Activity of transgene-produced B-domain-deleted factor VIII in human plasma following AAV5 gene therapy.

Adeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically use a B-domain-deleted FVIII transgene, such as human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) assays than in chromogenic-substrate (CS) assays, whereas recombinant FVIII-SQ products had lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (international units per milligram) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assay kits and clinical laboratories, suggesting that intrinsic molecular features are potential root causes. Further experiments in 2 participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from nonhemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate end point to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on clinicaltrials.gov as #NCT02576795 and #NCT03370913 and, respectively, on EudraCT (European Union Drug Regulating Authorities Clinical Trials Database; https://eudract.ema.europa.eu) as #2014-003880-38 and #2017-003215-19.

FIX potency of rFIX-Albumin fusion protein is underestimated by one-stage methods using silica-based APTT reagents.

INTRODUCTION: Higher potency is obtained with chromogenic substrate (CS) methods and one-stage (OS) method with SynthAFax vs silica-based OS methods on analysis of albutrepenonacog alpha (rFIX fused with albumin, rFIX-FP). AIM: Investigation of the effect of contact activator in search for explanation of discrepancy between methods. METHODS: Chromogenic Rox Factor IX method and OS methods with Pathromtin SL, SynthAFax or new OS method variants using different phospholipid emulsions and addition of either colloidal silica to create APTT reagents or addition of human FXIa together with calcium ions, in the latter case omitting contact activation. The effect of (a) adding different amounts of colloidal silica or (b) mixtures of Pathromtin SL and purified phospholipids immediately before addition of FXIa and calcium chloride was also explored. FIX activation via tissue factor/FVIIa was also made. RESULTS: FIX potency of rFIX-FP when using APTT reagents with pure phospholipid emulsions with added colloidal silica was similar to OS method with Pathromtin SL. In contrast, close to 80% higher FIX potency for rFIX-FP, and similar to OS method with SynthAFax and to the CS method, was obtained when FXIa replaced contact activation. No discrepancies were obtained for plasma-derived FIX. Gradual decrease of colloidal silica or decreasing proportion of Pathromtin SL added just before addition of FXIa raised rFIX-FP potency to that obtained with SynthAFax and Rox Factor IX. Supportive results were obtained with the tissue factor/FVIIa method. CONCLUSION: Colloidal silica and Pathromtin SL impair activation of rFIX-FP, causing underestimation of rFIX-FP potency.

New Tools to Study Contact Activation.

The recent availability of a sensitive chromogenic method approach for determination of FXIa activity has been explored for designing sensitive methods for FXIIa and kallikrein, both using FXa formation as the read-out. For both enzymes the assay range 1-10 nmol/L provides a resolution of about 0.8 absorbance units with a total assay time of about 20 min. For studies on activation kinetics, subsampling and extensive dilution can be performed in MES-bovine serum albumin (BSA) buffer pH 5.7 for quenching of enzyme activity and with ensuing determination of FXa generation in a chromogenic FXIa method. Optionally, suitable inhibitors such as aprotinin and/or corn trypsin inhibitor may be included. The stability of FXIa, FXIIa, and kallikrein in MES-BSA buffer was shown to be at least 5 h on ice. In conclusion, the use of a sensitive chromogenic FXIa method either per se or in combination with MES-BSA buffer pH 5.7 are new and potentially valuable tools for the study of contact factor enzymes and their inhibitors. So far, dose-response studies of FXIIa and kallikrein have been limited to purified systems, and hence more data are required to learn whether these new methods might or might not be applicable to the determination of FXIIa and kallikrein activities in plasma.

Characterization of thrombin derived from human recombinant prothrombin.

Thrombin (FIIa) is the key enzyme in haemostasis and acts on several substrates involved in clot formation, platelet activation and feed-back regulation of its own formation. During activation of blood coagulation, FIIa is formed by proteolytic cleavage of prothrombin (FII). In the production of recombinant human FII (rhFII), a key question is whether the thrombin formed has the same properties as endogenous thrombin. We have investigated whether FIIa formed from rhFII and plasma-derived human FII (pdhFII) have the same enzymatic and haemostatic properties against a number of substrates and the same haemostatic capacity in plasma, whole blood and on platelets. Pure FIIa was isolated from rhFII and pdhFII cleaved by recombinant ecarin, and analytical methods were developed to compare the activity of FIIa against different substrates. FIIa derived from rhFII and pdhFII were found to have very similar properties in activating FVIII, FXIII, protein C, platelet aggregation and plasma or whole blood coagulation. Further, the same turnover for S-2366 was found with similar KM. However, activation of FV with rhFIIa was approximately 25% more effective than with pdhFIIa and heparin-enhanced inhibition of rhFIIa by antithrombin was significantly more efficient compared with pdhFIIa with 10% higher inhibition both at steady state and at initial rate conditions. Although differences between the two FIIa preparations using ecarin cleavage were observed, FIIa derived from rhFII administered to human would likely be very similar in activity and function as FIIa formed from endogenous FII.

Criteria for specific measurement of plasminogen (enzymatic; procedure) in human plasma.

There is a lack of well-established criteria for the specific measurement of fibrinolytic variables. On behalf of the SSC the Subcommittee on Fibrinolysis started a process to develop such criteria. This report describes the criteria for the measurement of plasminogen (enzymatic; procedure) in human plasma. Apparently, the most specific methods for determination of plasminogen (enzymatic; procedure) adhere to the principle of streptokinase induced activation of plasminogen and recording of activity using a chromogenic substrate. Incorporation of fibrinogen attenuates the potential effect of elevated fibrinogen or fibrin(ogen) fragments in the plasma sample. The criteria for specific measurement of plasminogen(enzymatic; procedure) are based on this analytical principle. The kinetics and principles of the assay procedure are described, and criteria as well as test methods for criteria are detailed. Guidelines for standardization, quality assurance, analytical sensitivity and establishment of reference intervals are given. The pre-analytical conditions regarding preparation of the patient and the specimen are delineated. Nonstandard abbreviations: SSC, Scientific and Standardization Committee; ISTH, The International Society on Thrombosis and Haemostasis; IFCC, The International Federation of Clinical Chemistry; NCCLS, National Center for Clinical Laboratory Standards; HRG, histidine-rich glycoprotein; Lp(a), lipoprotein (a); SK, streptokinase.