Virus inactivation at moderately low pH varies with virus and buffer properties.

BACKGROUND: Virus inactivation is a critical operation in therapeutic protein manufacturing. Low pH buffers are a widely used strategy to ensure robust enveloped virus clearance. However, the choice of model virus can give varying results in viral clearance studies. Pseudorabies virus (SuHV) or herpes simplex virus-1 (HSV-1) are frequently chosen as model viruses to demonstrate the inactivation for the herpes family. RESULTS: In this study, SuHV, HSV-1, and equine arteritis virus (EAV) were used to compare the inactivation susceptibility at pH 4.0 and 4°C. SuHV and HSV-1 are from the same family, and EAV was chosen as a small, enveloped virus. Glycine, acetate, and citrate buffers at pH 4.0 and varying buffer strengths were studied. The inactivation susceptibility was found to be in the order of SuHV > HSV > EAV. The buffer effectiveness was found to be in the order of citrate > acetate > glycine. The smaller virus, EAV, remained stable and infectious in all the buffer types and compositions studied. CONCLUSION: The variation in inactivation susceptibility of herpes viruses indicated that SuHV and HSV cannot be interchangeably used as a virus model for inactivation studies. Smaller viruses might remain adventitiously infective at moderately low pH.

Physiochemical properties of enveloped viruses and arginine dictate inactivation.

BACKGROUND: Therapeutic protein manufacturing would benefit by having an arsenal of ways to inactivate viruses. There have been many publications on the virus inactivation ability of arginine at pH 4.0, but the mechanism of this inactivation is unknown. This study explored how virus structure and solution conditions enhance virus inactivation by arginine and leads to a better understanding of the mechanism of virus inactivation by arginine. RESULTS: Large diameter viruses from the Herpesviridae family (SuHV-1, HSV-1) with loosely packed lipids were highly inactivated by arginine, whereas small diameter, enveloped viruses (equine arteritis virus (EAV) and bovine viral diarrhea virus (BVDV)) with tightly packed lipids were negligibly inactivated by arginine. To increase the inactivation of viruses resistant to arginine, arginine-derivatives and arginine peptides were tested. Derivates and peptides demonstrated that a greater capacity for clustering and added hydrophobicity enhanced virus inactivation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) detected increases in virus size after arginine exposure, supporting the mechanism of lipid expansion. CONCLUSIONS: Arginine most likely interacts with the lipid membrane to cause inactivation. This is shown by larger viruses being more sensitive to inactivation and expansion of the viral size. The enhancement of arginine inactivation when increased hydrophobic molecules are present or arginine is clustered demonstrates a potential mechanism of how arginine interacts with the lipid membrane.