Optimization of cell culture‐derived influenza A virus particles purification using sulfated cellulose membrane adsorbers

Abstract

Downstream processing remains one of the biggest challenges in manufacturing of biologicals and vaccines. This work focuses on a Design of Experiments approach to understand factors influencing the performance of sulfated cellulose membrane adsorbers for the chromatographic purification of a cell culture-derived H1N1 influenza virus strain (A/Puerto Rico/8/34). Membranes with a medium ligand density together with low conductivity and a high virus titer in the feed stream resulted in optimum virus yields and low protein and DNA content in the product fraction. Flow rate and salt concentration in the buffer used for elution were of secondary importance while membrane permeability had no significant impact on separation performance. A virus loss of 2.1% in the flow through, a yield of 57.4% together with a contamination level of 5.1 pg DNA HAU −1 and 1.2 ng prot HAU −1 were experimentally confirmed for the optimal operating point predicted. The critical process parameters identified and their optimal settings should support the optimization of sulfated cellulose membrane adsorbers based purification trains for other influenza virus strains, streamlining cell culture-derived vaccine manufacturing.

Selection of chromatographic methods for the purification of cell culture-derived Orf virus for its application as a vaccine or viral vector

“…Alternatively, Wolff et al described the successful purification by pseudo-affinity stationary phases based on sulfated carbohydrates with virus yields exceeding 60 % ( Wolff et al, 2010a , b ) for MVA. Similar methods have previously been shown for the purification of Influenza A virus, using sulfated-cellulose membranes as a stationary phase ( Carvalho et al, 2018 ; Fortuna et al, 2018 , 2019 ) with recoveries of up to 81 %. As Scagliarini and co-workers described the affinity of ORFV to heparin ( Scagliarini et al, 2004 ), the application of sulfated cellulose ligands appears possible, as sulfated carbohydrates are known to mimic heparin ligands ( Gallagher et al, 2020 ; Paluck et al, 2016 ).…”

Development of a downstream process for the production of an inactivated whole hepatitis C virus vaccine

“…SCMA was applied for subsequent polishing, employing a pseudo affinity-based orthogonal technique. The method has been described for the purification of the Influenza A virus and the Modified Vaccinia Ankara virus [29][30][31][32][33] . It utilizes the heparin-mimicking effect of sulphated cellulose and should, thus, be widely applicable to viruses with an affinity to heparin.…”

Polysaccharide-based chromatographic adsorbents for virus purification and viral clearance

“…A virus yield of 64% was reached, with a residual DNA level of 3.8 ng DNA/µg HA that remained well above the regulatory recommendations for influenza vaccines (i.e., <10 ng DNA/dose for parenteral administration -knowing that a normal vaccine dose should contain 15 µg HA per IAV strain [131]. In a parallel study [171], the same group applied this DoE method specifically to the optimization of IAV purification using an industrial SCMA prototype [183]. They obtained comparable results regarding the virus yield and the removal of HC-related DNA and protein impurities.…”

“…However, the latest studies have been performed using sulfated CEL MAs [168,171,172,178]. These pseudo-affinity MAs are made of regenerated CEL crosslinked [183] or not [182] before sulfation.…”

“…The RC membrane and most Sart membranes were used in syringe filter-type units packed with 15 membrane layers (75 cm 2 total membrane area), except in the works by Peixoto et al [165] (total membrane areas: 90 cm 2 and 900 cm 2 ), Grein et al [164] (3 membrane layers, 15 cm 2 area), Nestola [177] and Weigel et al [178] where reduced membrane areas were used for small-scale experiments. For SCMA, 10 (50 cm 2 area) [43,178] or 15 (75 cm 2 area) [39] membrane layers were stacked in a stainless steel holder, while Carvalho et al [168] and Fortuna et al [171,172] used smaller membrane surfaces, i.e., 5.65 cm 2 (5 layers) [168] and 2.1 cm 2 (6 layers) [171] or 2.9 cm 2 (15 layers) [172]. c rAcMNPV, (recombinant) Autographa californica multicapsid nucleopolyhedrovirus (baculovirus); AdV-5, adenovirus type 5; rBV, (recombinant) baculovirus (derived from AcMNPV); AeDNV, Aedes aegypti densonucleosis virus; CAdV-2, canine adenovirus type 2; IAV/IBV, influenza A/influenza B virus; MVA, modified vaccinia Ankara virus.…”