Differences in the biochemical composition of three plasma derived human fibrinogen concentrates

•Usually, fibrinogen is the coagulation factor dropping below a critical level in severe bleeding, first.•Human fibrinogen concentrates are increasingly used to achieve and maintain hemostasis in bleeding patients.•Tested human fibrinogen concentrates differ in their factor XIII activity, fibronectin and von Willebrand factor content.•Whether found differences impact efficacy and safety of the fibrinogen concentrates has to be assessed in clinical studies. The use of human fibrinogen concentrates (HFC) is well established in the prevention and treatment of congenital fibrinogen-related bleeding disorders [[1]Casini A. de Moerloose P. Fibrinogen concentrates in hereditary fibrinogen disorder: past, present and future.Haemophilia. 2020; 26: 25-32Crossref PubMed Scopus (12) Google Scholar]. An increasing number of studies also recommend HFC for the treatment of haemorrhage in trauma, postpartum haemorrhage, and patients experiencing bleeding during major surgical interventions, particularly after cardiac surgery [2Raphael J. Mazer C.D. Subramani S. et al.Society of cardiovascular anesthesiologists clinical practice improvement advisory for management of perioperative bleeding and hemostasis in cardiac surgery patients.Anesth. Analg. 2019; 129: 1209-1221Crossref PubMed Scopus (46) Google Scholar, 3Muñoz M. Stensballe J. Ducloy-Bouthors A.S. et al.Patient blood management in obstetrics: prevention and treatment of postpartum haemorrhage. A NATA consensus statement.Blood Transfus. 2019; 17: 112-136PubMed Google Scholar]. However, there are also some studies with divergent results, therefore, a proven fibrinogen deficiency, determined by a quantitative, clot-based, functional assay or viscoelastic testing, and a goal-directed fibrinogen concentrate replacement therapy is recommended [[3]Muñoz M. Stensballe J. Ducloy-Bouthors A.S. et al.Patient blood management in obstetrics: prevention and treatment of postpartum haemorrhage. A NATA consensus statement.Blood Transfus. 2019; 17: 112-136PubMed Google Scholar]. The most widely used plasma derived HFC are fibryga® (Octapharma AG, Lachen, Switzerland), RiaSTAP®/Haemocomplettan® P (CSL Behring GmbH, Marburg, Germany), and FibClot®/Clottafact® (LFB, Les Ulis, France). Further available HFC are Fibrinogen HT (Benesis, Osaka, Japan), FibroRAAS (Shanghai RAAS, Shanghai, China) and GCC-Fibrinogen (GC Pharma, Yongin, South Korea) but they are not approved in Europe, USA, and/or Canada. So far, a direct comparison of the pharmacokinetic profile of fibryga® and RiaSTAP®/Haemocomplettan® P has been performed [[4]Ross C. Rangarajan S. Karimi M. et al.Pharmacokinetics, clot strength and safety of a new fibrinogen concentrate: randomized comparison with active control in congenital fibrinogen deficiency.J. Thromb. Haemost. 2018; 16: 253-261Crossref PubMed Scopus (20) Google Scholar] showing a larger area-under-the curve (normalized) and a slower clearance for fibryga®. Data comparing the effect of different HFC on global hemostasis assays such as thromboelastometry have been published, recently. In an in vitro model of crystalloid-induced dilutional coagulopathy [[5]Haas T. Cushing M.M. Asmis L.M. Comparison of the efficacy of two human fibrinogen concentrates to treat dilutional coagulopathy in vitro.Scand. J. Clin. Lab. Invest. 2018; 78: 230-235Crossref PubMed Scopus (11) Google Scholar], fibryga® led to a significantly higher maximum clot firmness (MCF) in the EXTEM assay, compared to Haemocomplettan® P. The result of this study has been confirmed by Groene et al. [[6]Groene P. Wiederkehr T. Kammerer T. et al.Comparison of two different fibrinogen concentrates in an in vitro model of dilutional coagulopathy.Transfus. Med. Hemother. 2020; 47: 167-174Crossref PubMed Scopus (2) Google Scholar] who have investigated in addition the influence of fibryga® and RiaSTAP®/Haemocomplettan® P on various colloid-induced coagulopathies. Depending on the diluent used, the two HFC showed differences in thrombin time, thromboelastometric coagulation time and MCF. Although similar purification and inactivation steps are included, the manufacturing process of fibrinogen concentrates remains specific for each product, leading to the question whether differences in the manufacturing processes might lead to small but potentially clinically relevant differences in composition. Therefore, the aim of our study was to perform a direct comparison of the biochemical composition of the main three available HFC as this has not been done so far. The products tested were 6 batches of fibryga®, 5 batches of RiaSTAP®/Haemocomplettan® P, and 2 batches of FibClot®/Clottafact®. Reconstitution was performed according to each product specific package insert, resulting in 1 g per vial reconstituted in 50 mL water for injection for fibryga® and RiaSTAP®/Haemocomplettan® P and 1.5 g per vial reconstituted in 100 mL water for injection for FibClot®/Clottafact®. The amount of different parameters, such as fibrinogen concentration and activity, accompanying proteins as well as activation and fibrinolysis markers has been determined. Accompanying proteins were quantified with commercially available ELISA test kits for the content of fibronectin (Technozym® Fibronectin ELISA, Technoclon, Vienna, Austria), von Willebrand Factor (VWF-Enzym-Immunoassay-Testkit VWF:Ag Asserachrom, Diagnostica Stago SAS, Asnières sur Seine, France), and vitronectin (Imubind Vitronectin ELISA Kit, Sekisui Diagnostics GmbH, Pfungstadt, Germany). An in-house ELISA method was performed for albumin. FXIII activity (FXIIIa) was measured using the Berichrom FXIII chromogenic ammonia release assay (Siemens Healthcare Diagnostics, Marburg, Germany). Activation and fibrinolysis markers tested were fibrinopeptide A (Zymutest human FPA, Hyphen Biomed, Neuville-sur-Oise, France), D-Dimer (Asserachrom D-Dimer Kit, Diagnostica Stago SAS, Asnières sur Seine, France), and plasminogen (HemosIL Plasminogen, Instrumentation Laboratory Company, Bedford, MA, USA). Molecular size distribution of proteins was analyzed by size exclusion chromatography using a Superdex 200 column (0.5 cm × 30 cm; Cytiva, Uppsala, Sweden) Phosphate buffered saline was used as running buffer; 20 μL of sample were injected and the elution pattern was followed by UV detection at 280 nm. Except fibrinogen activity, which was tested immediately after reconstitution, product aliquots were stored at −70 °C for all other analysis. Repeated freezing/thawing cycles of the samples were avoided. The results of the biochemical comparison per mL are summarized in Table 1. Taking into consideration that the most commonly suggested initial adult dose is 2–4 g, the amount of parameters available in a dosage of 3 g (mean dosage) has been calculated (Table 2).Table 1Concentration/activity of tested components per mL [mean and standard deviation (SD)]:Parameterfibryga®(n = 6)RiaSTAP®/Haemocomplettan® P(n = 5)FibClot®/Clottafact® (n = 2)MeanSDMeanSDMeanTotal protein [mg/mL]21.81.233.20.616.1Fibrinogen antigen [mg/mL]19.71.321.31.212.6Fibrinogen activityFibrinogen Clauss [mg/mL]24.81.225.80.713.1Clottable protein [mg/mL]19.31.720.90.614.0Specific activityaAccording to the European Pharmacopoeia 01/2008:0024 Specific activity [clottable protein/(total protein-albumin)]; VWF, Von Willebrand Factor; SEC, size exclusion chromatography. [%]90.37.789.14.587.0Accompanying proteinsFibronectin [μg/mL]14.34.7931.898.1128.0VWF antigen [U/mL]0.20.033.80.50.5Vitronectin [μg/mL]<0.013–0.040.010.12Albumin [mg/mL]0.420.119.650.350.0035FXIII activity [U/mL]3.90.21.10.32.1Activation and fibrinolysis markerD-dimer [μg/mL]0.070.020.360.050.12Fibrinopeptide A [ng/mL]6.82.275.021.797.0Plasminogen [U/mL]0.040.010.040.010.02Molecular size distributionSEC polymers [%]2.80.522.11.31.8a According to the European Pharmacopoeia 01/2008:0024 Specific activity [clottable protein/(total protein-albumin)]; VWF, Von Willebrand Factor; SEC, size exclusion chromatography. Open table in a new tab Table 2Amount/activity of tested components in a 3 g dosage.Parameterfibryga®RiaSTAP®/Haemocomplettan® PFibClot®/Clottafact®MeanMeanMeanTotal protein [g]3.35.13.2Fibrinogen antigen [g]3.03.32.6Fibrinogen activityFibrinogen Clauss [g]3.63.92.6Clottable protein [g]3.03.02.8Accompanying proteinsFibronectin [mg]2.114025.6VWF antigen [U]30570100Vitronectin [μg]2.16.024.0Albumin [mg]6314490.7FXIII activity [U]585165420Activation and fibrinolysis markerD-dimer [μg]115424Fibrinopeptide A [μg]0.911.419.4Plasminogen [U]664 Open table in a new tab When compared to each other and to human plasma as a reference, the performed biochemical comparison revealed differences in the composition of the three HFC tested. The differences found were put in relation to thrombus formation, stabilization, and firmness. Vitronectin, a thrombus stabilizer with a plasma concentration of 200–500 μg/mL, was available in trace amounts only in RiaSTAP®/Haemocomplettan® P and FibClot®/Clottafact® and even below the detection limit in fibryga®, therefore these differences might not have any impact on thrombus stabilization. The albumin concentration was different in all three HFC. The much higher concentration of albumin in RiaSTAP®/Haemocomplettan® P is related to the fact that albumin is added as a stabilizer within the manufacturing process. However, in all three HFC the albumin concentration was far below the lower limit of normal plasma (33–52 mg/mL). The same is valid for the D-dimer concentration which was in all three HFC below the cutoff value of ≤0.5 μg/mL fibrinogen equivalent units, defined to exclude thromboembolic events. Although fibrinopeptide A (FPA), a cleavage product of thrombin-induced proteolytic cleavage of fibrinogen, was 10 times and 13 times increased in RiaSTAP®/Haemocomplettan® P and FibClot®/Clottafact®, respectively, compared to human plasma (< 7.6 ng/mL), a higher concentration might have no impact on clot stability as FPA has a very short half-life of 3–5 min. The concentration in fibryga® was below the limit of human plasma. Activated plasminogen produces plasmin which main function is blood clot fibrinolysis. As the plasminogen concentration in all three HFC was in the same range and very low compared to human plasma (1 U/mL), there might be no impact on thrombus formation in a patient at all. In comparison to the results of the tested parameters above that have most likely no clinically relevant impact, main differences have been seen in the concentration/activity of fibronectin, VWF, and FXIII. The fibronectin concentration in fibryga® and FibClot®/Clottafact® was below the plasma concentration (~ 300 μg/mL) whereas the content in RiaSTAP®/Haemocomplettan® P is approximately 3 times higher. A higher amount of fibronectin in RiaSTAP®/Haemocomplettan® P compared to fibryga® has already been shown by Haas et al. [[5]Haas T. Cushing M.M. Asmis L.M. Comparison of the efficacy of two human fibrinogen concentrates to treat dilutional coagulopathy in vitro.Scand. J. Clin. Lab. Invest. 2018; 78: 230-235Crossref PubMed Scopus (11) Google Scholar]. Although elevated plasma fibronectin levels are potentially associated with atherothrombosis and venous thromboembolism [[7]Pecheniuk N.M. Elias D.J. Deguchi H. Averell P.M. Griffin J.H. Elevated plasma fibronectin levels associated with venous thromboembolism.Thromb. Haemost. 2008; 100: 224-228Crossref PubMed Scopus (14) Google Scholar], a higher fibronectin concentration in a HFC might have no impact on thrombus formation as fibronectin is catabolized with a half-life of 20–30 h. The same might be valid for Von Willebrand factor (VWF) who is involved in primary hemostasis. The amount of VWF:Ag in fibryga® is below, FibClot®/Clottafact® is equal to and RiaSTAP®/Haemocomplettan® P is at least 2.4 times higher than the amount in human plasma (0.36–1.57 U/mL). Although a higher VWF concentration can be an advantage in cardiac surgery (loss of VWF in patients with aortic stenosis and in patients with prolonged cardiopulmonary bypass time or need for extracorporeal membrane oxygenation), it is associated with an increased risk of thrombosis, e.g. in patients with cirrhosis undergoing liver transplantation [[8]Kalambokis G.N. Oikonomou A. Christou L. Baltayiannis G. High von willebrand factor antigen levels and procoagulant imbalance may be involved in both increasing severity of cirrhosis and portal vein thrombosis.Hepatology. 2016; 64: 1383-1385Crossref PubMed Scopus (9) Google Scholar]. However, a higher VWF concentration in a HFC might be of no clinical relevance as VWF has a relatively short half-life of approximately 15 h. This might be different for factor XIII, a protein complex circulating in plasma in an activity of 0.77–1.69 U/mL (mean value 1.2 ± 0.3 U/mL). Activated factor FXIII (FXIIIa) links fibronectin with fibrin, thereby enhancing the fibrin clot structure, stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. It has been demonstrated that with little or no FXIIIa, red blood cells are extruded from clots during clot contraction [[9]Byrnes J.R. Duval C. Wang Y. et al.Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin a-chain crosslinking.Blood. 2015; 126: 1940-1948Crossref PubMed Scopus (84) Google Scholar]. Comparing FXIII activity in the three HFC with the activity in human plasma, showed that RiaSTAP®/Haemocomplettan® P was equal to human plasma, whereas the activity in FibClot®/Clottafact® was 1.8 times and in fibryga® 3.2 times higher. A higher FXIII activity in fibryga® compared to RiaSTAP®/Haemocomplettan® P has already been reported by Haas et al. [[5]Haas T. Cushing M.M. Asmis L.M. Comparison of the efficacy of two human fibrinogen concentrates to treat dilutional coagulopathy in vitro.Scand. J. Clin. Lab. Invest. 2018; 78: 230-235Crossref PubMed Scopus (11) Google Scholar]. As FXIII has a long half-life of 11–14 days, it might be that a higher activity contributes, to a certain extent, to the stabilization of the clot. This assumption is supported by the study performed by Kaserer et al. [[10]Kaserer A. Casutt M. Sprengel K. et al.Comparison of two different coagulation algorithms on the use of allogenic blood products and coagulation factors in severely injured trauma patients: a retrospective, multicentre, observational study.Scand. J. Trauma Resusc. Emerg. Med. 2018; 26https://doi.org/10.1186/s13049-017-0463-0Crossref PubMed Scopus (9) Google Scholar] where 1250 U FXIII has been administered after each 6 g of fibrinogen concentrate or at a FXIIIa <60% to improve clot firmness and stability. Whether a higher concentration of fibronectin and/or VWF is related to a higher risk of thrombosis or a higher activity of FXIII improves clot firmness and stability can only be answered by clinical studies with a head-to-head comparison of the three HFC. A. Neisser-Svae study conception, data acquisition, data analysis and interpretation, manuscript writing, final approval. O. Hegener manuscript revision, final approval. K. Görlinger data interpretation, manuscript revision, final approval. Klaus Görlinger is employed by the company TEM Innovation GmbH. Oliver Hegener is employed by the company Octapharma AG. Andrea Neisser-Svae is employed by the company Octapharma PPGmbH.