Effective cell-based production processes of virus particles are the foundation for the global availability of classical vaccines, gene therapeutic vectors, and viral oncolytic treatments. Their pr...

Fucosylated chondroitin sulfate (FucCS) is a unique marine glycosaminoglycan that exhibits diverse biological functions, including antiviral and anticoagulant activity. In previous work, the FucCS derived from Pentacta pygmaea (PpFucCS) showed moderate anticoagulant effect but high inhibitory activity against the Wuhan strain of severe acute respiratory syndrome coronavirus (SARS-CoV-2). In this study, we perform free-radical depolymerization of PpFucCS by the copper-based Fenton method to generate low molecular weight (MW) oligosaccharides. PpFucCS oligosaccharides were structurally analyzed by ¹H nuclear magnetic resonance spectroscopy and were used to conduct structure–activity relationship studies regarding their effects against SARS-CoV-2 and clotting. Anticoagulant properties were measured by activated partial thromboplastin time, protease (factors Xa and IIa) inhibition by serine protease inhibitors (antithrombin [AT] and heparin cofactor II [HCII]), and competitive surface plasmon resonance (SPR) assay using AT, HCII, and IIa. Anti-SARS-CoV-2 properties were measured by the concentration-response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 Delta variant spike (S)-protein and competitive SPR assays using multiple S-proteins (Wuhan, N501Y [Alpha], K417T/E484K/N501Y [Gamma], L542R [Delta], and Omicron [BA.2 subvariant]). Cytotoxicity of native PpFucCS and oligosaccharides was also assessed. The PpFucCS-derived oligosaccharide fraction of the highest MW showed great anti-SARS-CoV-2 Delta activity and reduced anticoagulant properties. Results have indicated no cytotoxicity and MW dependency on both anti-SARS-CoV-2 and anticoagulant effects of PpFucCS, as both actions were reduced accordingly to the MW decrease of PpFucCS. Our results demonstrate that the high-MW structures of PpFucCS is a key structural element to achieve the maximal anti-SARS-CoV-2 and anticoagulant effects.

Steric exclusion chromatography (SXC) is a promising purification method for biological macromolecules such as the Orf virus (ORFV) vector. The method’s principle is closely related to conventional polyethylene glycol (PEG) precipitation, repeatedly implementing membranes as porous chromatographic media. In the past decade, several purification tasks with SXC showed exceptionally high yields and a high impurity removal. However, the effect of varying process parameters, on the precipitation success and its limitations to SXC, is not yet well understood. For this reason, the precipitation behavior and SXC adaptation for ORFV were investigated for the PEG/ORFV contact time, the membranes pore size, and the type and concentration of ions. All three parameters influenced the ORFV recoveries significantly. A small pore size and a long contact time induced filtration effects and inhibited a full virus recovery. The application of salts had complex concentration-dependent effects on precipitation and SXC yields, and ranged from a complete prevention of precipitation in the presence of kosmotropic substances to increased efficiencies with Mg²⁺ ions. The latter finding might be useful to reduce PEG concentrations while maintaining high yields. With this knowledge, we hope to clarify several limitations of SXC operations and improve the tool-set for a successful process adaptation.

The steric exclusion chromatography (SXC) is a rather new method for the purification of large biomolecules and biological nanoparticles based on the principles of precipitation. The mutual steric exclusion of a nonionic organic polymer, i.e., polyethylene glycol (PEG), induces target precipitation and leads to their retention on the chromatographic stationary phase. In this work, we investigated the application of latex particles in the SXC by altering the particle’s surface charge as well as the PEG concentration and correlated both with their aggregation behavior. The parameters of interest were offline precipitation kinetics, the product recovery and yield, and the chromatographic column blockage. Sulfated and hydroxylated polystyrene particles were first characterized concerning their aggregation behavior and charge in the presence of PEG and different pH conditions. Subsequently, the SXC performance was evaluated based on the preliminary tests. The studies showed (1) that the SXC process with latex particles was limited by aggregation and pore blockage, while (2) not the aggregate size itself, but rather the aggregation kinetics dominated the recoveries, and (3) functionalized polystyrene particles were only suitable to a limited extent to represent biological nanoparticles of comparable size and charge.