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

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.

Would 20 nm Filtered Fetal Bovine Serum-Supplemented Media Support Growth of CHO and HEK-293 Cells?

“…For brevity, the detailed procedures for filter-making and characterization can be found in previous publications from our group in open access sources. 24 , 25 , 27 …”

“…For brevity, the detailed procedures for listed proteomic characterizations can be found in previous publications from our group in open access sources. 24 , 25 , 27 Additionally, for the determination of levels of specific protein biomarkers, i.e. LC3B and β-actin, Western blotting was performed with immunoblotting antiobodies.…”

“…The nanocellulose-based filters were prepared by hot-pressing a nonwoven cellulose wet-cake mass formed by draining a nanocellulose dispersion over a supporting membrane. For brevity, the detailed procedures for filter-making and characterization can be found in previous publications from our group in open access sources. ,, …”

“…To evaluate the nanocellulose-based filter’s applicability for serum-supplied cell culture applications, FBS (1:20, v/v) solution was filtered with these filter papers. The nanocellulose-based filter paper is a nonwoven virus removal size-exclusion filter ,− that was previously shown useful for upstream bioprocessing of basal and chemically defined media supplemented with insulin-transferrin-selenite (ITS). , Considering the high complexity of FBS, the prefiltration with 11-μm thick filter paper was performed prior to the filtration with 33-μm thick filter paper as described previously for the nanofiltration of plasma-derived prothrombin complex concentrate and human serum albumin …”

Aggregate Removal Nanofiltration of Human Serum Albumin Solution Using Nanocellulose-Based Filter Paper

“…When applied in downstream bioprocessing, the filter exhibited 5-6 LRV of ΦX174 (28 nm) or MS2 (27 nm) phages during the filtration of spiked human plasma-derived intravenous immunoglobulin (IVIG) at 3 bar [40]. Recently, the filtration of plasma-derived human coagulation factor IX-rich prothrombin complex with mille-feuille filter paper was shown [41].…”