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2021 FDA Science Forum

Development of Immobilized Von Willebrand Factor (VWF) Affinity Chromatography to Characterize a Protein Fraction Unable to Bind VWF in Recombinant Factor VIII Products

Download the Poster (PDF; 0.56 MB)
Authors:
Poster Author(s)
Chun, Haarin, FDA/CBER; Pettersson, John R, FDA/CBER; Shestopal, Svetlana A, FDA/CBER; Wu, Wells W, FDA/CBER; Marakasova, Ekaterina S, FDA/CBER; Olivares, Philip, FDA/CBER; Surov, Stepan S, FDA/CBER; Ovanesov, Mikhail V, FDA/CBER; Shen, Rong-Fong, FDA/CBER; Sarafanov, Andrey G, FDA/CBER
Center:
Contributing Office
Center for Biologics Evaluation and Research

Abstract

Poster Abstract

Introduction

Therapeutic products with coagulation factor VIII (FVIII), used to treat FVIII deficiency (Hemophilia A), have a wide range of specific activities, implying presence of FVIII molecules with altered structures. Previous studies showed that recombinant FVIII products contain a fraction unable to bind VWF, reported to lack activity. The level of FVIIIFT and its clinical relevance should be examined more closely in rFVIII products.

Methods

Protein fractions unable and able (FVIIIEL) to bind VWF were isolated from rFVIII products (FVIIITP) using Immobilized VWF Affinity Chromatography (IVAC) and characterized by gel electrophoresis, immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and plasma clearance in mice.

Results

An IVAC methodology was developed and applied for analysis of ten rFVIII products marketed in the USA. FVIIIFT was found at various contents (0.4 - 21.5%) in all tested products. Compared to FVIIIEL, FVIIIFT had similar patterns of polypeptide bands by gel electrophoresis, but lower chromogenic assay activity. In several products, FVIIIFT was found to have reduced sulfation at Tyr1680, which is important for VWF binding; decreased interaction with a low-density lipoprotein receptor related protein 1 (LRP1) fragment; and faster plasma clearance in mice.

Conclusion

These findings provide some basic characterization of FVIIIFT and demonstrate a potential for IVAC to control FVIIIFT fraction in rFVIII products. This may result in improving quality and efficiency of rFVIII products, thus improving care of Hemophilia A.

Disclaimer: This is an informal communication and it represents the authors' own best judgment. These comments do not bind or obligate FDA.

Poster Image
Development of Immobilized Von Willebrand Factor (VWF) Affinity Chromatography to Characterize a Protein Fraction Unable to Bind VWF in Recombinant Factor VIII Products

Download the Poster (PDF; 0.56 MB)

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