Two-Step Size-Exclusion Nanofiltration of Prothrombin Complex Concentrate Using Nanocellulose-Based Filter Paper.

Levon Manukyan,Albert Mihranyan,Athanasios Mantas,Eugen Golubev,Mikhail Razumikhin,Andrey Katalevsky

doi:10.3390/biomedicines8040069

Abstract

Coagulation Factor IX-rich protrhombin complex concentrate (FIX-PCC) is a therapeutic biologic product that consists of a mixture of several human plasma-derived proteins, useful for treating hemophilia B. Due to its complex composition, FIX-PCC is very challenging to bioprocess through virus removing nanofilters in order to ensure its biosafety. This article describes a two-step filtration process of FIX-PCC using a nanocellulose-based filter paper with tailored porosity. The filters were characterized with scanning electron microscopy (SEM), cryoporometry with differential scanning calorimetry, and nitrogen gas sorption. Furthermore, in order to probe the filter’s cut-off size rejection threshold, removal of small- and large-size model viruses, i.e., ΦX174 (28 nm) and PR772 (70 nm), was evaluated. The feed, pre-filtrate, and permeate solutions were characterized with mass-spectrometric proteomic analysis, dynamic light scattering (DLS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analytical size-exclusion high-performance liquid chromatography (SEHPLC). By sequential filtration through 11 μm pre-filter and 33 μm virus removal filter paper, it was possible to achieve high product throughput and high virus removal capacity. The presented approach could potentially be applied for bioprocessing other protein-based drugs.

Highlights

Replacement therapy using plasma-derived Factor IX (FIX) products is a life-saving treatment for patients with hemophilia B
High purity FIX is obtained from prothrombin complex concentrate (PCC), which is a mixture of vitamin K-dependent clotting factors, e.g., factor II, V, VII, IX, and X, and clotting inhibitors, e.g., protein C, Z, and S [2]
FIX-rich PCC was provided by National Center for Hematology, Moscow, Russia, as lyophilized powder

Summary

Introduction

Replacement therapy using plasma-derived Factor IX (FIX) products is a life-saving treatment for patients with hemophilia B. Both recombinant and plasma-derived FIX show high efficacy in clinical trials [1]. High purity FIX is obtained from prothrombin complex concentrate (PCC), which is a mixture of vitamin K-dependent clotting factors, e.g., factor II (prothrombin), V, VII, IX, and X, and clotting inhibitors, e.g., protein C, Z, and S [2]. PCC preparation is a highly complex mixture of proteins and may contain up to 50% of proteins other than. Both highly purified FIX and PCC can be used for hemophilia B treatment [4]. PCC preparation may be useful for prevention of bleeding due to overdose of oral anticoagulants and liver dysfunctions [2,3]

Methods

Discussion

Conclusion