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

Abstract

This study is dedicated to the rapid removal of protein aggregates and viruses from plasma-derived human serum albumin (HSA) product to reduce the risk of viral contamination and increase biosafety. A two-step filtration approach was implemented to first remove HSA aggregates and then achieve high model virus clearance using a nanocellulose-based filter paper of different thicknesses, i.e., 11 μm (prefilter) and 22 μm (virus filter) at pH 7.4 and room temperature. The pore size distribution of these filters was characterized by nitrogen gas sorption analysis. Dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography (SE-HPLC) were performed to analyze the presence of HSA aggregates in process intermediates. The virus filter showed high clearance of a small-size model virus, i.e., log10 reduction value (LRV) > 5, when operated at 3 and 5 bar, but a distinct decrease in LRV was detected at 1 bar, i.e., LRV 2.65–3.75. The throughput of HSA was also dependent on applied transmembrane pressure as was seen by Vmax values of 110 ± 2.5 L m−2 and 63.6 ± 5.8 L m−2 at 3 bar and 5 bar, respectively. Protein loss was low, i.e., recovery > 90%. A distribution of pore sizes between 40 nm and 60 nm, which was present in the prefilter and absent in the virus filter, played a crucial part in removing the HSA aggregates and minimizing the risk of virus filter fouling. The presented results enable the application of virus removal nanofiltration of HSA in bioprocessing as an alternative to virus inactivation methods based, e.g., on heat treatment.

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. ,, …”

“… 19 , 26 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 25 and human serum albumin. 27 …”

“…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 …”