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With the increased prevalence of new SARS-CoV-2 variants of concern, such as Delta and Omicron, the COVID-19 pandemic has become an ongoing human health disaster, killing millions worldwide. SARS-CoV-2 invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition, heparan sulfate (HS) on the surface of host cells plays an important role as a co-receptor for this viral pathogen–host cell interaction. Our previous studies demonstrated that many sulfated glycans, such as heparin, fucoidans, and rhamnan sulfate have anti-SARS-CoV-2 activities. In the current study, a small library of sulfated glycans and highly negatively charged compounds, including pentosan polysulfate (PPS), mucopolysaccharide polysulfate (MPS), sulfated lactobionic acid, sulodexide, and defibrotide, was assembled and evaluated for binding to the S-proteins and inhibition of viral infectivity in vitro. These compounds inhibited the interaction of the S-protein receptor-binding domain (RBD) (wild type and different variants) with immobilized heparin, a highly sulfated HS, as determined using surface plasmon resonance (SPR). PPS and MPS showed the strongest inhibition of interaction of heparin and S-protein RBD. The competitive binding studies showed that the IC50 of PPS and MPS against the S-protein RBD binding to immobilized heparin was ~35 nM and ~9 nM, respectively, much lower than the IC50 for soluble heparin (IC50 = 56 nM). Both PPS and MPS showed stronger inhibition than heparin on the S-protein RBD or spike pseudotyped lentiviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, PPS and MPS exhibited strong antiviral activities against pseudotyped viral particles of SARS-CoV-2 containing wild-type or Delta S-proteins. 

Background: Although most biologics are produced using recombinant technologies, heparin persists as a product purified from animal tissues. A cell based system for production of heparin would eliminate risk of supply shortage and contamination. Additionally, genetic engineering could yield heparin with improved qualities such as reduced risk of heparin-induced thrombocytopenia. Aims: This work is focused on engineering mammalian cell lines and bioprocess methods to produce recombinant heparin. Methods: The heparan sulfate biosynthetic pathway of mastocytoma cells was genetically engineered to alter the expression of heparan sulfate sulfotransferases. The resulting cell lines were screened for production of anti-FXa activity. Heparan sulfate production from a candidate cell line was tested in chemically defined medium. The recombinant product was characterized structurally and in clotting, anti-protease and heparin induced thrombocytopenia assays. Results: Engineered cells produced heparan sulfate in chemically defined medium with anti-Xa and anti-IIa activity exceeding the requirement for unfractionated heparin despite having lower sulfate content. Chain length was longer than unfractionated heparin. Additionally, binding to platelet factor 4 was reduced compared to unfractionated heparin, suggesting less risk of heparin-induced thrombocytopenia. Conclusion: These results demonstrate the feasibility of producing a substitute for unfractionated heparin from recombinant cell culture. Additionally, recombinant technology may allow production of heparin substitutes with improved properties such as reduced side effects.