(189s) Arginine Interactions Resulting in Virus Inactivation

Therapeutic proteins treat a range of diseases including cancers, infections, autoimmunity/inflammation, and genetic disorders. To guarantee the effectiveness and safety of the final product, advancements in the manufacturing process are desired. For example, the low ph and/or surfactant treatment used to inactivate enveloped viruses during therapeutic protein manufacturing can cause aggregation or fragmentation of the final protein product. To maintain the stability and structure of proteins during viral clearance, it is favorable to keep pH levels above 4.0 or detergent concentrations low; however, established processes do not meet these criteria. One solution to increase protein stability and yields is to use the synergistic effects of arginine. Arginine can inactivate enveloped viruses at a less acidic pH (≥ 4) or lower temperature (30-40°C) than conventional methods. However, the mechanisms and optimal conditions for inactivation are not understood, and therefore, synergistic arginine viral inactivation is not widespread.

Optimal solution conditions for viral inactivation found in literature are high arginine concentrations (0.7-1M), a time of 60 minutes, and a synergistic factor of moderate temperature (40°C), low pH ( pH 4), or Tris buffer (5 mM). However, full inactivation does not occur over all viruses tested. Viruses that are resistant to arginine inactivation contain membrane proteins with increased stability or a matrix protein that stabilizes the lipids bilayer. Therefore, increasing the stability of protein or lipids limits the effects of arginine. To determine if arginine inactivation relies on protein and/or lipid destabilization, herpes simplex virus-1 (HSV-1), bovine viral diarrhea virus (BVDV), equine arteritis viral (EAV), and pseudorabies virus (PRV) were subjected to arginine inactivation. Similar to literature, inactivation varied across different viruses. For example, HSV-1 inactivated to higher levels than BVDV across all solutions conditions. HSV-1 is a large enveloped virus with a diameter of 100-300 nm while BVDV has a diameter of 50-65 nm. The smaller diameter of BVDV increases lipid packing density, likely opposing membrane deformations and manipulations by arginine. The correlation between low viral inactivation and tightly packed lipids supports the mechanism of lipid destabilization by arginine. In addition to testing several viruses, a range of solution conditions were tested to optimize viral inactivation and determine arginine mechanisms. Agmatine, a derivative of arginine, was tested to understand the influence of charge on inactivation. Pyrenebutyrate, a hydrophobic cation, was added to arginine solutions to evaluate added hydrophobicity on inactivation. When the mechanisms are known, viral inactivation by arginine may be further enhanced by the addition of functional groups, charges, or molecules to completely inactivate all enveloped viruses.