Characterization of protein unable to bind von Willebrand factor in recombinant factor VIII products

Abstract

Background: Therapeutic products with coagulation factor VIII (FVIII) have a wide range of specific activities, implying presence of protein with altered structure. Previous studies showed that recombinant FVIII products (rFVIII) contain a fraction (FVIII FT) unable to bind von Willebrand factor (VWF) and reported to lack activity. Because of loss of function(s), FVIII FT can be defined as a product-related impurity, whose properties and levels in rFVIII products should be investigated. Objective: To isolate and characterize the FVIII FT fraction in rFVIII products. Methods: Protein fractions unable (FVIII FT) and able (FVIII EL) to bind VWF were isolated from rFVIII products using immobilized VWF affinity chromatography (IVAC) and characterized by gel electrophoresis, immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. Results and Conclusions: A robust IVAC methodology was developed and applied for analysis of 10 rFVIII products marketed in the United States. FVIII FT was found at various contents (0.4%-21.5%) in all products. Compared with FVIII EL , FVIII FT had similar patterns of polypeptide bands by gel electrophoresis, but lower functional activity. In several representative products, FVIII FT was found to have reduced sulfation at Tyr1680, important for VWF binding, decreased interaction with a low-density lipoprotein receptor-related protein 1 fragment, and faster plasma clearance in mice. These findings provide basic characterization of FVIII FT and demonstrate a potential for IVAC to control this impurity in rFVIII products to improve their efficacy in therapy of hemophilia A.

Characterization of interaction between blood coagulation factor VIII and LRP1 suggests dynamic binding by alternating complex contacts

“…For quantification of LRP1 and LDLR expression, immunoblot band intensities were determined by Image Studio Lite (LI‐COR Biosciences). 56 …”

“…Chameleon Duo Pre‐Stained Protein Ladder (LI‐COR) was used as control. For quantification of LRP1 and LDLR expression, immunoblot band intensities were determined by Image Studio Lite (LI‐COR Biosciences) 56 …”

Shifting Paradigms and Arising Concerns in Severe Hemophilia A Treatment

“…This impurity raises concerns about the product's efficacy and may increase the risk of forming FVIII inhibitors ( Table 3 ). 81 Besides increased FVIII inhibitors, other adverse events such as skin reactions, headache, arthralgia, pyrexia, upper respiratory tract infection, and nasopharyngitis are also associated with rFVIII concentrates. 82…”

Plasma Clearance of Coagulation Factor VIII and Extension of Its Half-Life for the Therapy of Hemophilia A: A Critical Review of the Current State of Research and Practice

“…The potential and used FVIII designs involve (i) shielding its LRP1/LDLR-binding sites on the LCh, together with (ii) shielding its VWF-binding site (to decouple FVIII from the VWF-dependent plasma clearance pathway), (iii) increasing FVIII affinity to VWF, (iv) affecting FVIII carbohydrate moieties, and (v) making FVIII single-chained (SCh) which stabilizes the molecule by preventing its dissociation during purification, etc. Notably, the SCh form retains FVIII major functions [79] and was found to be present in all preparations of rFVIII [80]. In many designs, (vi) removal of the large, heavily glycosylated B-domain, believed dispensable for FVIII activity [81], is considered favorable for both the manufacturing process and gene therapy applications.…”

“…Notably, all rFVIII products contain a protein unable to bind endogenous VWF, in contrast to pdFVIII. This fraction may constitute ≤20% of total protein and, having fast plasma clearance rate, may affect its overall plasma half-life upon infusion [80].…”