomaterials: Formation, Clinical Use, and Molecular Engineering. Macromolecular Bioscience 2020, 20 (1), 1900283. 38. Soria, J.; Mirshahi, S.; Mirshahi, S. Q.; Varin, R.; Pritchard, L. L.; Soria, C.; Mirshahi, M., Fibrinogen αC domain: Its importance in physiopathology. Res Pract Thromb Haemost 2019, 3 (2), 173-183. 39. (prev. 9) Cantero, M.; Rojas, H.; Angles-Cano, E.; Marchi, R., Fibrin γ / γ' influences the secretion of fibrinolytic components and clot structure. BMC Molecular and Cell Biology 2019, 20 (1), 47. 40. Greenberg, C. S.; Miraglia, C. C.; Rickles, F. R.; Shuman, M. A., Cleavage of blood coagulation factor XIII and fibrinogen by thrombin during in vitro clotting. J Clin Invest 1985, 75 (5), 1463-70. 41. Riedel, T.; Suttnar, J.; Brynda, E.; Houska, M.; Medved, L.; Dyr, J. E., Fibrinopeptides A and B release in the process of surface fibrin formation. Blood 2011, 117 (5), 1700-1706. 42. Joo, J. Y.; Amin, M. L.; Rajangam, T.; An, S. S. A., Fibrinogen as a promising material for various biomedical applications. Molecular & Cellular Toxicology 2015, 11 (1), 1-9. 43. Solovieva, E. V.; Fedotov, A. Y.; Mamonov, V. E.; Komlev, V. S.; Panteleyev, A. A., Fibrinogen-modified sodium alginate as a scaffold material for skin tissue engineering. Biomed Mater 2018, 13 (2), 025007. 44. Spotnitz, W. D., Fibrin Sealant: Past, Present, and Future: A Brief Review. World Journal of Surgery 2010, 34 (4), 632-634. 45. Jackson, M. R., Fibrin sealants in surgical practice: An overview. Am J Surg 2001, 182 (2 Suppl), 1s-7s. 46. Sahni, A.; Odrljin, T.; Francis, C. W., Binding of basic fibroblast growth factor to fibrinogen and fibrin. J Biol Chem 1998, 273 (13), 7554-9. 47. (prev. 18) Taboada, G. M.; Yang, K.; Pereira, M. J. N.; Liu, S. S.; Hu, Y.; Karp, J. M.; Artzi, N.; Lee, Y., Overcoming the translational barriers of tissue adhesives. Nature Reviews Materials 2020, 5 (4), 310-329. 48. Keragala, C. B.; Medcalf, R. L., Plasminogen: an enigmatic zymogen. Blood 2021, 137 (21), 2881-2889. 49. Lorentz, K. M.; Kontos, S.; Frey, P.; Hubbell, J. A., Engineered aprotinin for improved stability of fibrin biomaterials. Biomaterials 2011, 32 (2), 430-8. 50. Redman, C., Xia, H., Fibrinolysis and Proteolysis 2000, 14, 198-205. 51. H. Sandberg, D. Lutkemeyer, S. Kuprin, M. Wrangel, A. Almstedt, P. Persson, V. Ek, M. Mikaelsson, Biotechnology and Bioengineering 2006, 95, 961-971. 52. O. Hiller, A. Lichte, A. Oberpichler, A. Kocourek, H. Tschesche, Journal of Biological Chemistry 2000, 275, 33008-33013.

Claims

**Claim 1** An isolated recombinant fibrinogen comprising two fibrinogen alpha chains, two fibrinogen beta chains and two fibrinogen gamma chains and having an amino acid sequence derived from human fibrinogen, wherein: a) in the fibrinogen alpha chain, with respect to each position in SEQ ID NO: 2, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R38, R42, K97, K100, R114, R123, R129, R135, K210, R216, R218, K225, K227, K238, K243, K249, R258, R271, R287, R308, R353, R367, R425, R426, K432, K437, K440, R443, K446, K448, R458, R459, K463, K467, K476, K480, R510, R512, K527, R547, K558, R573, K575, K581, R591, K599, K602, K620, R621, K625, R687 and R847 are substituted or deleted; and / or b) in the fibrinogen beta chain, with respect to each position in SEQ ID NO: 3, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R44, K51, K52, R53, R60, R72, K77, K83, R87, K152, R158, K160, K163, K178, R285, K328, R334, K348, K353, K367, K374, K458 and K471 are substituted or deleted; and / or c) in the fibrinogen gamma chain, with respect to each position in SEQ ID NO: 4, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R40, K79, K84, K88, K111, K114, K185, K199, K238, K292, R301, K382 and K399 are substituted or deleted An isolated recombinant fibrinogen, characterized by the above. **Claim 2** i. One or more substituted or deleted K and R residues in the fibrinogen alpha chain comprise, or consist of, one or more of R135, K210, K243, R287, R308, R425, R426, K432, K437, K440, K446, K448, R458, R459, K463, K467, K480, R512, R547, K558, R573, K575, R591, K599, K620, R621, K625 and combinations thereof; and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain comprise, or consist of, one or more of R44, K51, R53, K77, R158, K160, K178, R334, K348, K353, K367, K374, K458, K471, and combinations thereof; and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain comprise, or consist of, K292, R301, or both; and / or ii. One or more substituted or deleted K and R residues in the fibrinogen alpha chain comprise, or consist of, one or more of R38, R42, K97, K100, R114, R123, R129, R135, R216, K225, K238, K249, R258, R271, R443, R510, K527 and K602; and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain comprise, or consist of, one or more of K52, R72, K83, K152 and K163; and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain comprise, or consist of, one or more of K79, K84, K88, K111, K114, K185, K382 and K399, preferably one or more of K79, K88, K111, K382 and K399; and iii. Optionally, all K and R residues shown in i) and / or ii) are deleted or substituted, preferably substituted, The isolated recombinant fibrinogen according to claim 1.

3. One or more substituted or deleted K and R residues in the fibrinogen alpha chain include, or consist of, K100, R114, R123, K225, K238, K249, R258, R271, R287, R308, R425, R426, K432, R443, R458, K467, R510, K527, R547, K558, K575, K599, K602, K621 and combinations thereof; and / or one or more substituted or deleted K and R residues in the fibrinogen beta chain include, or consist of, R44, K51, K52, R72, K83, K152, R158, K160, K163 and combinations thereof; and / or one or more substituted or deleted K and R residues in the fibrinogen gamma chain include, or consist of, one or more of K79, K88, K111, K382, K399 and combinations thereof, the isolated recombinant fibrinogen according to claim 1.

4. The isolated recombinant fibrinogen according to any one of claims 1 to 3, wherein the fibrinogen alpha, beta, and gamma chains contain only substitutions of K or R residues and do not contain deletions.

5. One or more K or R residues are alanine (A), histidine (H), N6-[(2-azidoethoxy)carbonyl]-L-lysine, propargyl-L-lysine, azidohomoalanine, trans-cyclooct-2-en-L-lysine, exo BCN-L-lysine, trans-cyclooct-4-en-L-lysine, pyrrolysine and pyrrolysine analogs, N ε -cyclopentyloxycarbonyl-L-lysine, lysine-nitrobenzyl-oxycarbonyl-N ε -L-lysine, O-methyl-L-tyrosine and analogs, methionine (M), isoleucine (I), leucine (L), phenylalanine (F), tryptophan (W), preferably selected from the group consisting of alanine (A), histidine (H), N6-[(2-azidoethoxy)carbonyl]-L-lysine, propargyl-L-lysine, azidohomoalanine, methionine (M), isoleucine (I), leucine (L); more preferably selected from alanine (A) or histidine (H), an amino acid or an isotope of these amino acids, the isolated recombinant fibrinogen according to any one of the preceding claims.

6. In the fibrinogen alpha chain and / or fibrinogen beta chain and / or fibrinogen gamma chain, at least two, preferably at least three, more preferably at least four, even more preferably at least five, at least six, at least seven or at least eight, or all of the K and R residues are deleted or substituted, preferably substituted, the isolated recombinant fibrinogen according to any one of the preceding claims.

7. i. Each fibrinogen alpha chain preferably excludes one or more substituted or deleted K and R residues and has an amino acid sequence that is identical to any of SEQ ID NOs: 6, 7, 8, 41, 42, 43, 45, 46, 47, 48, 49, and 50, preferably SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 41, or SEQ ID NO: 42, or SEQ ID NO: 43, more preferably SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, with at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% identity, or consists of such an amino acid sequence; and / or ii. Each fibrinogen beta chain preferably excludes one or more substituted or deleted K and R residues and has an amino acid sequence that is identical to SEQ ID NO: 9, or SEQ ID NO: 44, preferably SEQ ID NO: 9, with at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% identity, or consists of such an amino acid sequence; and / or iii. Each fibrinogen gamma chain preferably excludes one or more substituted or deleted K and R residues and has an amino acid sequence that is identical to SEQ ID NO: 10, or SEQ ID NO: 11, with at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% identity, or consists of such an amino acid sequence. The isolated recombinant fibrinogen according to any one of the preceding claims.

8. A fibrin sealant comprising the isolated recombinant fibrinogen according to any one of claims 1 to 7 and optionally thrombin.

9. The fibrin sealant according to claim 8, which does not contain a plasmin inhibitor.

10. i) A container containing the isolated recombinant fibrinogen according to any one of claims 1 to 7; and ii) A container containing thrombin, preferably derived from human A fibrin sealant kit comprising.

11. The fibrin sealant according to claim 8 or 9, or the fibrin sealant kit according to claim 10, further comprising at least one additive selected from the group consisting of prototransglutaminase, preferably factor XIII (FXIII), calcium salt, preferably calcium chloride, polyphosphate (PolyP), Zn2+, fibronectin, hydroxyapatite, growth factor, preferably VEGF, one or more monosaccharides and / or polysaccharides, and cells, preferably stem cells.

12. A eukaryotic cell comprising an exogenous nucleotide sequence encoding one or more recombinant fibrinogen alpha chains, one or more recombinant fibrinogen beta chains, and one or more recombinant fibrinogen gamma chains; i. Preferably, each of the one or more fibrinogen alpha chains preferably contains, except for K and R residues that are preferably substituted or deleted, any of SEQ ID NOs: 6, 7, 8, 41, 42, 43, 45, 46, 47, 48, 49, and 50, preferably SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 41, or SEQ ID NO: 42, or SEQ ID NO: 43, more preferably SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, and has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% amino acid sequence identity, or consists of; and / or ii. Preferably, each of the one or more fibrinogen beta chains, preferably excluding one or more substituted or deleted K and R residues, comprises, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% identity to SEQ ID NO: 9, or SEQ ID NO: 44, preferably SEQ ID NO: 9; and / or iii. Preferably, each of the one or more fibrinogen gamma chains, preferably excluding one or more substituted or deleted K and R residues, comprises, or consists of, an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5% identity to SEQ ID NO: 10, or SEQ ID NO: 11; (a) In the fibrinogen alpha chain, for each position in SEQ ID NO: 2, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R38, R42, K97, K100, R114, R123, R129, R135, K210, R216, R218, K225, K227, K238, K243, K249, R258, R271, R287, R308, R353, R367, R425, R426, K432, K437, K440, R443, K446, K448, R458, R459, K463, K467, K480, R510, R512, K527, R547, K558, R573, K575, K581, R591, K599, K602, K620, R621, K625, R687 and R847; and / or (b) in the fibrinogen beta chain, with respect to each position in SEQ ID NO: 3, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R44, K51, K52, R53, R60, R72, K77, K83, R87, K152, R158, K160, K163, K178, R285, K328, R334, K348, K353, K367, K374, K458 and K471 are substituted or deleted; and / or (c) in the fibrinogen gamma chain, with respect to each position in SEQ ID NO: 4, one or more lysine (K) and / or arginine (R) residues selected from the group consisting of R40, K79, K84, K88, K111, K114, K185, K199, K238, K292, R301, K382 and K399 are substituted or deleted characterized in that; a eukaryotic cell that produces a functional recombinant fibrinogen when cultured under conditions in which fibrinogen is produced eukaryotic cell

13. A method for producing an isolated recombinant fibrinogen, comprising A) culturing, in a culture medium, under conditions in which fibrinogen is produced, a eukaryotic cell comprising an exogenous nucleotide sequence encoding at least one fibrinogen alpha chain, at least one fibrinogen beta chain, and at least one fibrinogen gamma chain, preferably derived from mammalian, more preferably human, fibrinogen; and B) recovering the produced recombinant fibrinogen comprising; step A) is preferably performed in the presence of at least one matrix metalloproteinase (MMP) inhibitor selected from the group consisting of 1,10-phenanthroline, UK 370106, GM6001, and combinations thereof; optionally, the isolated recombinant fibrinogen is the isolated recombinant fibrinogen according to any one of claims 1 to 7 method

14. The eukaryotic cell is selected from mammalian cell culture cells, preferably Chinese hamster ovary (CHO) cells, PER.C6 cells, or human embryonic kidney (HEK) cells, or mouse myeloma cells, or baby hamster kidney (BHK) cells, or rat myeloma cells, the eukaryotic cell according to claim 12, and / or the method for producing the recombinant fibrinogen according to claim 13.

15. A method for identifying a plasmin-resistant recombinant fibrinogen variant, comprising: A) i) incubating a fibrin gel at a density of preferably 10 to 30 mg / ml, more preferably 20 mg / ml, preferably with a plasmin solution at 0.001 to 0.01 mU / ml; ii) collecting the supernatant at different time points after the addition of the plasmin solution; iii) determining the cleavage sites in fibrinogen, preferably using mass spectrometry, to obtain a time-resolved cleavage pattern of fibrinogen; and iv) identifying lysine (K) and / or arginine (R) residues immediately before and optionally immediately after the cleavage sites in the amino acid sequence of fibrinogen Steps for identifying plasmin cleavage sites in fibrinogen; and B) Optionally, for substitution or deletion, preferably based on the time-resolved cleavage pattern, selecting one or more of the identified lysine (K) and / or arginine (R) residues in the amino acid sequence of fibrinogen to obtain a new recombinant fibrinogen variant; and optionally, incubating the new recombinant fibrinogen variant with plasmin to test its sensitivity to protein cleavage by plasmin Including; Optionally, in step A) iii), an internal standard, preferably angiotensin II, is used to analyze the cleavage pattern. Method.

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